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Eur. J. Biochem.

269, 397412 (2002) FEBS 2002

REVIEW ARTICLE

Plant a-amylase inhibitors and their interaction with insect a-amylases


Structure, function and potential for crop protection
Octavio L. Franco1,2,3, Daniel J. Rigden1, Francislete R. Melo1,3 and Maria F. Grossi-de-Sa1
1

Centro Nacional de Recursos Geneticos e Biotecnologia, Cenargen/Embrapa, Braslia-DF, Brazil; 2Universidade Catolica de Braslia, Braslia-DF, Brazil; 3Depto. de Biologia Celular, Braslia-DF, Brazil

Insect pests and pathogens (fungi, bacteria and viruses) are responsible for severe crop losses. Insects feed directly on the plant tissues, while the pathogens lead to damage or death of the plant. Plants have evolved a certain degree of resistance through the production of defence compounds, which may be aproteic, e.g. antibiotics, alkaloids, terpenes, cyanogenic glucosides or proteic, e.g. chitinases, b-1,3-glucanases, lectins, arcelins, vicilins, systemins and enzyme inhibitors. The enzyme inhibitors impede digestion through their action on insect gut digestive a-amylases and proteinases, which play a key role in the digestion of plant starch and proteins. The natural defences of crop plants may be improved through the use of transgenic technology. Current research in the area focuses particularly on weevils as these are highly dependent on starch for their energy supply. Six dierent a-amylase inhibitor classes, lectin-like, knottin-like, cereal-type, Kunitz-like, c-purothionin-like and thaumatin-like could be used in pest control. These classes of inhibitors show remarkable structural variety leading to dierent modes of inhibition and dierent

specicity proles against diverse a-amylases. Specicity of inhibition is an important issue as the introduced inhibitor must not adversely aect the plant's own a-amylases, nor the nutritional value of the crop. Of particular interest are some bifunctional inhibitors with additional favourable properties, such as proteinase inhibitory activity or chitinase activity. The area has beneted from the recent determination of many structures of a-amylases, inhibitors and complexes. These structures highlight the remarkable variety in structural modes of a-amylase inhibition. The continuing discovery of new classes of a-amylase inhibitor ensures that exciting discoveries remain to be made. In this review, we summarize existing knowledge of insect a-amylases, plant a-amylase inhibitors and their interaction. Positive results recently obtained for transgenic plants and future prospects in the area are reviewed. Keywords: a-amylase inhibitors; knottin-like; lectin-like; thaumatin-like; Kunitz; cereal-type; bean weevils; bifunctional inhibitors.

Insect pests and pathogens such as fungi, bacteria and viruses are together, responsible for severe crop losses. Worldwide, losses in agricultural production due to pest attack are around 37%, with small-scale farmers hardest hit [1]. Starchy leguminous seeds are an important staple food

Correspondence to O. L. Franco, Centro Nacional de Recursos Geneticos e Biotecnologia, Cenargen/Embrapa, S.A.I.N. Parque Rural, Final W5, Asa Norte, Biotecnologia, Laboratory 05, CEP: 70770900, Bras lia-DF, Brazil, Fax: + 55 61 340 3624, Tel.: + 55 61 448 4705, E-mail: ocfranco@cenargen.embrapa.br Abbreviations: AAI, Amaranthus a-amylase inhibitor; a-AI1 and a-AI2, a-amylase inhibitors 1 and 2 from the common bean; AMY1 and AMY2, a-amylases from barley seeds; BASI, barley a-amylase subtilisin inhibitor; BLA, Bacillus licheniformis a-amylase; CAI, cowpea a-amylase inhibitor; CHFI, corn Hageman factor inhibitor; HSA, human salivary a-amylase; LCAI, Lachrima jobi chitinase/ a-amylase inhibitor; PAI, pigeonpea a-amylase inhibitor; PPA, porcine pancreatic a-amylase; RASI, rice a-amylase/subtilisin inhibitor; RBI, ragi bifunctional inhibitor; SIa1, SIa2 and SIa3, Sorghum a-amylase inhibitors 13; TASI, triticale a-amylase/subtilisin inhibitor; TMA, Tenebrio molitor a-amylase; WASI, wheat a-amylase subtilisin inhibitor; ZSA, Zabrotes subfasciatus a-amylase. (Received 28 August 2001, accepted 6 November 2001)

and a source of dietary protein in many countries. These seeds are rich in protein, carbohydrate and lipid and therefore suffer extensive predation by bruchids (weevils) and other pests. The larvae of the weevil burrow into the seedpods and seeds and the insects usually continue to multiply during seed storage. The damage causes extensive losses, especially if the seeds are stored for long periods. In general, plants contain a certain degree of resistance against insect predation, which is reected in the limited number of insects capable of feeding on a given plant. This resistance is the result of a set of defence mechanisms acquired by plants during evolution [2]. It is only recently that many secondary chemical compounds have been denitively associated with plant defence, for example through their synthesis in response to pest or pathogen attack. Plant defences are, however, incomplete, as bruchids and other insects are able to infest seeds and different plant tissues despite the presence of plant defence compounds. Two factors seem to have contributed to this phenomenon. First, many plants suffer reductions in defence compounds during domestication [3]. Thus, the selection of bettertasting plants with better nutritional value has led, concomitantly, to crops that are more susceptible to predation. Secondly, just as plants evolve defences, their predators evolve means to evade those defence mechanisms; this is the

398 O. L. Franco et al. (Eur. J. Biochem. 269)

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phenomenon of host-parasite coevolution, as described by Ehrlich & Raven [4]. Among these means are detoxication or excretion of the defence compound, or simple adaptation of the predator so that the toxin no longer has any effect. The relationship between leguminous seed plants and seed weevils provides an excellent example of host-parasite coevolution. The seeds are rich in defence compounds so that the majority of possible predators cannot eat them, yet seed weevils thrive on the same seeds. Plant defence compounds include antibiotics, alkaloids, terpenes, cyanogenic glucosides and some proteins. Among these proteins are chitinase and b-1,3glucanase enzymes, lectins, arcelins, vicilins, systemins and enzyme inhibitors [58]. The enzyme inhibitors act on key insect gut digestive hydrolases, the a-amylases and proteinases. Several kinds of a-amylase and proteinase inhibitors, present in seeds and vegetative organs, act to regulate numbers of phytophagous insects [911]. a-Amylase inhibitors are attractive candidates for the control of seed weevils as these insects are highly dependent on starch as an energy source. The use of a-amylase inhibitors, through plant genetic engineering, for weevil control will be the focus of this review. The properties of insect a-amylases and available inhibitors will be reviewed and issues affecting their specicity of interaction addressed.

INSECT a-AMYLASES
a-Amylases (a-1,4-glucan-4-glucanohydrolases, EC 3.2.1.1) constitute a family of endo-amylases that catalyse the hydrolysis of a-D-(1 4)-glucan linkages in starch components, glycogen and other carbohydrates. The enzyme plays a key role in carbohydrate metabolism of microorganisms, plants and animals. Moreover, several insects, especially those similar to the seed weevils that feed on starchy seeds during larval and/or adult stages, depend on their a-amylases for survival. Research on starch digestion as a target for control of starch-dependent insects was stimulated in recent years after results showed that a-amylase inhibitors from Phaseolus vulgaris seeds are detrimental to the development of cowpea weevil Callosobruchus maculatus and Azuki bean weevil Callosobruchus chinensis [12,13]. The carbohydrate digestion of bruchid weevils, such as the Mexican bean weevil Zabrotes subfasciatus and the cowpea weevil C. maculatus, occurs mainly in the lumen of the midgut. High enzymatic activities against starch, maltose, maltodextrins and galactosyl oligosaccharides were found in the luminal uid, while only aminopeptidase activity was predominantly associated with gut membrane [14]. In the yellow mealworm Tenebrio molitor, the a-amylases are synthesized in anterior midgut cells and packed in the Golgi area into secretory vesicles that undergo fusion, as they migrate to the cell apex. At the same time, the cell apex undergoes structural disorganization with the disappearance of cell organelles. Eventually, the apical cytoplasm with the large amylase-containing membranous structure is discharged into the midgut lumen. After extruding the apical cytoplasm, the cell apparently remains functional, as cells are found to lack the cell apex, but have all the other normal ultra structural features [15]. To validate insect a-amylases as targets for crop protection, it is important to research their variety and understand how the expression of different forms is controlled. Studies

in this area are at an early stage, although some important observations have been made. The presence of different forms of a-amylases in the insect midgut lumen has been observed in C. maculatus and Z. subfasciatus [14,16]. Patterns of a-amylase expression vary in Z. subfasciatus fed on different diets, apparently in response to the presence of antimetabolic proteins such as a-amylase inhibitors, rather than as a response to structural differences in the starch granules. Bean bruchids, such as the Mexican bean weevil larvae, also have the ability to modulate the concentration of a-glucosidases and a-amylases when reared on different diets [14]. Although the sequences of several insect a-amylases are known [17,18], the only three-dimensional insect a-amylase yet determined is that of the T. molitor enzyme (TMA). This enzyme is well adapted to the slightly acidic physiological environment of the larval midgut with a pH optimum of 5.8 for the cleavage of starch [19]. The structure of TMA comprises a single polypeptide chain of 471 amino-acid residues, one calcium ion, one chloride ion and 261 water molecules (Fig. 1 [20]). The protein folds into three distinct domains, named A, B and C (Fig. 1). Domain A, the major structural unit of TMA is composed of two segments (residues 197 and 160379; green in Fig. 1) and forms a (b/a)8-barrel; an eight-stranded, parallel b-barrel embraced by a concentric circle of eight helical segments (seven a helices and one 310-helix). This domain contains the

Fig. 1. Ribbon three-dimensional structure of Tenebrio molitor a-amylase (PDB code 1tmq). The domain A, B and C are coloured in green, red and orange, respectively. The structure contains one calcium ion (yellow) and one chloride ion (cyan). The gure was made using MOLSCRIPT 2 [148] as were all other gures except Fig. 2A.

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Plant a-amylase inhibitors (Eur. J. Biochem. 269) 399

catalytic site and the ligand binding residues [20,21]. Domain B is globular and is inserted into domain A. It is formed by several extended segments and a short a helix (residue 98159; red in Fig. 1). This domain forms a cavity against the b barrel of domain A in which the calcium ion is bound. This cation is of fundamental importance for the structural integrity of a-amylases [20,22]. Finally, domain C is located exactly opposite to domain B on the other side of domain A. The C domain comprises the C-terminal residues 380471 (orange in Fig. 1) and forms a separated folding unit, exclusively made of b sheet. Eight of the 10 strands fold into a b sandwich structure with Greek key topology. The conservation of the interface of A and C domains among a-amylases from different sources suggests an important role for enzyme activity, stability and folding [20]. In the porcine pancreatic a-amylase (PPA), the interface between C and A domains contains a secondary starch-binding site, occupied by maltose in one crystal structure [23], but it remains to be seen if this is also the case for TMA. TMA, in common with almost all determined a-amylase structures (the exception being that of calcium-depleted Bacillus licheniformis a-amylase (BLA) [21], contains a calcium ion at a conserved position (yellow in Fig. 1). The removal of the calcium ion in BLA causes local disorder around the Ca2+-binding site, resulting in an inactive enzyme [24]. The calcium-binding site of TMA is located at the interface between domains A and B (Fig. 1), near to the catalytic centre. The Ca2+ ion is important for activity due its contact with His189. This histidine residue interacts with the fourth sugar of the substrate, bound in the active site, forming a hinge between the catalytic-site and the Ca2+-binding site [20]. TMA crystal structures also contain a chloride anion (cyan in Fig. 1). The chloride may be capable of allosterically activating TMA [19] due to its proximity to a water molecule, which probably initiates

substrate cleavage [25]. The nucleophilicity of this water molecule might be enhanced by the negative charge of the chloride anion. The enzymatic mechanism of a-amylases has not yet been completely elucidated. It is likely that different a-amylases have a similar mechanism of action with catalytic residues conserved among all the enzymes [26,27]. Three acidic side chains in PPA (Asp197, Glu233 and Asp300) (Fig. 2B), corresponding to Asp185, Glu222 and Asp287 in TMA [20] are directly involved in catalysis [28]. The polysaccharide-binding groove of a-amylases can accommodate at least six sugar units, as observed crystallographically in PPA [28], and cleavage occurs between the third and the fourth pyranose residues. The reaction is believed to proceed by a double displacement mechanism [27].

a-AMYLASE INHIBITORS
Nonproteinaceous inhibitors The class of nonproteinaceous inhibitors contains diverse types of organic compounds such as acarbose, isoacarbose, acarviosine-glucose, hibiscus acid and the cyclodextrins (Fig. 2A). The two hibiscus acid forms, puried from Roselle tea (Hibiscus sabdaria), the acarviosine-glucose, the isoacarbose and a-, b- and c-cyclodextrins are highly active against porcine and human pancreatic a-amylase (PPA and HPA) [2932]. The inhibitory activity of these compounds against a-amylases is due in part to their cyclic structures, which resemble a-amylase substrates and therefore bind to a-amylase catalytic sites. In previous X-ray crystallography studies [33], three a-cyclodextrins molecules I-III bound to PPA. a-Cyclodextrin I and II bound near to the catalytic binding cleft, while a-cyclodextrin III biound at

Fig. 2. The structures of nonproteinaceous a-amylases and acarbose green bound to the catalytic site of PPA. (A) Nonproteinaceous a-amylase inhibitors. (B) The structure of acarbose green bound to the catalytic site of PPA. Only enzyme residues making hydrogen bonds (dashed lines) or hydrophobic contacts with the inhibitor are shown. The three catalytic acidic residues are labeled.

400 O. L. Franco et al. (Eur. J. Biochem. 269)

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an accessory site. The a- and b-cyclodextrin are not hydrolyzed to any signicant extent by a-amylases, except by fungi amylases [34,35]. In contrast, human saliva a-amylase (HSA) and PPA are capable of hydrolysing c-cyclodextrin [36]. The cyclodextrin mechanism of PPA inhibition is pH-, temperature- and substrate-dependent. When amylose is used as substrate, the inhibition is of the competitive type, but when maltopentose is used, the inhibition becomes noncompetitive [37]. PPA inhibition by acarbose, in contrast, is noncompetitive, irrespective of substrate [38]. The structure of PPA with acarbose, a pseudotetrasaccharide (Fig. 2B), bound to the active site has been determined [39]. Two linked identical acarbose fragments occupy the PPA active site (coloured green in Fig. 2B), hindering substrate hydrolysis. These acarbose fragments are bonded to residues from the a-amylase active site by a hydrogen-bonding network (Fig. 2B). No displacement of acarbose-binding residues is required for acarbose binding, compared to their positions in the empty enzyme structure, enhancing the effectiveness of the inhibition [39]. The valienamine ring (Fig. 2A) of acarbose is considered to be crucial in the inhibition mechanism of a-glucosidases, a-amylases and other amylolytic enzymes [40,41]. Its unsaturated structure and half-chair conformation are reminiscent of a planar oxocarbonium ion, proposed as either a transition state or an intermediate during the hydrolytic pathway of glucosidases [42]. The properties of nonproteinaceous inhibitors make them interesting in the eld of medicine, both for treatment [43] and in diagnostic procedures [44]. Nevertheless, the use of nonproteinaceous inhibitors for production of insect resistant transgenic plants is much more difcult. The production of acarbose or organic acids in plants is very complex and several metabolic pathways are involved. Hence, the presence of multiple expressed transgenes would be required in order to confer protection. In this area, the proteinaceous inhibitors, coded by a single gene, are more suitable. Proteinaceous inhibitors Proteinaceous a-amylase inhibitors are found in microorganisms, plants and animals [5,4547]. In plants, proteinaceous inhibitors are mainly present in cereals such as wheat Triticum aestivum [46,48,49], barley Hordeum vulgareum [50], sorghum Sorghum bicolor [51], rye Secale cereale [47,52] and rice Oryza sativa [53] but also in leguminosae such as pigeonpea Cajanus cajan [54], cowpea Vigna unguiculata [55] and bean P. vulgaris [56,57]. These inhibitors have showed monomeric molecular masses of 5 kDa [51], 9 kDa [55] and 13 kDa [49], homodimeric and heterodimeric masses of  26 kDa [49,57] and tetrameric masses of 50 kDa [58]. Different plant a-amylase inhibitors exhibit different specicities against a-amylases from diverse sources (Table 1). Determination of specicity of inhibition is the important rst step towards the discovery of an inhibitor that could be useful for generating insect-resistant transgenic plants. In some cases, the a-amylase inhibitors act only against mammalian a-amylases or, on the contrary, just against insect a-amylases. In the latter case, this provides a highly specic potential weapon in plant defence. a-AI2, AAI and some wheat inhibitors are among those naturally possessing favourable inhibition proles (Table 1). However, in general, a-amylase inhibitors inhibit several a-amylases from

different sources. In these cases, an improved understanding of the structural bases for inhibition proles (as discussed later) may enable the rational design of mutants with more desirable characteristics. As proposed by Richardson [59], a-amylase inhibitors may be conveniently classied by their tertiary structure (Table 2) into six classes: lectin-like, knottin-like, cereal-type, Kunitz-like, c-purothionin-like and thaumatin-like. Lectin like a-amylase inhibitors a-AIs has been puried and characterized from different accessions and varieties of the common bean P. vulgaris, including the white, red and black kidney beans [58,60 62]. The best-characterized isoform, known as a-AI1, was cloned and identied as an a-amylase inhibitor homologous to phytohemagglutinin (PHA) [63]. A second variant of a-AI, called a-AI2, is found in some wild accessions of the common bean [56]. These two allelic variants have different inhibition specicities. a-AI1 inhibits PPA as well as the a-amylases of the C. maculatus and C. chinensis, but it does not inhibit the a-amylases of the Z. subfasciatus (ZSA). In contrast, a-AI2 does not inhibit the rst three amylases mentioned above but it does inhibit ZSA [56]. To reach their active mature form, comprising two noncovalently bound glycopeptide subunits, a and b, of 7.8 and 14 kDa, respectively [57,64], bean a-AIs are posttranslationally modied. The proteolysis leading to the activation of a-AI1 has been studied by mass spectrometry. A simple cleavage at the carboxyl side of Asn77, presumably by an Asn-specic seed protease of previously demonstrated importance in legume lectin processing [65], is made and Asn79 is removed apparently by the action of a carboxypeptidase. Furthermore, 19 residues at the C-terminus of the b chain of a-AI1 are clipped. a-AI2 shows similar cleavages to a-AI1, but a somewhat different glycosylation pattern [57]. Both variant inhibitors in their mature form have a heterotetrameric structure of two a chains and two b chains [58,66] and are highly glycosylated [57]. a-AIs contain glycans attached to Asn63 and Asn67 [57] but these may not be necessary for inhibitory activity [67]. A third isoform, a-AIL (also known as a-AI3), isolated from P. vulgaris cv Rico 23 is a single-chain a-amylase inhibitor-like protein completely inactive towards all a-amylases tested [68]. This protein, as with the insecticidal isoform Grp29 [69], may represent an evolutionary intermediate between phytohaemagglutinnins, arcelins and a-amylase inhibitors [68,157,158]. Interestingly, another noncleaved member of this inhibitor group specically inhibits fungal a-amylases [70] and additionally possesses hemagglutination activity, showing that these two activities are not mutually exclusive and that cleavage probably is not a prerequisite for a-amylase inhibition. The formation of the inhibitorenzyme complex for this class of a-amylase inhibitors is pH-, time- and concentration-dependent [56,62]. One heterotetramer of a-AI1 binds to and inhibits two molecules of PPA with KD 10)10 M [58]. To elucidate the inhibitory mechanism of these inhibitors, the structures of the common bean a-AI1 in complex with PPA [71] and TMA [72] have been determined. Structural analysis demonstrated that two hairpin

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Plant a-amylase inhibitors (Eur. J. Biochem. 269) 401

Table 1. Activity of amylase inhibitors from dierent plant sources against mammalian and insect a-amylases. Low activity represents less than 40% of the maximum activity. Inhibitory activities Inhibitors a-AI1 Source P. vulgaris Mammalian PPA Insect Callosobruchus maculatus Callosobruchus chinensis Diabrotica virgifera virgifera Hypothenemus hampei Tenebrio molitor Zabrotes subfasciatus Diabrotica virgifera virgifera Lygus hesperus Lygus lineoralis Diabrotica virgifera virgifera Callosobruchus maculatus Zabrotes subfasciatus Acanthoscelides obtectus Tenebrio molitor Sitophilus oryzae Tribolium castaneum Tenebrio molitor Callosobruchus maculatus Zabrotes subfasciatus Acanthoscelides obtectus Tenebrio molitor Sitophilus oryzae Tribolium castaneum Tenebrio molitor Callosobruchus maculatus Zabrotes subfasciatus Tenebrio molitor Sitophilus oryzae Tribolium castaneum Callosobruchus maculatus Tenebrio molitor (low) Sitophilus oryzae Tenebrio molitor Zabrotes subfasciatus Acanthoscelides obtectus Tenebrio molitor Hypothenemus hampei Prostephanus truncatus Callosobruchus maculatus (low) Helicoverpa armigera (low) Tribolium castaneum Sitophilus zeamais Rhyzoperta dominica Locusta migratoria Periplaneta americana References [12,18,31,149]

a-AI2 Wheat Extract 0.19

P. vulgaris T. aestivum

None activity PPA and HSA

[56,150] [49,151]

T. aestivum

PPA and HSA

[18,46,49,97]

0.53

T. aestivum

HSA and PPA (low)

[46,90,97]

0.28 WRP25

T. aestivum T. aestivum

PPA and HSA None

[97] [46,49]

WRP26

T. aestivum

None

[46,49]

WRP27 1,2 and 3 BIII AAI

T. aestivum S. cereale S. cereale A. hypochondriacus

None HSA HSA and PPA None activity

[49] [52] [47] [77,82,149,152]

CAI PAI Zeamatin

V.unguiculata C. cajan Z mays

None HSA and PPA None activity

[55] [54] [112,113]

SIa1, SIa2 and SIa3

S. bicolor

HSA (low)

[51]

loops of a-AI1 (residues 2946 and 171189) were inserted into the TMA reactive site (Fig. 3A), blocking substrate binding and establishing a hydrogen bond network with the residues of the substrate-binding region. The catalytic residues are strongly bonded to the inhibitor residues Tyr186 and Tyr37 that occupies the catalytic pocket. When compared to results obtained in the PPAa-AI1 complex, the strong contacts in the catalytic clefts are highly conserved and only slight modications occur in the extended proteinprotein interaction [71,72]. Bean amylase

inhibitors have been extensively used in transgenic plants due to their insecticidal properties. Kidney bean crude extracts containing lectin-like a-amylase inhibitors originally found in vivo, were used as starch blockers in the early 1980s, for the control of human noninsulin-dependent diabetes mellitus and obesity [43,44,73,74]. Those early attempts were unsuccessful due to the undesirable presence of PHAs and proteinase inhibitors in the extract. Later work with puried a-AI in diabetic patients met with more success [73]. More

402 O. L. Franco et al. (Eur. J. Biochem. 269)


This inhibitor class was not classied by CATH program. bOrengo et al. [154]. c1, 2, and 3 from CATH code represents mainly a helix, mainly b sheets and mixed a helix and b sheets, respectively.

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AA1 0.19, 0.53, 0.28 WRP25, WRP26, WRP27 & RBI BASI, WASI & RASI

a-A11 & a-A12

SIa1, SIa2 & SIa3

recently this class of inhibitors has been used for its insecticidal properties to protect seeds for insect predation [13,75,76]. Knottin-type a-amylase inhibitors The a-amylase inhibitor from Amaranthus hypocondriacus seeds (AAI) is the smallest proteinaceous inhibitor of a-amylases yet described, with just 32 residues and three disulde bonds [77]. The structure of its inhibitor, as determined by NMR [78,79], contains a knottin fold; three antiparallel b strands and a characteristic disulde topology. It revealed structural similarity to other proteins such as the proteinase inhibitor from Cucubirta maxima [80], charybdotoxin and conotoxins [81]. AAI specically inhibits insect a-amylases and is inactive against mammalian a-amylases ([77]; Table 1). The structure of its inhibitor in complex revealed that inhibition, as with the lectin-like inhibitors, is through blockage of the catalytic site ([82], Fig. 3B). The inhibitor binds in the active site crevice interacting with catalytic residues from the A and B domains of TMA (Fig. 1 [82]). The residue Asp287, one of the catalytic residues of its enzyme, forms a salt bridge directly with Arg7 of AAI. The other two enzymatic catalytic residues, as well other conserved residues involved in substrate recognition and orientation, are connected to AAI via an intricate water-mediated hydrogen-bonding network [82]. The TMAAAI complex is characterized by a high complementarity of the interaction surfaces (Fig. 3B). Structural comparisons of the inhibitor structure in solution [79] to the X-ray structure of AAI bounded to TMA [82] demonstrate that both backbone and side conformations are only slightly adjusted on formation of the complex [79]. The specic activity of AAI against insect a-amylases makes it an attractive candidate for the development of insectresistant transgenic plants. Cereal-type a-amylase inhibitors a-Amylase inhibitors of the cereal family are composed of 120160 amino-acid residues forming ve disulde bonds (Table 2) [46,83,84]. These inhibitors are also known as sensitizing agents in humans upon repeated exposure, causing allergy, dermatitis and baker's asthma associated with cereal our [85,86]. N-glycosylation is involved in the reactivity of the most reactive allergen, an inhibitor from rye [87]. The exogenous wheat a-amylase inhibitor coded 0.19 [46,49] and the bifunctional inhibitor from Indian nger millet RBI [88,89], are the most studied inhibitors from this family. The a-amylase inhibitor 0.19, named according to its gel electrophoretic mobility relative to bromophenol blue, inhibits a-amylases from birds, Bacilli, insects and mammals (Table 1). Its inhibition of human salivary a-amylase is characterized by Ki 0.29 nM [160]. It has 124 amino-acid residues and is homologous to RBI [90]. Mass spectrometry results [46] clearly demonstrated the presence of homodimers of 0.19 with smaller quantities of other multimers, in accord with sedimentation [49] and X-ray crystallography results [91]. Nevertheless, some cereal inhibitors act as monomers, such the wheat inhibitors 0.28, WRP25, WRP26 and WRP27 [46]. The dimeric a-amylase inhibitor 0.19 was crystallized [92] and its structure determined. It contains ve

Names

Concanavalin A-like lectins/glucanases Knottins Bifunctional inhibitor/ lipid-transfer protein/ seed storage 2S albumin b-Trefoil 2.60.120.60c Lectin NDa 1.10.120.10 Cereal inhibitor 3 5 12

CATHb code and Family

SCOP fold

Table 2. Dierent structural classes of a-amylase inhibitors, based on a classication by Richardson [59].

Disulde bonds

58 173235 Maize [112,113,156] Thaumatin type

Residue numbers

32 124160

240250

176181

Source and references

Amaranth [78,79] Wheat [46], barley [59] & Indian nger millet [153]

Common beans [31,56]

Barley [102], wheat [99] & rice [100]

Legume lectin type

Structural class

Knottin type Cereal type

c-Purothionin type

Kunitz type

Sorghum [51]

4748

2.80.10.50.6 Proteinase inhibitor 2.60.110.10 Sweet tasting protein 3.30.30.10 Antibacterial protein

Osmotin Thaumatin-like proteins Knottins

Zeamatin

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Fig. 3. The various known modes of a-amylase inhibition. A standard colouring scheme is used with enzyme drawn in magenta and inhibitor helix, strand and coil drawn in red, cyan and yellow, respectively. The calcium ion common to all structures is drawn in orange. The structures are (A) TMA bound to a-AI1 (PDB code 1viw) (B) TMA-AAI (1clv) (C) TMA-RBI (1tmq) and (D) AMY2-BASI (1ava). In (D) the calcium ion bound at the enzymeinhibitor interface (see text for details) is drawn in green.

a-helices arranged in an up-and-down manner, satisfying favorable packing modes, with all 10 cysteine residues forming disulde bonds [91]. The bifunctional a-amylase/trypsin inhibitor (RBI) is another prototype of the cereal inhibitor family. This inhibitor is a stable monomer of 122 amino acids with ve disulde bonds, which is resistant to urea, guanidine hydrochloride and thermal denaturation [93]. This bifunctional inhibitor presents a three-dimensional structure very similar to that of 0.19 inhibitor [91], with a globular fold with four a helices in a simple up-and-down topology and a small antiparallel b sheet ([94]; Fig. 3C). Like other inhibitors of this class, it can competitively inhibit a variety of a-amylases, including PPA and TMA. This latter enzyme is inhibited with a Ki 15 2 nM [88,89,95].

As the structure of RBITMA complex reveals, the inhibitor binds to the enzyme active site, once again impeding substrate binding (Fig. 3C). Two RBI segments are responsible for the interaction with TMA. Segment 1, comprising the N-terminal residues Ser1Ala11 and the residues Pro52Cys55, protrudes like an arrow head into TMA's substrate-binding groove and directly targets the active site of the enzyme. The N-terminus forms the arrow tip, adopting a 310-helical conformation in complex. The Ser1 residue makes several hydrogen bonds with the catalytic Asp185 and Glu222 from enzyme while Val2 and Ser5 from inhibitor interact with the third conserved acidic residue of the catalytic site, Asp287. The second binding segment comprises several residues, which form a collar around the upper part of the arrow head and stabilize

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the complex by further interactions with this enzyme [88]. Tests with a peptide containing the N-terminal 10 residues of RBI showed that only segment 1 is necessary for a-amylase inhibition. Synthetic peptides containing mutated N-terminal RBI sequences demonstrated different inhibitory potentials [89]. Alam et al. [89] also showed that this inhibitor binds to soluble amylase substrate, reducing the apparent afnity of the enzyme for the substrate. Inhibitorsubstrate interactions could explain the differences in the type of inhibition observed for different substrates with the same enzyme [37,89]. Exploiting the overall structural similarity between RBI and 0.19, molecular models of 0.19TMA and 0.19HSA complexes have been constructed [46]. Multiple genes encode the members of the cereal-type inhibitor family [96]. Their different sequences yield a remarkable array of inhibition specicity proles (Table 1 [46,52]). In wheat, some a-amylases inhibitors genes may be silent or expressed at a much lower level [96]. It can be envisaged that the lack of the pertinent predator in the ecological niche could silent the respective inhibitor gene [97]. Kunitz-like a-amylase inhibitors The Kunitz-like a-amylase inhibitors contain around 180 residues and four cystines (Table 2). They are present in cereals such as barley [98], wheat [99] and rice [100]. The best-characterized a-amylase inhibitor from the Kunitz class is the barley a-amylase/subtilisin inhibitor (BASI), a bifunctional double-headed inhibitor with a fast tight inhibitory reaction with cereal a-amylase AMY2 (Ki 0.22 nM) and serine proteinases of the subtilisin family [50,101]. The structure of BASI [102] revealed two disulde bonds and a b trefoil topology (Fig. 3D) shared with the homologous wheat a-amylase subtilisin inhibitor (WASI [103]), the Erythrina cara trypsin inhibitor [104] and the ricin B chain [105]. In the cases of a-AI1, AAI and RBI, inhibition involves the insertion of inhibitor loops into the a-amylase active site, thereby establishing a network of hydrogen bonds with catalytic and substrate-binding residues (Fig. 3AC). The mechanism of inhibition of barley a-amylase 2 (AMY2) by BASI [102] is different, in that the inhibitor does not interact directly with any catalytic acidic residues of the enzyme. Nevertheless, this inhibitor interacts strongly with both the A and B domains near the catalytic site, through the formation of 12 hydrogen bonds, two salt bridges and multiple van der Waal's contacts, and thereby prevents substrate access (Fig. 3D). A cavity at the enzymeinhibitor interface contains a trapped calcium ion whose presence is suggested to electrostatically enhance the network of water molecules at the complex interface and thereby raises the stability of the complex. BASI is involved in regulating the degradation of seed carbohydrate, preventing the endogenous a-amylase 2 from hydrolysing starch during premature sprouting [41]. Additionally, it protects the seeds against exogenous proteinases and a-amylases produced by various pathogens and pests [106]. BASI inhibits the endogenous enzyme with a stoichiometry of 1 : 1 [107], but, interestingly, is unable to inhibit barley a-amylase 1 (AMY1), which bears 74% sequence identity to AMY2 [101].

Thaumatin-like a-amylase inhibitors type The thaumatin-like inhibitors are proteins with molecular masses of  22 kDa with signicant sequence similarity to pathogenesis-related group 5 (PR-5) proteins and to thaumatin, an intensively sweet protein from Thaumatococcus danielli fruit [108,109]. The best-characterized inhibitor from this class is zeamatin, a bifunctional inhibitor from Zea mays that is homologous to the sweet protein thaumatin. Zeamatin has a total of 13 b strands, 11 of which form a b sandwich at the core of protein ([110]; Fig. 4A). Several loops extend from this inhibitor core and are secured by one or more of the

Fig. 4. The structural classes of a-amylase inhibitors whose modes of inhibition are not yet known (A) zeamatin (PDB code 1 dl5) and (B) SIa1 (1gpt). The coordinates of SIa1 have not been deposited in the PDB so that (B) shows the structure of the homologous c-thionin [124].

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eight disulde bonds. Electrostatic modelling of zeamatin reveals an electrostatically polarized surface, heavily populated with Arg and Lys residues [110]. This maize inhibitor is not sweet, despite its similarity to thaumatin, probably due to changes in a putative receptor-binding site [111]. Zeamatin was able to inhibit porcine pancreatic trypsin and digestive a-amylases of the insects T. castaneum, Sitophilus zeamays and Rizopherta dominica [112,113]. Other proteins from this class, such as the thaumatin-like proteins R and S from barley seeds, did not show any inhibitory activity against trypsin or a-amylases [114] despite their highly similar N-terminal sequences. Zeamatin is mainly known for its antifungal activity, but this is not related to inhibition of hydrolyic enzymes as this protein does not inhibit fungal a-amylases [112] and fungi do not contain trypsin. Zeamatin binds to b-1,3-glucans [115] and permeabilizes fungal-cells leading to cell death [116] but the antifungal mode of action of this protein is still a matter of debate. For these properties, zeamatin could be used as a medical agent, acting on vaginal murine candidosis cells [117] or in transgenic plants, increasing their resistance against pests and pathogens. c-Purothionins-like a-amylase inhibitors The a-amylase inhibitors of this family have 47 or 48 residues, are sulfur-rich and form part of the c-thionin superfamily (Table 2). Members of this superfamily are involved in plant defence through a remarkable variety of mechanisms: modication of membrane permeability [118,119], inhibition of protein synthesis [120] and proteinase inhibition [121] Inhibition of insect a-amylases has been observed for three isoforms from Sorghum bicolor called SIa-1, SIa-2 and SIa-3 [51]. These molecules strongly inhibited the digestive a-amylases of guts of locust and cockroach, poorly inhibited a-amylases from A. oryzae and human saliva and failed to inhibit the a-amylases from porcine pancreas, barley and Bacillus sp. [51]. The structure of SIa-1 has been solved by NMR [122] revealing a a + b sandwich structure [123] with a nine-residue helix packed tightly against the sheet (Fig. 4B). The helix is held in place by two disulde bridges, which link sequential turns of the helix to residues 41 and 43 in the middle of strand b3, the so-called cysteine-stabilized helix (CSH) motif [122]. As expected from sequence comparison, the structure is similar to those of wheat c1-purothionin [124] and scorpion toxins [122].

BIFUNCTIONAL INHIBITORS
As the above discussion highlights, bifunctional a-amylase/ proteinase inhibitors are relatively common (Table 3). As inhibition of predatory insect digestive proteinases is another attractive route for plant protection, the combination of a-amylase and proteinase inhibition is potentially very useful. It is therefore important to know whether simultaneous inhibition of proteinase and a-amylase is possible for these inhibitors. In the case of RBI, with two independent inhibition sites, formation of a stable a-amylaseRBItrypsin complex has been observed [95]. The N-terminal site is responsible for a-amylase inhibition, as previously discussed, while on the opposite side a canonical substrate-like trypsin inhibitor region is present [89,94,125,126]. In this inhibitor, the exposed trypsin-binding loop is located between two a-helices, contains the residues Gly32Tyr37 and also contains Arg34 that confers trypsin-specicity. Modelling of the TMARBItrypsin complex (Fig. 5; [88]), conrms

Fig. 5. A model of a ternary complex formed by TMA (magenta), RBI (blue) and pancreatic bovine trypsin (green). Constructed as described in the text.

Table 3. Activity of bifunctional inhibitors from dierent plant sources against mammalian and insect a-amylases. Low activity represents approximately 40% of a total activity. a-Amylase inhibition Inhibitor BASI RASI WASI CHIF Zeamatin RBI LCAI Source Barley Rice Wheat Maize Maize Finger millet Jobi tear's seeds Insect + + ND + + + + Fungal ND ND ND ND ND ND Plant + + + ND ND ND ND Mammalian ND ND ND Low + References [50,102] [53,100] [99,103] [128] [112,113] [88] [132] Other activity Subtilisin Inhibition Subtilisin Inhibition Subtilisin Inhibition Trypsin Inhibition Trypsin Inhibition Trypsin Inhibition Chitinase

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that no steric clashes prevent simultaneous inhibition of both enzymes. The homologous bifunctional corn Hageman factor inhibitor (CHIF) inhibits mammalian trypsin, Factor XIIa (Hageman factor) of the contact pathway of coagulation as well a-amylases from several insects [127]. Its structure has been solved revealing a similar proteinase inhibitory site to that in RBI [128]. As with RBI, a-amylase inhibition requires the N-terminal region [129]. Kunitz-like inhibitors are also bifunctional, this time possessing inhibitory activity against the subtilisin class pf proteinases. Among them, the BASI and WASI are the best characterized, and both structures have been determined by X-ray crystallography [102,103]. It has been suggested that WASI has two different sites because the activity against a-amylases is retained after incubation with proteinases K [130]. A complex of WASIproteinase K showed that a loop containing the Gly66 and Ala67 is crucial to proteinase inhibition [131]. This class of bifunctional inhibitors inhibits insect and endogenous plant a-amylases but not mammalian a-amylases [53,100,101]. They are also inactive against different classes of proteinases such as trypsins and chymotrypsins [53]. BASI is deposited during grain lling and therefore found in the seed prior to AMY2, which is synthesized de novo during germination. This inhibitor has been proposed to control the activity of AMY2 in the case of premature sprouting or to act in plant defence [159]. The inhibitor RASI could also help to regulate seed development by inhibiting a development-specic a-amylase [53]. This a-amylase inhibition specicity is in agreement with a dual role in starch control of the storage tissues at plant developmental stages and as defensive agents in response to pest attack. Zeamatin represents a third bifunctional a-amylase/ proteinase inhibitor [112,113] with a known crystal structure [110]. However, the observation of trypsin inhibition, albeit weak, was only made recently [113] and it remains to be seen whether or not zeamatin possesses independent sites for proteinase and a-amylase inhibition. In addition to bifunctional a-amylase/proteinase inhibition, a single report has found chitinase activity present in an insect a-amylase inhibitor isolated from Lachrima jobi seeds [132]. Chitinase activity is another recognized plant defence [7] so that this double activity is of great potential biotechnological interest. However, further characterization of the inhibitor is clearly required.

ISSUES OF a-AMYLASE INHIBITOR SPECIFICITY


In order to be of practical use for the production of transgenic plants, a-amylase inhibitors should have appropriate specicity proles. On the one hand, they should ideally be effective against the full range of potential predatory insects. However, they must not interfere with the action of endogenous a-amylases, which are of demonstrated importance in, for example, germination [41]. They should also lack activity against the mammalian enzymes, although this is in general a lesser issue as cooking would denature any inhibitors before ingestion. These simple considerations, in combination with biochemical data in the literature (Tables 1 and 3), already highlight some inhibitors as more promising candidates than others. For example, the cereal bifunctional a-amylase/subtilisin inhibitors have strong

afnity for plant enzymes [107] deriving from their role in regulation of starch metabolism, and are therefore less favoured. The known a-amylase inhibitors selective for insect enzymes and inactive against mammalian enzymes include WRP25, WRP26 and WRP27 from the cereal-type class [46], the Amaranthus a-amylase inhibitor [77] and zeamatin [113]. As well as differences in afnity for broad groups of a-amylases, some remarkable examples of ne specicity exist. Among several interesting specicity differences in the cereal-type inhibitors is the inability of WRP26 to inhibit ZSA, while WRP25, 98% identical in sequence to WRP26, is an effective inhibitor [46]. Given the remarkable structural and functional variety naturally found among a-amylase inhibitors (Tables 13), screening for inhibitors with desirable characteristics is a viable option. An attractive alternative, however, would be the rational redesign of known inhibitors in order to confer upon them the required specicity prole. Although conceivably more rapid than the screening approach, inhibitor redesign clearly requires a full understanding of amylaseinhibitor interaction structural bases. With the availability of an ever-increasing number of crystal structures a number of recent studies have addressed experimentally observed issues of amylase-inhibitor specicity, generally through sequence analysis and modelling, sometimes supported by mutagenesis studies [46,62,82,133,134]. Two independent studies [62,133] address the specicity of a-AI1 for PPA, not inhibiting ZSA and the opposite specicity of a-AI2 for ZSA over PPA [56]. The reliance on simple counting of hydrogen bonds weakens the conclusions of Le Berre-Anton et al. [135] regarding the a-AI1/a-AI2 comparison but they show that the bulkier, single chain a-AIL is sterically impeded from binding either ZSA or PPA. In another study, analysis of other factors, including electrostatics and hydrophobic interactions, fails to lead to a simple explanation of a-AI1/a-AI2 specicity and the authors concluded that specicity was conferred by multiple factors [133]. In studies aimed at explaining the ability of BASI to inhibit AMY2 but not AMY1, previous indications of the importance of electrostatic interactions [136] have been examined through site-directed mutagenesis [134]. Mutations of AMY2 residues known to make electrostatic interactions at the interface with this inhibitor, Arg128 and Asp142, were made, weakening the enzyme inhibitor interaction and reducing the effect of charge screening on the interaction. Remarkably, the introduction of just two AMY2 residues, Arg128 and Pro129, into the AMY1 environment was enough to enhance BASI sensitivity at least 100-fold. As well as the electrostatic characteristics of the Arg, the conformational properties of the proline, which forms a cis peptide in AMY2, are implicated in the AMY1/AMY2 specicity. The lysine, which replaces Pro129 in AMY1 is unlikely to form a cis peptide bond, with consequent changes in the conformation of neighbouring Arg128 and other residues at the interface. The varied inhibition specicity proles of a family of cereal a-amylase inhibitors have been addressed by sequence analysis and model building [46]. In the absence of crystal structures for any of the analysed inhibitors in complex with a-amylase, the complex of TMA with the more distantly related RBI [88] was used as the basis for model construc-

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tion. Differences in behaviour between inhibitors sharing as much as 98% sequence identity were successfully explained, enhancing condence in the model. Different factors were found to be involved in conferring specicity towards different amylases, among the size and electrostatic properties of key residues. The different loop lengths present in mammalian and insect a-amylases were found to inuence specicity in some cases but not in others. The conformational differences presumed to result from the presence of either proline or cysteine at a certain position was also suggested to be important. Finally, the report of the structure of TMAAAI was accompanied by an explanation of the inability of AAI to inhibit PPA [82]. Amino-acid differences between insect and mammalian enzymes at the interface leading to reduce hydrogen-bonding capability were suggested as being wholly responsible for specicity, in the absence of any obvious steric impediment to formation of the PPAAAI complex. The consequences of the predicted loss of six of the 10 hydrogen bonds observed at the TMAAAI interface would be exacerbated by the relatively small size of the interface, the absence of signicant hydrophobic interactions and the single favourable electrostatic interaction across the interface [82]. Even with the limited number of investigations of a-amylase/inhibitor specicity so far published, it is clear that a wide variety of factors are probably involved; steric factors [46,134], electrostatic properties [46,134], hydrogen bonding capability [82], the special conformational properties of proline [134] and the disulde bonding of cysteine [46]. It is also important to remember that in vivo conditions may crucially modulate a-amylase specicity. For example, the acidic optimum pH for inhibition by a-AI2 may be responsible for their inhibition of amylases in Coleoptera, whose intestinal contents are acidic, but not of amylases from Lepidoptera, where the intestinal contents are alkaline [137]. The cleavage of inhibitors by insect digestive proteinases [138] may complicate pest control by a-amylase inhibitors and could explain the low in vivo efciency of some inhibitors against insect pests [16]. The a-amylase diversity found in a single insect [14] indicates that unless an a-amylase inhibitor has reasonably broad specicity, being capable of inhibiting all the a-amylases produced by the insect, its incorporation in articial seeds or its expression in transgenic plants would probably have no impact on starch digestion and therefore would not constitute a deterrent against predation of these seeds. Despite the complexity of the problem and the possible in vivo complications, the success of the rational redesign of AMY1, conferring BASI inhibition [134], emphasizes the feasibility of inhibitor redesign and should encourage further analyses.

PRACTICAL ASPECTS OF TRANSGENIC PLANTS EXPRESSING a-AMYLASE INHIBITORS


The transgenic plant approach provides an attractive alternative to the use of chemical pesticides and insecticides and could contribute to the production of crop varieties that are inherently tolerant/resistant to their major target insect pests. Besides the benet on agricultural crop production, the use of genes that encode insecticidal proteins in

transgenic crops also has the potential to benet the environment. The rst reports of transgenic plants appeared in 1984 [139], and since then there has been rapid progress using this new technology for crop improvement. Several different classes of plant proteins have been shown to be insecticidal towards a range of economically important insect pests when tested in articial diets or transgenic plants [11,140]. a-Amylase inhibitors show particular promise against bruchids of stored grains that depend to a large extent on a-amylase activity for survival [10,56,138]. The rst practical demonstration involving a-amylase inhibitors used a-AI1, which specically inhibits the a-amylases of the three Old World bruchids; the pea weevil Bruchus pisorum, the cowpea weevil and the azuki bean weevil. In transgenic pea plants, complete resistance against these bruchids was observed for a-AI1 levels in the range 0.81.0%, with complete larval mortality of the rst or second instars [13,75]. Similar observations were made under eld conditions [76]. Similarly, azuki bean plants expressing a-AI1 were completely resistant to the azuki bean weevil [141]. As a-AI2 is a much less effective inhibitor of pea weevil a-amylase, this inhibitor was only partially effective in protecting eld-grown transgenic peas against pea weevils [76]. Nevertheless, feeding tests carried out with articial diets containing proteinaceous extracts of this a-AI2-expressing transgenic pea showed complete effective ness against Z. subfasciatus (Grossi de Sa, O. L. Franco, F. R. Melo & C. P. Magalhaes, unpublished results). At an earlier stage of development, wheat inhibitors, as potent in vitro inhibitors of the gut hydrolytic enzymes from the larvae of seed storage weevils, are promising future weapons against these pests [46,142144]. Just as important as the proof of protection of transgenic crops against pests is the demonstration that the new crops present no health risk to consumers. This appears to be less of a problem in the case of human consumption as such crops would be cooked before human consumption, with concomitant protein denaturation and inactivation. However, such crops may also be used as animal feed so that potential differences between uncooked normal and transgenic crops should be evaluated. A recent study has addressed this issue by feeding rats with transgenic peas expressing high levels of a-AI1 and monitoring possible effects on intestinal metabolism, growth and starch and protein digestibility [145]. The minimal nutritional differences seen up to a dietary level of 300 g per kg of transgenic pea should encourage the use of transgenic crops as animal feed. A further important factor affecting the practical development of transgenic plants expressing a-amylase inhibitors lies outside the purely scientic arena. The degree of social acceptance of transgenic crops depends on consumer reactions, which in turn depend on the social context of the transgenic technology. Although rigorous specications have been established to ensure the safety of the transgenic products for human health and for the environment, severe consumer doubts remain about whether the transgenic technology is necessary or if it could causes human diseases. For example, if transgenic development is believed to be for the benet of industry and not the consumer, public acceptance is likely to be lower [146]. In addition, the genetic modication of animals is more acceptable if it is applied within a medical

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7. Bishop, J.G., Dean, A.M. & Mitchell-Olds, T. (2000) Rapid evolution in plant chitinases: molecular targets of selection in plant-pathogen co-evolution. Proc. Natl Acad. Sci. USA 97, 53225327. 8. Sales, M.P., Gerhardt, I.R., Grossi-de-Sa, M.F. & Xavier-Filho, J. (2000) Do legumes storage proteins play a role in defending seeds against bruchids? Plant Physiol. 124, 515522. 9. Konarev, A.V. (1996) Interaction of insect digestive enzymes with plant protein inhibitors and host-parasite co-evolution. Euphytica 92, 8994. 10. Chrispeels, M.J., Grossi-de-Sa, M.F. & Higgins, T.J.V. (1998) Genetic engineering with a-amylase inhibitors seeds resistant to bruchids. Seed Sci. Res. 8, 257263. 11. Gatehouse, A.M.R. & Gatehouse, J.A. (1998) Identifying proteins with insecticidal activity: use of encoding genes to produce insect-resistant transgenic crops. Pest. Sci. 52, 165175. 12. Ishimoto, M. & Kitamura, K. (1989) Growth inhibitory eects of an a-amylase inhibitor from kidney bean, Phaseolus vulgaris (L.) on three species of bruchids (Coleoptera: Bruchidae). Appl. Ent. Zool. 24, 281286. 13. Shade, R.E., Schroeder, H.E., Pueyo, J.J., Tabe, L.L., Murdock, T.J.V., Higgins, M.J. & Chrispeels, M.J. (1994) Transgenic pea seeds expressing the a-amylase inhibitor of the common bean are resistant to bruchid beetles. Bio/Technol. 12, 793796. 14. Silva, C.P., Terra, W.R., Xavier-Filho, J., Grossi-de-Sa, M.F., Lopes, A.R. & Pontes, E.G. (1999) Digestion in larvae of Callosobruchus maculatus and Zabrotes subfasciatus (Coleoptera: Bruchidae) with emphasis on a-amylases and oligosaccaridases. Insect Biochem. Mol. Biol. 29, 355366. 15. Cristofoletti, P.T., Ribeiro, A.F. & Terra, W.R. (2001) Apocrine secretion of amylase and exocytosis of trypsin along the midgut of Tenebrio molitor larvae. J. Insect Physiol. 47, 143155. 16. Campos, F.A.P., Xavier-Filho, J., Silva, C.P. & Ary, M.B. (1989) Resolution and partial characterization of proteinases and a-amylases from midguts of larvae of the bruchid beetle Callosobruchus maculatus (F.). Comp. Biochem. Physiol. B 92, 5157. 17. Grossi de Sa, M.F. & Chrispeels, M.J. (1997) Molecular cloning of bruchid (Zabrotes subfasciatus) a-amylase cDNA and interactions of the expressed enzyme with bean amylase inhibitors. Insect Biochem. Mol. Biol. 27, 271281. 18. Titarenko, E. & Chrispeels, M.J. (2000) cDNA cloning, biochemical characterization and inhibition by plant inhibitors of the a-amylases of the Western corn rootworm, Diabrotica virgifera virgifera. Insect Biochem. Mol. Biol. 30, 979990. 19. Buonocore, V., Poerio, E., Pace, W., Petrucci, T., Silano, V. & Tomasi, M. (1976) Interaction of Tenebrio molitor L. a-amylase with wheat our protein inhibitor. FEBS Lett. 67, 202206. 20. Strobl, S., Maskos, K., Betz, M., Wiegand, G., Huber, R., Gomis-Ruth, F.X. & Glockshuber, R. (1998) Crystal structure of yellow mealworm a-amylase at 1.64 A resolution. J. Mol. Biol. 278, 617628. 21. Machius, M., Wiegand, G. & Huber, R. (1995) Crystal structure of calcium-depleted Bacillus licheniformis a-amylase at 2.2 A resolution. J. Mol. Biol. 246, 545559. 22. Valee, B.L., Stein, E.A., Summerwell, W.N. & Fisher, E.H. (1959) Metal content of a-amylases of various origins. J. Biol. Chem. 234, 29012905. 23. Qian, M., Haser, R. & Payan, F. (1995) Carbohydrate binding site in a pancreatic a-amylase-substrate complex, derived from X-ray structure analysis at 2.1 A resolution. Protein Sci. 4, 747 755. 24. Hwang, K.Y., Song, H.K., Chang, C., Lee, J., Lee, S.Y., Kim, K.K., Choe, S., Sweet, R.M. & Suh, S.W. (1997) Crystal structure of thermostable a-amylase from Bacillus licheniformis rened at 1.7 A resolution. Mol. Cells 7, 251258. 25. Mazur, A.K., Haser, R. & Payan, F. (1994) The catalytic mechanism of a-amylases based upon enzyme crystal structures

context than a food-related context [147]. Bearing in mind these factors, it is essential to create an informed consumer who is able to make rational decisions regarding consumption of products and provide input into the strategic development of the science.

CONCLUSIONS AND PERSPECTIVES


The a-amylase inhibitors described in this review have long been proposed as possibly important weapons against insect pests whose diets make them highly dependent on a-amylase activity. In vitro and in vivo trials, including those made under eld conditions, have now fully conrmed this potential, raising the possibility of signicantly increased yields. Equally important, the nutritional value of transgenic strains seems minimally different to natural crops. The structural variety of the a-amylase inhibitors so far characterized is striking, encompassing proteins with mainly alpha, beta and mixed folds. The continuing discovery of new a-amylase inhibitors suggests that the list of a-amylase inhibitors is far from complete. As has been emphasized, consideration of a-amylase inhibitor specicity is of prime importance. The inhibition spectra of inhibitors so far characterized are remarkably variable and include some with the desirable combination of activity against insect enzymes and inactivity towards mammalian enzymes. Equally notable, from a growing number of studies attempting to explain these specicity proles, is the range of biophysical factors, which inuence a-amylase/inhibitor specicity. Although biochemical screening will continue to play an important role in the search for inhibitors with desirable characteristics, a thorough understanding of the structural bases of a-amylaseinhibitor interactions will also enable site directed mutagenesis of existing inhibitors, or design of synthetic peptides, to yield a-amylase inhibitors specic to a small number of pests. Possible environmental side-effects of the transgenic technology will thereby be minimized, helping to achieve consumer acceptance for the transgenic crop.

ACKNOWLEDGEMENTS
The authors thank Charles Dayler for his technical support. This work was supported by Embrapa/Cenargen, CAPES and CNPq, Brazil.

REFERENCES
1. Gatehouse, A.M.R., Boulter, D. & Hilder, V.A. (1992) Potential of plant-derived genes in the genetic manipulation of crops for insect resistance. In Biotechnology in Agriculture, Plant Genetic Manipulation for Crop Protection. Vol. 7, pp. 155181. CAB International, Wallingford, UK. 2. Schuler, H.T., Poppy, M.G., Kerry, B.R. & Denholm, L. (1998) Insect-resistant transgenic plants. Trends Biotechnol. 16, 168174. 3. Hilder, V., Gatehouse, A., Sheerman, S., Barker, R. & Boulter, D. (1987) A novel mechanism of insect resistance engineered into tobacco. Nature 330, 160163. 4. Erlich, P.R. & Raven, P.H. (1964) Butteries and plants: a study in co-evolution. Evolution 1, 18. 5. Ryan, C.A. (1990) Protease inhibitors in plants: genes for improving defenses against insects and pathogens. Annu. Rev. Phytopathol. 28, 425449. 6. Ryan, C.A. & Pearce, G. (1998) Systemin: a polypeptide signal for plant defensive genes. Annu. Rev. Cell Dev. Biol. 14, 117.

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and model binding calculations. Biochem. Bioph. Res. Com. 204, 297302. Svensson, B. (1994) Protein engineering in the a-amylase family: catalytic mechanism, substrate specicity and stability. Plant Mol. Biol. 25, 141157. MacGregor, E.A., Janececk, S. & Svensson, B. (2001) Relationship of sequence and structure to specicity in the a-amylase family of enzymes. Biochem. Biophys. Acta 1546, 120. Machius, M., Vertesy, L., Huber, R. & Wiegand, G. (1996) Carbohydrate and protein-based inhibitors of porcine pancreatic a-amylase: structure analysis and comparison of their binding characteristics. J. Mol. Biol. 260, 409421. Kim, M.-J., Lee, S.-B., Lee, H.-B., Lee, S.-Y., Baek, J.-S., Kim, D., Moon, T.-W., Robyt, J.F. & Park, K.-H. (1999) Comparative study of the inhibition of a-glucosidase, a-amylase, and cyclomaltodextrin glucanosyltransferase by acarbose, isoacarbose, and acarviosine-glucose. Arch. Biochem. Biophys. 371, 277283. Hansawasdi, C., Kawabata, J. & Kasai, T. (2000) a-Amylase inhibitors from Roselle (Hibiscus sabdaria Linn.) tea. Bioscienci. Biotechnol. Biochem. 64, 10411043. Nahoum, V., Roux, G., Anton, V., Rouge, P., Puigserver, A., Bischo, H., Henrissat, B. & Payan, F. (2000) Crystal structures of human pancreatic a-amylase in complex with carbohydrate and proteinaceous inhibitors. Biochem. J. 346, 201208. Qian, M., Nahoum, V., Bonicel, J., Bisho, H., Henrissat, B. & Payan, F. (2001) Enzyme-catalyzed condensation reaction in a mammalian alpha-amylase. High resolution structural analysis of an enzyme inhibitor complex. Biochemistry 40, 77007709. Larson, S.B., Greenwood, A., Cascio, D., Day, J. & McPherson, A. (1994) Rened molecular structure of pig pancreatic a-amy lase at 2.1 A resolution. J. Mol. Biol. 231, 15601584. Suetsugu, N., Koyama, S., Takeo, K. & Kuge, T. (1974) Kinetic studies on the hydrolases of a-, b- and c-cyclodextrins by Takaamylase A. J. Biochem. 76, 5763. Kamitori, S., Kondo, S., Okuyama, K., Yokota, T., Shimura, Y., Tonozuka, T. & Sakano, Y. (1999) Crystal structure of Termo-actynomices vulgaris R.-47 a-amylase II (TVA II) hydrolyzing cyclodextrins and Pullulan at 2.6 A resolution. J. Mol. Biol. 287, 907921. Kondo, H., Nakatami, H. & Hiromi, K. (1990) In vitro action of human and porcine a-amylase on cyclomalto-oligosaccharides. Carbohydrate Res. 204, 207213. Koukiekoulo, R., Desseaux, V., Moreau, Y., Marchis-Mouren, G. & Santimone, M. (2001) Mechanism of porcine pancreatic a-amylase. Inhibition of amylose and maltopentaose hydrolysis by a-, b- and c-cyclodextrins. Eur. J. Biochem. 268, 841848. Koukiekoulo, R., Le Berre-Anton, V., Desseaux, V., Moreau, Y., Rouge, P., Marchis-Mouren, G. & Santimone, M. (1999) Mechanism of porcine pancreatic a-amylase. Inhibition of amylose and maltopentaose hydrolysis by kidney bean (Phaseolus vulgaris) inhibitor and comparison with that by acarbose. Eur. J. Biochem. 265, 2026. Gilles, C., Astier, J.P., Marchis-Mouren, G., Cambillau, C. & Payan, F. (1996) Crystal structure of pig pancreatic a-amylase isoenzyme II, in complex with the carbohydrate inhibitor acarbose. Eur. J. Biochem. 238, 561569. Wilcox, E.R. & Whitaker, J.R. (1984) Some aspects of the mechanism of complexation of red kidney bean a-amylase inhibitor and a-amylase. Biochemistry 3, 17831791. Kadziola, A., Sogaard, M., Svensson, B. & Haser, R. (1998) Molecular structure of a barley a-amylase inhibitor complex: implications for starch binding and catalysis. J. Mol. Biol. 278, 205217. Sinnott, M.L. (1990) Catalytic mechanisms of enzymatic glycosyl transfer. Chem. Rev. 90, 11711202.

Plant a-amylase inhibitors (Eur. J. Biochem. 269) 409


43. Bischo, H., Ahr, H.J., Schmidt, D. & Stoltefuss, J. (1994) Acarbose-ein neues Wirkprinzip in der Diabetes-Therapie. Nachr. Chem. Techn Laboratory 42, 11191128. 44. O'Donnell, M.D., Fitzgerald, O. & McGeeney, K.F. (1997) Dierential serum amylase determination by use of an inhibitor and design of a routine procedure. Clin. Chem. 23, 560566. 45. Silano, V. (1987) a-Amylase inhibitors. In Enzymes and Their Role in Cereal Technology (Kruger, J. & Lineback, D., eds), pp. 141199. American Association of Cereal Chemists, St. Paul, MD, USA. 46. Franco, O.L., Rigden, D.J., Melo, F.R., Bloch Jr, C., Silva, C.P. & Grossi de Sa, M.F. (2000) Activity of wheat a-amylase inhibitors towards bruchid a-amylases and structural explanation of observed specicities. Eur. J. Biochem. 267 (8), 1466 1473. 47. Iulek, J., Franco, O.L., Silva, M., Slivinski, C.T., Bloch Jr, C., Rigden, D.J. & Grossi de Sa, M.F. (2000) Purication, biochemical characterisation and partial primary structure of a new a-amylase inhibitor from Secale cereale (Rye). Inter. J. Biochem. Cell Physiol. 32, 11951204. 48. Petrucci, T., Rab, A., Tomasi, M. & Silano, V. (1976) Further characterization studies of the alpha-amylase protein inhibitor of gel eletrophoretic mobility 0. 19 from the wheat kernel. Biochim. Biophys. Acta 420, 288297. 49. Feng, G.H., Richardson, M., Chen, M.S., Kramer, K.J., Morgan, T.D. & Reeck, G.R. (1996) a-Amylase inhibitors from wheat: a sequences and patterns of inhibition of insect and human a-amylases. Insect Biochem. Mol. Biol. 26, 419426. 50. Abe, J.I., Sidenius, U. & Svensson, B. (1993) Arginine is essential for the a-amylase inhibitory activity of the a-amylase/subtilisin inhibitor (BASI) from barley seeds. Biochem. J. 293, 151155. 51. Bloch Jr, C. & Richardson, M. (1991) A new family of small (5 kD) protein inhibitors of insect a-amylase from seeds of sorghum (Sorghum bicolor (L.) Moench) have sequence homologies with wheat d-purothionins. FEBS Lett. 279, 101104. 52. Garcia-Casado, G.L., Sanchez-Monge, R., Lopez-Otin, C. & Salcedo, G. (1994) Rye inhibitors of animal a-amylases shown dierent specicities, aggregative properties and IgE-binding capacities than their homologues from wheat and barley. Eur. J. Biochem. 224, 525531. 53. Yamagata, H., Kunimatsu, K., Kamasaka, H., Kuramoto, T. & Iwasaki, T. (1998) Rice bifunctional a-amylase/subtilisin inhibitor: characterization, localization, and changes in developing and germinating seeds. Biosc. Biotechnol. Biochem. 62, 978985. 54. Giri, A.P. & Kachole, M.V. (1998) Amylase inhibitors of pigeonpea (Cajanus cajan) seeds. Phytochemistry 47, 197202. 55. Melo, F.R., Sales, M.P., Silva, L.S., Franco, O.L., Bloch,C. Jr & Ary, M.B. (1999) a-Amylase from cowpea seeds. Prot. Pept. Lett. 6, 387392. 56. Grossi de Sa, M.F., Mirkov, T.E., Ishimoto, M., Colucci, G., Bateman, K.S. & Chrispeels, M.J. (1997) Molecular characterization of a bean a-amylase inhibitor that inhibits the a-amylase of the Mexican bean weevil Zabrotes subfasciatus. Planta 203, 295303. 57. Young, N.M., Thibault, P., Watson, D.C. & Chrispeels, M.J. (1999) Post-translational processing of two a-amylase inhibitors and an arcelin from the common bean, Phaseolus vulgaris. FEBS Lett. 446, 203206. 58. Kasahara, K., Hayashi, K., Arakawa, T., Philo, J.S., Wen, J., Hara, S. & Yamaguchi, H. (1996) Complete sequence, subunit structure and complexes with pancreatic a-amylase of an a-amylase inhibitor from Phaseolus vulgaris white kidney beans. J. Biochem. 120, 177183. 59. Richardson, M. (1990) Seed storage proteins: the enzyme inhibitors. In Methods in Plant Biochemistry (Rogers, L., ed.) 5, pp. 261307. Academic Press, London, UK.

26. 27. 28.

29.

30. 31.

32.

33. 34. 35.

36. 37.

38.

39.

40. 41.

42.

410 O. L. Franco et al. (Eur. J. Biochem. 269)


60. Marshall, J.J. & Lauda, C.M. (1975) Purication and properties of phaseolamin, an inhibitor of a-amylase, from the kidney bean, Phaseolus vulgaris. J. Biol. Chem. 250, 80308037. 61. Wilcox, E.R. & Whitaker, J.R. (1984) Characterization of two amylase inhibitors from black bean (Phaseolus vulgaris). J. Food Biochem. 8, 189213. 62. Le Berre-Anton, V., Bompard-Gilles, C., Payan, F. & Rouge, P. (1997) Characterization and functional properties of the a-amylase inhibitor (a-AI) from kidney bean (Phaseolus vulgaris) seeds. Biochem. Biophys. Act. 1343, 3140. 63. Moreno, J. & Chrispeels, M.J. (1989) A lectin gene encodes the a-amylase inhibitor of the common bean. Proc. Natl Acad. Sci. USA 86, 78857889. 64. Yamaguchi, H. (1993) Isolation and characterization of the subunits of a heat labile a-amylase inhibitor from Phaseolus vulgaris white kidney bean. Biosci. Biotechnol. Biochem. 57, 297302. 65. Young, N.M., Watson, D.C., Yaguchi, M., Adar, R., Arango, R., Rodriguez-Arango, E., Sharon, N., Blay, P.K.S. & Thibault, P. (1995) C-Terminal post-translational proteolysis of plant lectins and their recombinant forms expressed in Escherichia coli. Characterization of ragged ends by mass spectrometry. J. Biol. Chem. 270, 25632570. 66. Nakaguchi, T., Arakawa, T., Philo, J.S., Wen, J., Ishimoto, M. & Yamaguchi, H. (1997) Structural characterization of an a-amylase inhibitor from a wild common bean (Phaseolus vulgaris): insight into the common structural features of leguminous a-amylase inhibitors. J. Biochem. 121, 350354. 67. Pueyo, J.J., Hunt, D.C. & Chrispeels, M.J. (1993) Activation of bean (Phaseolus vulgaris) a-amylase inhibitor requires proteolytic processing of the pro-protein. Plant Physiol. 101, 13411348. 68. Finardi-Filho, F., Mirkov, T.E. & Chrispeels, M.J. (1996) A putative precursor protein in the evolution of the bean alphaamylase inhibitor. Phytochemistry 43, 5762. 69. Ishimoto, M., Yamada, T. & Kaga, A. (1999) Insecticidal activity of an a-amylase inhibitor-like protein resembling a putative precursor of a-amylase inhibitor in the common bean, Phaseolus vulgaris L. Biochim. Biophys. Acta 1432, 104112. 70. Fakhoury, A.M. & Woloshuk, C.P. (2001) Inhibition of growth of Aspergillus avus and fungal alpha-amylases by a lectin-like protein. Mol. Plant-Microbe Interact. 14, 955961. 71. Bompard-Gilles, C., Rousseau, P., Rouge, P. & Payan, F. (1996) Substrate mimicry in the active centre of a mammalian a-amylase: structural analysis of an enzyme inhibitor complex. Structure 4, 14411452. 72. Nahoum, V., Farisei, F., Le-Berre-Anton, V., Eglo, M.P., Rouge, P., Poerio, E. & Payan, F. (1998) A plant-seed inhibitor of two classes of alpha-amylases: X-ray analysis of Tenebrio molitor larvae alpha-amylase in complex with the bean Phaseolus vulgaris inhibitor. Acta Biol. Crystal. D. 55, 360362. 73. Layer, P., Carlson, G.L. & DiMagno, E.P. (1985) Partially puried white bean amylase inhibitor reduces starch digestion in vitro and inactivates intraduodenal amylase in humans. Gastroenterology 88, 18951902. 74. Turcotte, G.E., Nadeau, L., Forest, J.C., Douville, P., Leclerc, P., Bergeron, J. & Laclos, B.F. (1994) A new rapid immunoinhibition pancreatic amylase assay: diagnostic value for pancreatitis. Clin. Biochem. 27, 133139. 75. Schroeder, H.E., Gollash, S., Moore, A., Tabe, L.M., Craig, S., Hardie, D., Chrispeels, M.J., Spencer, D. & Higgins, T.J.V. (1995) Bean a-amylase inhibitor confers resistance to the pea weevil, Bruchus pisorum, in genetically engineered peas (Pisum sativum L.). Plant Physiol. 107, 12331239. 76. Morton, R.L., Schroeder, H.E., Bateman, K.S., Chrispeels, M.J., Armstrong, E. & Higgins, T.J.V. (2000) Bean alpha-amylase inhibitor 1 in transgenic peas (Pisum sativum) provides

FEBS 2002
complete protection from pea weevil (Bruchus pisorum) under eld conditions. Proc. Natl Acad. Sci. USA 97 (8), 3820 3825. Chagolla-Lopez, A., Blanco-Labra, A., Patthy, A., Sanchez, R. & Pongor, S. (1994) A novel a-amylase inhibitor from Amaranth (Amaranthus hypocondriacus) seeds. J. Biol. Chem. 269, 23675 23680. Lu, S., Deng, P., Liu, X., Luo, J., Han, R., Gu, X., Liang, S., Wang, X., Li, F., Lozanov, V., Patthy, A. & Pongor, S. (1999) Solution structure of the major a-amylase inhibitor of the crop plant amaranth. J. Biol. Chem. 274, 2047320478. Martins, J.C., Enassar, M., Willen, R., Wieruzeski, J.M., Lippens, G. & Wodak, S.J. (2001) Solution structure of the main a-amylase inhibitor from amaranth seeds. Eur. J. Biochem. 268, 23792389. Bode, W., Geyerling, H.J., Huber, R., Otleweski, J. & Wilusz, T. (1989) The rened 2.0 A X-ray crystal structure of the complex formed between bovine b-trypsin and CMTI-I, a trypsin inhibitor from squash seeds (Cucurbita maxima). Topological similarity of the squash seed inhibitors with the carboxypeptidase A inhibitor from potatoes. FEBS Lett. 242, 285292. Park, C.S. & Miller, C. (1992) Mapping function to structure in a channel-blocking peptide: electrostatic mutants of charybdotoxin. Biochemistry 31, 77497755. Pereira, P.J.B., Lozanov, V., Patthy, A., Huber, R., Bode, W., Pongor, S. & Strobl, S. (1999) Specic inhibition of insect a-amylases: yellow meal worm a-amylase in complex with the Amaranth a-amylase inhibitor at 2.0 A resolution. Structure 7, 10791088. Buonocore, V., Petrucci, T. & Silano, V. (1977) Wheat protein inhibitors of a-amylase. Phytochemistry 16, 811820. Lyons, A., Richardson, M., Tatham, A.S. & Shewry, P.R. (1987) Characterization of homologous inhibitors of trypsin and a-amylase. Biochim. Biophys. Acta 915, 305313. Garcia-Casado, G., Sanchez-Monge, R., Chrispeels, M.J., Armentia, A., Salcedo, G. & Gomez, L. (1996) Role of complex asparagine-linked glycans in the allergenicity of plant glycoproteins. Glycobiology 6, 471477. Kusaba-Nakayama, M., Ki, M., Iwamoto, M., Shibata, R., Sato, M. & Imaizumi, K. (2000) CM-3, one of the wheat a-amylase inhibitor subunits, and binding of IgE in sera from Japanese with atopic dermatitis related to wheat. Food Chem. Tox. 38, 179185. Garcia-Casado, G., Armentia, A., Sanchez-Monge, R., Sanchez, L.M., Lopez-Otin, C. & Salcedo, G. (1995) A major baker's asthma allergen from rye our is considerably more active than its barley counterpart. FEBS Lett. 364, 3640. Strobl, S., Maskos, K., Wiegand, G., Huber, R., Gomis-Ruth, F.X. & Glockshuber, R. (1998) A novel strategy for inhibition of a-amylases: yellow meal worm a-amylase in complex with the Ragi bifunctional inhibitor at 2.5 A resolution. Structure 6, 911 921. Alam, N., Gourinath, S., Dey, S., Srinivasan, A. & Singh, T.P. (2001) Substrateinhibitor interactions in the kinetics of a-amylase inhibition by Ragi a-amylase/trypsin inhibitor (RATI) and its various N-terminal fragments. Biochemistry 40, 42294233. Maeda, K., Hase, T. & Matsubara, H. (1983) Complete amino acid sequence of an alpha-amylase inhibitor in wheat kernel. Biochim. Biophys. Acta 743, 5257. Oda, Y., Matsunaga, T., Fukuyiama, K., Miyasaki, T. & Morimoto, J.T. (1997) Tertiary and quaternary structures of 0.19 a-amylase inhibitor from wheat kernel determined by X-ray analysis at 2.06 A resolution. Biochemistry 36, 1350313511. Miyazaki, T., Morimoto, T., Fukuyama, K. & Matsubara, H. (1994) Crystallization and preliminary X-ray diraction studies

77.

78.

79.

80.

81. 82.

83. 84. 85.

86.

87.

88.

89.

90. 91.

92.

FEBS 2002
of the a-amylase inhibitor coded 0.19 from wheat kernel. J. Biochem. 115, 179181. Alagiri, S. & Singh, T.P. (1993) Stability and kinetics of a bifunctional amylase/trypsin inhibitor. Biochim. Biophys. Acta 1203, 7784. Strobl, S., Muhlhahn, P., Bernstein, R., Wiltscheck, R., Maskos, K., Wunderlich, M., Huber, R., Glockshuber, R. & Holak, T.A. (1995) Determination of the three dimensional structure of the bifunctional a-amylase/trypsin inhibitors from Ragi seeds by NMR spectroscopy. Biochemistry 34, 82818293. Maskos, K., Huber-Wunderlich, M. & Glockshuber, R. (1996) RBI, a one-domain a-amylase/trypsin inhibitor with completely independent binding sites. FEBS Lett. 397, 1116. Garcia-Maroto, F., Marana, C., Mena, M., Garcia-Olmedo, F. & Carbonero, P. (1990) Cloning of cDNA and chromosomal location of genes encoding the three types of subunits of the wheat tetrameric inhibitor of insect a-amylases. Plant Mol. Biol. 14, 845853. Sanchez-Monge, R., Gomez, L., Garcia-Olmedo, F. & Salcedo, G. (1989) New dimeric inhibitor of heterologous a-amylases encoded by a duplicated gene in the short arm of chromosome 3B of wheat (Triticum aestivum L.). Eur. J. Biochem. 183, 3740. Rodenburg, K.W., Varallyay, E., Svendsen, I. & Svensson, B. (1995) Arg-27, Arg-127 and Arg-155 in the b-trefoil protein barley a-amylase/subtilisin inhibitor are interface residues in the complex with barley a-amylase 2. Biochem. J. 309, 969976. Gvozdeva, E.L., Valueva, T.A. & Mosolov, V.V. (1993) Enzymatic oxidation of the bifunctional wheat inhibitor of subtilisin and endogenous a-amylase. FEBS Lett. 334, 7274. Ohtsubo, K. & Richardson, M. (1992) The amino acid sequence of a 20-kDa bifunctional subtilisin/a-amylase inhibitor from grain of rice (Oryza sativa L.) seeds. FEBS Lett. 309, 6872. Mundy, J., Svendsen, I. & Hejgaard, J. (1983) Barley a-amylase/ subtilisin inhibitor. Isolation and characterization. Carlsberg Res. Commun. 48, 8190. Valle, F., Kadziola, A., Bourne, Y., Juy, M., Rodenburg, K.W., Svensson, B. & Haser, R. (1998) Barley a-amylase bound to its endogenous protein inhibitor BASI: crystal structure of the complex at 1.9 A resolution. Structure 6, 649659. Zemke, K.J., Muller-Fahrnow, A., Jany, K.-D., Pal, G.P. & Saenger, W. (1991) The three-dimensional structure of the bifunctional proteinase K/a-amylase inhibitor from wheat (PK13) at 2.5 A resolution. FEBS Lett. 279, 240242. Onesti, S., Brick, P. & Blow, D.M. (1991) Crystal structure of a Kunitz-type trypsin inhibitor from Erythrina cara seeds. J. Mol. Biol. 217, 153176. Rutenber, E. & Robertus, J.D. (1991) Structure of ricin B chain at 2.5 A resolution. Proteins 10, 260269. Garcia-Olmedo, F., Salcedo, G., Sanchez-Monge, R., Hernandez-Lucas, C., Carmona, M.J., Lopez-Fando, J.J., Fernandez, J.A., Gomez, L., Royo, J., Garcia-Maroto, F., Castagnaro, A. & Carbonero, P. (1992) Trypsin/a-amylase inhibitors and thionins: possible defense proteins from barley. In Barley: Genetics, Biochemistry, Molecular Biology and Biotechnology (Shewry, P.R., ed.) pp. 335350, CAB International, Wallingford, UK. Sidenius, U., Olsen, K., Svensson, B. & Christensen, U. (1995) Stopped-ow kinetic studies of the reaction of barley a-amylase/ subtilisin inhibitor and the high pI barley a-amylase. FEBS Lett. 361, 250254. Cornelissen, B.J.C., Hooft Van Huijsduijnen, R.A.M. & Bol, J.F. (1986) A tobacco mosaic virus-induced tobacco protein is homologous to the swett-tasting protein thaumatin. Nature 231, 531532. Vigers, A., Roberts, W. & Sellitrenniko, C.P. (1991) A new family of plant antifungal proteins. Mol. Plant Microb. Interact. 4, 315323.

Plant a-amylase inhibitors (Eur. J. Biochem. 269) 411


110. Batalia, M.A., Monzingo, A.F., Ernst, S., Roberts, W. & Robertus, J.D. (1996) The crystal structure of the antifungal protein zeamatin, a member of the thaumatin-like, PR-5 proteinfamily. Nat. Struct. Biol. 3, 1923. 111. Roberts, W.K. & Selitrennikof, C.P. (1990) Zeamatin, an antifungal protein from maize with membrane-permeabilizing activity. J. General Microbiol. 136, 17711778. 112. Blanco-Labra, A. & Iturbe-Chinas, F.A. (1980) Purication and characterization of an a-amylases inhibitor from maize (Zea mays). J. Food Biochem. 193, 112. 113. Schimoler-O'Rourke, R., Richardson, M. & Selitrenniko, C.P. (2001) Zeamatin inhibits trypsin and a-amylase activities. Appl. Environ. Microbiol. 67, 23652366. 114. Hejgaard, J., Jacobsen, S. & Svendsen, I. (1991) Two antifungal thaumatin-like proteins from barley grain. FEBS Lett. 291, 127131. 115. Trudel, J., Grenier, J., Ptovin, C. & Asselin, A. (1998) Several thaumatin-like proteins bind to b-1,3-glucans. Plant Physiol. 118, 14311438. 116. Abad, L.R., D'Urzo, M.P., Lin, D., Narasimhan, M.L., Renveni, M., Zhu, J.K., Niu, X., Singh, N.K., Hasegawa, P.M. & Bressan, R.A. (1996) Anti-fungal activity of tobacco osmotin has specicity and involves plasma membrane permeabilization. Plant Sci. 118, 1123. 117. Selitrenniko, C.P., Wilson, S.J., Clemons, K.V. & Stevens, D.A. (2000) Zeamatin, an antifungal protein. Curr. Opin. AntiInfective Drugs 2, 368374. 118. Castro, M.D., Fontes, W., Morhy, L. & Bloch Jr, C. (1996) Complete amino acide sequence from g-thionins from maize (Zea mays L.) seeds. Protein Pept. Lett. 3, 267274. 119. Thevissen, K., Ghazi, A., De Samblanx, G., Brownlee, C., Osborn, R.W. & Broekaert, W.F. (1996) Fungal membrane responses induced by plant defensines and thionins. J. Biol. Chem. 271, 1501815025. 120. Mendez, E., Rocher, A., Calero, M., Girbes, T., Citores, L. & Soriano, F. (1996) Primary structure of x-hordothionin, a member of a novel family of thionins from barley endosperm, and its inhibition of protein synthesis in eukaryotic and prokaryotic cell-free systems. Eur. J. Biochem. 239, 6773. 121. Wijaya, R., Neumann, G.M., Condron, R., Hughes, A.B. & Polya, G.M. (2000) Defense proteins from seed of Cassia stula include a lipid transfer protein homologue and a protease inhibitory plant defensin. Plant Sci. 159, 243255. 122. Bloch, C. Jr, Patel, S.U., Baud, F., Zvelebil, M.J.J.M., Carr, M.D., Sadler, P.J. & Thornton, J.M. (1998) H-NMR structure of an antifungal c-thionin protein Sia1: similarity to scorpion toxins. Proteins 32, 334349. 123. Orengo, C.A. & Thornton, J.M. (1993) Alpha plus beta folds revisited: some favoured motifs. Structure 1, 105120. 124. Bruix, M., Jimenz, M.A., Santoro, J., Gonzalez, C., Colilla, F.J., Mendez, E. & Rico, M. (1993) Solution structure of c1-H and c1-P thionins from barley and wheat endosperm determined by 1 H-NMR: a structural motif common to toxic arthropod proteins. Biochemistry 32, 715724. 125. Tyndall, J.D.A. & Fairlie, D.P. (1999) Conformational homogeneity in molecular recognition by proteolytic enzymes. J. Mol. Recog. 12, 363370. 126. Bode, W. & Huber, R. (2000) Structural basis of the endoproteinaseprotein inhibitor interaction. Biochim. Biophys. Acta 1477, 241252. 127. Chen, M.S., Feng, G., Zen, K.C., Richardson, M., ValdesRodriguez, S., Reeck, G.R. & Kramer, K.J. (1992) a-amylase from three species of stored grain coleoptera and their inhibition by wheat and corn proteinaceous inhibitor. Insect Biochem. Mol. Biol. 22, 261268. 128. Behnke, C.A., Yee, V.C., Trong, I.L., Pedersen, L.C., Stenkamp, R.E., Kim, S.S., Reeck, G.R. & Teller, D.C. (1998) Structural

93. 94.

95. 96.

97.

98.

99. 100. 101. 102.

103.

104. 105. 106.

107.

108.

109.

412 O. L. Franco et al. (Eur. J. Biochem. 269)


determinants of the bifunctional corn Hageman factor inhibitor: X-ray crystal structure at 1.95 A resolution. Biochemistry 37, 1527715288. Hazegh-Azam, M., Kim, S.S., Masoud, S., Andersson, L., White, F., Johnson, L., Muthukrishnan, S. & Reeck, G. (1998) The corn inhibitor of activated Hageman factor: purication and properties of two rcombinant forms of the protein. Prot. Expres. Purif. 13, 143149. Betzel, C., Pal, G.P. & Saenger, W. (1988) Synchrotron X-ray data collection and restrained least-squares renement of the crystal structure of proteinase K at 1.5 A resolution. Acta Crystallogr. 44, 163172. Pal, G.P., Betzel, C.H., Jane, K.D. & Saenger, W. (1986) Crystallization of the bifunctional proteinase/amylase inhibitor PKI-3 and of its complex with proteinase K. FEBS Lett. 197, 111114. Ary, M.B., Richardson, M. & Shewry, P.R. (1989) Purication and characterization of an insect a-amylase inhibitor/endochitinase from seeds of Job's Tears (Coix Lachryma-jobi). Biochim. Biophys. Acta 913, 260266. da Silva, M.C.M., Grossi de Sa, M.F., Chrispeels, M.J., Togawa & Neshich, G. (2000) Analysis of structural and physico-chemical parameters involved in the specicity of binding between a-amylase and their inhibitors. Prot. Eng. 13, 167177. Rodenburg, K.W., Vallee, F., Juge, N., Aghajari, N., Guo, X., Haser, R. & Svensson, B. (2000) Specic inhibition of barley a-amylase 2 by barley a-amylase/subtilisin inhibitor depends on charge interaction and can be conferred to isozyme 1 by mutation. Eur. J. Biochem. 267, 10191029. Le Berre-Anton, V., Nahoum, V., Payan, F. & Rouge, P. (2000) Molecular basis for the specic binding of dierent a-amylase inhibitors from Phaseolus vulgaris sedes to the active site of a-amylase. Plant Physiol. Biochem. 38, 657665. Matsui, I. & Svensson, B. (1997) Improved activity and modulated action pattern obtained by random mutagenesis at the fourth beta-alpha loop involved in substrate binding to the catalytic (beta/alpha) 8-barrel domain of barley alpha-amylase 1. J. Biol. Chem. 272, 2245622463. La-Jolo, F.M., Finardi-Filho, F. & Menezes, E.W. (1991) Amylase inhibitors in Phaseolus vulgaris bean. Food Technol. 8, 119121. Ishimoto, M. & Chrispeels, M.J. (1996) Protective mechanism of the Mexican bean weevil against high levels of a-amylase inhibitor in the common bean. Plant Physiol. 111, 393401. Horsh, R.B., Fraley, R.T., Rogers, S.G., Sanders, P.R., Lloyd, A. & Homan, H. (1984) Inheritance of functional genes in plants. Science 223, 496498. Jouanin, L., Bonade-Bottino, M., Girard, C., Morrot, G. & Giband, M. (1998) Transgenic plants for insect resistance. Plant Sci. 131, 111. Ishimoto, M., Sato, T., Chrispeels, M.J. & Kitamura, K. (1996) Bruchid resistance of transgenic azuki bean expressing seed a-amylase inhibitor of common bean. Entomol. Exp. Appl. 79, 309315. Kashlan, N. & Richardson, M. (1981) The complete amino acid sequence of a major wheat protein inhibitor of a-amylase. Phytochemistry 20, 17811784. Garcia-Maroto, F., Carbonero, P. & Garcia-Olmedo, F. (1991) Site-directed mutagenesis and expression in Escherichia coli of WMAI-1, a wheat monomeric inhibitor of insect a-amylase. Plant Mol. Biol. 17, 10051011. Okuda, M., Satoh, T., Sakurai, N., Shibuya, K., Kaji, H. & Samejima, T. (1997) Overexpression in E. coli of chemically synthesized gene for active 0.19 a-amylase inhibitor from wheat kernel. J. Biochem. 122, 918926.

FEBS 2002
145. Pusztai, A., Bardocz, G.G., Alonso, R., Chrispeels, M.J., Schroeder, H.E., Tabe, L.M. & Higgins, T.J. (1999) Expression of the insecticidal bean alpha-amylase inhibitor transgene has minimal detrimental eect on the nutritional value of peas fed to rats at 30% of the diet. J. Nutrit. 129, 15971603. 146. Foreman, C.T. (1990) Food safety and quality for the consumer: policies and communication. In Animal Biotechnology in 1990s: Opportunities and Challenges (MacDonald, J.F., ed.), pp. 121126. National Agricultural Biotechnology Council, Ithica, NY. 147. Sparks, P., Shepherd, R. & Frewer, L.J. (1995) Assessing and structuring attitudes towards the use of gene technology in food production: the role of perceived ethical obligation. J. Bas. Appl. Soc. Psych. 16, 267285. 148. Kraulis, P.J. (1991) MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946950. 149. Valencia, A., Bustillo, A.E., Ossa, G.E. & Chrispeels, M.J. (2000) a-Amylases of the coee berry borer (Hypothenemus hampei) and their inhibition by two plant amylase inhibitors. Insect Biochem. Mol. Biol. 30, 207213. 150. Silva, C.P., Terra, W.R., Xavier-Filho, J., Grossi-de-Sa, M.F., Isejima, E.M., Damatta, R.A., Miguens, F.C. & Bifano, T.D. (2001) Digestion of legume starch granules by larvae of Zabrotes subfasciatus (Coleoptera: Bruchidae) and the induction of a-amylases in response to dierent diets. Insect Biochem. Mol. Biol. 31, 4150. 151. Zeng, F. & Cohen, A.C. (2000) Partial characterization of a-amylase in the salivary glands of Lygus hesperus and L. lineoralis. Comp. Biochem. Physiol. B 126, 916. 152. Mendiola-Olaya, E., Valencia-Jimenez, A., Valdes-Rodrigues, S., Delano-Frier, J. & Blanco-Labra, A. (2000) Digestive amylase from the larger grain borre, Prostephnus truncatus Horn. Comp. Biochem. Physiol. B 126, 425433. 153. Campos, F.A.P. & Richardson, M. (1983) The complete amino acid sequence of the bifunctional a-amylase/trypsin inhibitor from seeds of ragi (Indian nger millet, Eleusine coracana Gaertneri.). FEBS Lett. 152, 300304. 154. Orengo, C.A., Michie, A.D., Jones, S., Jones, D.T., Swindells, M.B. & Thornton, J.M. (1997) CATH-A hierarchic classication of protein domain structures. Structure 5, 10931108. 155. Murzin, A.G., Brenner, S.E., Hubbard, T. & Chothia, C. (1995) SCOP: a structural classication of proteins database for the investigation of sequences and structures. J. Mol Biol. 247, 536540. 156. Richardson, M., Valdes-Rodriguez, S. & Blanco-Labra, A. (1987) A possible function for thaumatin and a TMV-induced protein suggested homology to a maize inhibitor. Nature 327, 432434. 157. Mirkov, T.E., Whalstrom, J.M., Hagiwara, K., Finardi-Filho, F., Kjemtrup, S. & Chrispeels, M.J. (1994) Evolutionary relationships among proteins in the phytohemagglutinin-arcelina-amylase inhibitor family of the common bean and its relatives. Plant Mol. Biol. 26, 11031113. 158. Wato, S., Kamei, K., Arakawa, T., Philo, J., Wen, J., Hara, S. & Yamaguchi, H. (2000) A chimera-like a-amylase inhibitor suggesting the evolution of Phaseolus vulgaris a-amylase inhibitor. J. Biochem. 128, 139144. 159. Jones, R.L. & Jacobsen, J.V. (1991) Regulation of synthesis and transport of secreted proteins in cereal aleurone. Int. Rev. Cytol. 126, 4988. 160. Go, D.J. & Kull, F.J. (1995) The inhibition of human salivary alpha-amylase by type II alpha-amylase inhibitor from Triticum aestivum is competitive, slow and tight-binding. J. Enzyme Inhib. 9, 163170.

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