Review

TRENDS in Biotechnology

Vol.25 No.5

Enzyme promiscuity: mechanism and

applications
Karl Hult and Per Berglund
School of Biotechnology, Department of Biochemistry, Royal Institute of Technology (KTH), AlbaNova University Center,
SE-106 91 Stockholm, Sweden

Introductory courses in biochemistry teach that

enzymes are specific for their substrates and the reactions they catalyze. Enzymes diverging from this statement are sometimes called promiscuous. It has been
suggested that relaxed substrate and reaction specificities can have an important role in enzyme evolution;
however, enzyme promiscuity also has an applied
aspect. Enzyme condition promiscuity has, for a long
time, been used to run reactions under conditions of low
water activity that favor ester synthesis instead of
hydrolysis. Together with enzyme substrate promiscuity, it is exploited in numerous synthetic applications,
from the laboratory to industrial scale. Furthermore,
enzyme catalytic promiscuity, where enzymes catalyze
accidental or induced new reactions, has begun to be
recognized as a valuable research and synthesis tool.
Exploiting enzyme catalytic promiscuity might lead to
improvements in existing catalysts and provide novel
synthesis pathways that are currently not available.
Introduction
A modern term in enzymology is promiscuity, but what
does it mean? As with many modern words, it is used
without a well-defined meaning or with different meanings, depending on the author. In this overview, we will
define a promiscuous enzyme as one that does things it is
not expected to do, and we will focus on the enzyme
promiscuity that has been exploited to expand the use of
enzymes in the synthesis of compounds, whereas promiscuity connected to enzyme evolution will only be mentioned
briefly.
Promiscuity comes in many different forms, and we
would like to introduce the classification of promiscuity
into condition promiscuity, substrate promiscuity and
catalytic promiscuity (Box 1). The classification is sometimes not straightforward, and we will see that different
types of promiscuity can be combined. One example illustrating this problem is amide formation catalyzed by a
lipase: amines are, undoubtedly, promiscuous substrates
for a lipase, but is this also an example of catalytic promiscuity and thereby a new reaction for a lipase? Some
scientists think so, whereas others regard the two transition states as being too similar to be classified as different
reactions. Even if the term promiscuity is new in enzymology, there are numerous old examples in the literature that
live up to the definition. An example of promiscuity was
Corresponding author: Hult, K. (kalle@biotech.kth.se).
Available online 26 March 2007.
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demonstrated in the reversal of hydrolytic enzyme activity

shown for maltase 1898 [1] and for pancreatic lipase in
1900 [2]. Condition promiscuity was explored by Rona et al.
in the early 1930s in a series of experiments using pig liver
lipase and pancreatic lipase in organic solvents for resolution of chiral alcohols and esters [3]. A good example of
both substrate and catalytic promiscuity is the use of
pyruvate decarboxylase to form carboncarbon bonds; this
was first studied in 1921 and is, today, an industrial
activity [4].
Enzyme promiscuity has become popular in terms of
enzyme evolution, enzyme engineering and biocatalysis
[57]. Some scientists regard the promiscuity of enzymes
as the starting point for enzyme divergent evolution; thus,
low promiscuous activity towards an unnatural substrate
might, by a single point mutation, turn the enzyme into a
much more proficient catalyst, affording a survival benefit
to the organism. Following the first beneficial mutation,
the enzyme then has time to evolve into an even better
catalyst [8]. Furthermore, in the laboratory, a promiscuous
function can be the starting point for the creation of a new
enzyme activity [9]. The evolutionary aspect of promiscuity
has recently been discussed in more detail by Tawfik et al.
they propose that a new promiscuous activity can evolve
without negative trade-offs in the native activity, leading
to a generalist enzyme that can later on become a specialist
[10]. Understanding of substrate and transition state
recognition in enzyme catalysis offers great opportunities
in the development of new catalytic functions in the scaffold of old enzymes.
In nature, enzymes appear to develop to a point at which
they are good enough for their task, meaning that the
substrate and catalytic specificities have reached a point
that satisfies the needs of the cell. There are, however,
important exceptions. Rubisco (ribulose-1,5-bisphosphate
carboxylase/oxygenase) evolved under low oxygen pressure
and is unable to discriminate between carbon dioxide and
oxygen. The consequence is an oxygenase reaction that
creates byproducts that the cell has to cope with. In our
opinion, this is a case of natural promiscuity that the cell
has to live with in the absence of a better alternative. Also,
as mentioned above and pointed out by OBrien and Herschlag, promiscuity can be a part of the evolution of new
enzyme activity [11]. Gerlt et al. has shown that some
enzymes with similar reaction intermediates are able to
catalyze each others reactions, sometimes with astonishingly high efficiency [12,13]. Furthermore, these enzymes
probably originated from the same gene.

0167-7799/$ see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2007.03.002

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Enzymes taken out of the cell can be exposed to reaction

conditions and substrates not seen in vivo that will challenge their specificity and might force them to handle
substrates and catalyze reactions they were not designed
for. This is why there is a low barrier to exploit enzymes as
catalysts for unnatural reactions in the laboratory and
industry. This could be called accidental in vitro promiscuity [14].
We will now describe the various types of promiscuity,
focusing on examples of interest in applied synthesis
(Table 1).

exploitation of lipases using modified substrates in organic

solvents is particularly pronounced; this combination of
substrate promiscuity and condition promiscuity has lead
to several industrial applications [15].
Solidgas bioreactors represent another interesting
example of condition promiscuity. There is no liquid phase;
instead, the enzyme is immobilized in a dry form on a solid
support, and the substrates and products are present in the
gas phase only. The system enables individual fine-tuning
of the thermodynamic activities of substrates and water,
and is of great theoretical interest. In addition, the high
productivity of the system enables the lipase-catalyzed
production of esters at a commercial scale [16].
Early important examples of engineering the condition
promiscuity of enzymes come from the group of Frances
Arnold at the beginning of the 1990s. They explored the
stability and activity of the serine protease subtilisin E in
the polar organic solvent DMF where water hydrolysis is
suppressed and improved the performance of the enzyme
for peptide synthesis with random mutagenesis. The initial
enzyme was further improved with three rounds of
directed evolution, leading to an enzyme with ten amino
acid substitutions [17]. This enzyme showed a 256-fold
higher activity than the wild type in 60% DMF. The
important interactions contributing to protein stability
in non-aqueous solvents have been summarized as design
rules by Arnold [18].

Condition promiscuity
Both condition promiscuity and substrate promiscuity
have been explored and used in enzyme-catalyzed synthesis. There are thousands of examples of these, such
as reactions performed in organic solvents, in absence of
solvents, at high temperature, or at extreme pH. The

Substrate promiscuity
As mentioned above, substrate promiscuity has been
frequently exploited in the past. Here, we will mention
examples of enzymes with strict substrate specificity, which
can be destroyed by simple point mutations, and enzymes
with an intrinsic high substrate promiscuity, which can be

Box 1. Types of enzyme promiscuity

 Enzyme condition promiscuity
Shown by enzymes with catalytic activity in various reaction
conditions different from their natural ones, such as anhydrous
media, extreme temperature or pH.
 Enzyme substrate promiscuity
Shown by enzymes with relaxed or broad substrate specificity.
 Enzyme catalytic promiscuity
Shown by enzymes catalyzing distinctly different chemical transformations with different transition states. Enzyme catalytic promiscuity can be either:
(i) accidental a side reaction catalyzed by the wild-type enzyme;
(ii) induced a new reaction established by one or several
mutations rerouting the reaction catalyzed by the wild-type
enzyme.

Table 1. Examples of promiscuity

Example

Maltase: reversal of reaction (1898)

Pancreatic lipase: reversal of reaction (1900)
Pig liver lipase: reactions in solvent (1931)
Pyruvate decarboxylate: used for synthesis of CC bonds (1921)
Lipase: transacylation in gas phase
Subtilisin E: solventtolerant mutants
Alanine dehydrogenase: cofactor usage
Phosphite dehydrogenase: cofactor usage
Lipase: resolution of chiral acids
Lipase: resolution of chiral alcohols
Enolase super family: promiscuous activities as part of evolution
Acylases catalyzed Markovnicov additions: CN bond formation
Lipase catalyzed aldol addition: CC bond formation
Serine hydrolases catalyzed Michael addition: CO, CS and CN bond
formation
Protease catalyzed conjugate additions: CN bond formation
Lipase catalyzed conjugate additions: CN bond formation
Lipase catalyzed Michael additions: CN and CS bond formation
Lipase catalyzed Michael additions: CC bond formation
Carbonic anhydrase as a peroxidase
Aminopeptidase as an oxidase
Aminopeptidase hydrolyzing phosphodiesters
Pyridoxalphosphatedependent racemase catalyzed aminotransferase
Pyridoxalphosphatedependent racemase catalyzed retroaldol
reaction
Glycosidase catalyzed synthase reactions
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Type of promiscuity
Condition
Substrate

Refs
Catalytic
Accidental

Induced

x
x
x
x

x
x
x
x
x
x
x
x
x
x
x
x
x
x

[1]
[2]
[3]
[4]
[15]
[17]
[19]
[20]
[21]
[24]
[26]
[2729]
[31]
[32]

x
x

[3334]
[35]
[36]
[37]
[38]
[39]
[40]
[45]
[46]

[4751]

x
x
x
x

Review

TRENDS in Biotechnology

increased even more. These examples will show that even if

enzymes intrinsically have high or low substrate specificity,
they often can be engineered into a promiscuous substrate
behavior that suits a specific application.
Coenzyme promiscuity
For the living cell it is important to keep the NADPH
NADP+ pool in a reduced state, whereas the pool of NADH
NAD+ must be oxidized. Consequently, dehydrogenases
must be able to distinguish between the two cofactor pairs.
This is an example of where the cell does not permit
promiscuity, even if a few point mutations can induce it.
Ashida et al. describe an interesting case in which one point
mutation can destroy the NAD+ selectivity or even reverse
it to favor NADP+ [19]. Alanine dehydrogenase from Shewanella sp. Ac10 shows a high preference for NAD+ compared with NADP+, with a ratio of specificity constants of
2500. The mutation Asp198Ala creates an enzyme with a
decreased KM and an increased kcat for NADP+, leading to
specificity for NADP+ of 14 compared with NAD+
altogether, a change of 35 000-times without a drop in
total activity. A similar mutation, Asp198Gly, creates an
enzyme almost without cofactor specificity. Other dehydrogenases are equally sensitive to simple point mutations.
For example, the NAD+ specificity of phosphite dehydrogenase can be abolished by one point mutation and turned
into NADP+ specificity by two [20].
Lipase substrate promiscuity
Lipases are promiscuous with regard to substrates: they
accept a wide range of esters and are extensively used in
the resolution of racemic acids and alcohols, although they
normally show low enantioselectivity towards chiral acids.
The research group around Reetz showed that it was
possible to evolve lipases with preference for either the
R- or S-enantiomer. They started with the lipase from
Pseudomonas aeruginosa, which has no enantioselectivity
towards 2-methyldecanoic acid p-nitrophenol ester, and
subjected its gene to random mutagenesis to obtain, first,
a lipase with an enantiomeric ratio of 51 favoring the Senantiomer and, later on, a new mutant showing an Rpreference with an enantiomeric ratio of 38 [21]. These
experiments were performed without knowledge of the
enzyme structure but were later followed up by a paper
describing the structure and the effects of the mutations
[22].
It has been determined that all lipases have the same
preference for secondary alcohols, where the preferred
enantiomers have the same geometrical configuration
called the Kazlauskas rule [23]. Lipase B from Candida
antarctica has a stereospecificity pocket in which the
chiral alcohol binds during catalysis. Through a single
point mutation, Magnusson et al. were able to change the
enantioselectivity 8  106-fold and reverse it from R- to Sselectivity, mainly by increasing the activity towards the
S-enantiomer [24]. Interestingly, the change was only
because of an increase in the entropic term favoring
the S-enantiomer. Although only a small change in
the protein is needed to obtain a lipase with inverted
enantioselectivity, no enzyme is known from natural
sources.
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Catalytic promiscuity
Enzyme catalytic promiscuity refers to the ability of an
enzyme active site to catalyze more than one different
chemical transformation. Kazlauskas proposes that transformations are different if the types of bonds cleaved and/or
made are different, and if the transition states of the two
reactions are different [25]. A detailed mechanistic insight
can be used to predict promiscuous reactions in an existing
enzyme active site. One challenge in current enzyme redesign is to turn a low promiscuous activity of an enzyme into
the main activity or to use part of the existing mechanism
and divert the reaction path to new products. We will
discuss a few such examples in this section.
Catalytic promiscuity and enzyme evolution
The enzymes mandelate racemase, muconate lactonizing
enzyme and enolase share a common partial reaction,
namely the Mg2+-assisted a-proton abstraction and enolization of carboxylic acids (Figure 1). The enol intermediate
subsequently reacts differently in each enzyme to produce
their specific products [14]. The enzymes show a similar bidomain structure in which the active site has a conserved
location. With these common features it is easy to accept
the hypothesis that these enzymes have evolved by divergent evolution from a common progenitor. In some cases,
the evolution has not only changed the substrate specificity
but also the reaction specificity. Gerlt et al. found
one member of the enolase super-family that had a promiscuous activity as a N-acylamino acid racemase, with
a kcat/KM equal to 590 M1s1 for the best substrate,
N-acetylmethionine, compared with 2  105 M1s1 for
the natural reaction as o-succinylbenzoate synthase. The
discovery of enzymes with dual activity indicates that at
least some keep their original activity at the same time as
new activities start to develop, resulting in an enzyme
showing natural catalytic promiscuity [26].
Markovnikov additions catalyzed by a hydrolase
accidental catalytic promiscuity
The Markovnikov addition is an important reaction in
synthetic chemistry. Wu et al. have demonstrated the
promiscuous addition of nitrogen-containing heterocycles
to vinyl esters, catalyzed by the enzymes D-aminoacylase
[27], penicillin G acylase [28] and acylase from Aspergillus
oryzae [29] in an organic solvent. Several different
N-heterocyclic compounds were used, including purines
and pyrimidines, and several reactions reached close to
a quantitative yield. No product was observed when using
vinyl ethers. This suggests that the carbonyl in the substrate is needed, and the authors propose that its oxygen
is bound in an oxyanion hole or, alternatively, coordinated

Figure 1. The common Mg2+ assisted a-proton abstraction and enolization of

carboxylic acids in the enolase super family, which can produce racemase,
lactonizing enzyme and enolase activities.

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to the zinc ion in the enzyme active site, which, according to

them, would polarize the vinyl double bond. The heterocyclic nucleophile is deprotonated by a general base and
simultaneously attacks the partial positive charge of the
C=C double bond. It is somewhat surprising to note that
none or low enantioselectivity was observed in the Markovnikov additions. This is unexpected and hard to
explain.
Aldol and Michael additions as accidental and
induced catalytic promiscuity in a lipase
In the reaction mechanism of serine hydrolases, the
oxyanion hole is an important feature: it polarizes the
carbonyl double bond in the ester substrate and renders
the carbonyl carbon susceptible for nucleophilic attack by
the active-site serine (Figure 2a). If the serine is removed
by mutation, the oxyanion hole is still able to activate
carbonyl functions. This is the basis for several of the
promiscuous reactions that are shown to take place in
the active site of serine hydrolases.
Carboncarbon bond formation is important in synthesis,
and many biotransformation examples exist [30]. Branneby
et al. have shown that lipase B from Candida antarctica
catalyzes carboncarbon bond formation through aldol
addition, with the substrates hexanal and propanal [31].
The proposed reaction mechanism involves binding of the
carbonyl in the oxyanion hole of the lipase while the activesite histidine acts as a base to abstract a a-proton so that a
carboncarbon bond can be formed with the second substrate (Figure 2b). The specific activity increased by a factor
of four when the active-site serine was replaced with an
alanine to avoid the nucleophilic attack present in the
natural reaction. Wild-type lipase covalently inhibited with
methyl p-nitrophenyl n-hexylphosphonate was completely
inactive, confirming that the active site is needed for catalysis of this reaction. The aldolase reaction was carried out in
organic solvent, with unnatural substrates and catalyzed by
a lipase; it is, therefore, an example that combines all three
types of promiscuity: condition, substrate and catalytic.
Conjugate addition on a,b-unsaturated carbonyl
compounds is long known to be catalyzed by serine hydrolases [32]. Recently, conjugate additions by N-nucleophiles
in organic solvents have been studied when catalyzed by a
protease [33,34] or by a lipase [35]. Thiols were mainly
used as nucleophiles by Carlqvist et al., who proposed that
the mechanism involves the active-site histidine as a base
that activates the nucleophile for attack at the b-carbon of

the substrate, with the carbonyl oxygen hydrogen of the

substrate bonded in the oxyanion hole of the enzyme
(Figure 2c) [36]. This was supported by quantum chemical
calculations on a minimal model system for C. antarctica
lipase B, based on docking studies and molecular dynamics
simulations. Furthermore, in that study, the Ser105Ala
mutant of C. antarctica lipase B was found to catalyze
conjugate additions of thiols and amines on a,b-unsaturated aldehydes, ketones and methyl esters in cyclohexane,
toluene or diethyl ether. The reactions followed saturation
kinetics with kcat values in the range of 0.001 to
4.000 min1. The enzyme proficiency was 107 for the
Ser105Ala mutant, a figure approaching that of natural
enzymes. The conjugate-addition activity was, in many
cases, improved by the Ser105Ala mutation for the tested
substrates.
Svedendahl et al. has recently shown that Michael
additions forming a carboncarbon bond between a 1,3dicarbonyl compound and an a/b-unsaturated aldehyde or
ketone are efficient promiscuous reactions for Candida
antarctica lipase B [37]. The reactions were run with
various substrates in cyclohexane or in absence of solvent,
with specific rates recorded in the range of 0.011000 s1.
The wild type showed, in all cases, 100-fold lower specific
rate than the mutant Ser105Ala.
It is interesting to note that the wild type enzyme was
able to catalyze all the reactions mentioned above, which
could be a good example of accidental promiscuity found in
vitro. It is, although, highly improbable that those reactions would occur in nature because it is unlikely that the
lipase would encounter such substrates. The increased
efficiency of the active-site serine-deficient mutant is an
example of induced catalytic promiscuity.
Induced catalytic promiscuity in artificial
metalloenzymes
There are several interesting examples of artificial
metalloenzymes that can catalyze enantioselective oxidation. Okrasa and Kazlauskas made a thorough study
of manganese-substituted carbonic-anhydrase-catalyzed
oxidation of styrene analogues. They created the manganese enzyme by dialysis, where the natural Zn2+ was
changed to Mn2+. The reaction was run in a buffer containing bicarbonate using hydrogen peroxide as oxidant. The
reaction rate was lower for the manganese-substituted
carbonic anhydrase than for free manganese or bicarbonate; however, the produced epoxide had enantiomeric

Figure 2. In serine hydrolases, the carbonyl of the substrate ester is coordinated in the oxyanion hole and attacked by the active site serine (a). Mutation of the active site
serine opens up the possibility for other reaction types that depend on polarization of the carbonyl function, such as aldol addition (b) or Michael type additions (c). The
oxyanion hole of Candida antarctica lipase B can form three hydrogen bonds to the oxygen of the substrate in the transition state, whereas, in ground state, only two would
be developed.
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235

Figure 3. The first part of the reaction mechanism of pyridoxal-phosphate (PLP)-dependent enzymes. The mechanism then diverges, depending on the type of enzyme.

excesses of up to 67%, and no aldehyde product could be

seen. Unfortunately, the conversion was low owing to
degradation of the anhydrase by reactive intermediates
generated during catalysis [38].
Catechol oxidase activity was introduced in an
aminopetidase by da Silva and Ming. They prepared a
di-Cu2+-substituted aminopeptidase that oxidized catechol
with hydrogen peroxide, recording a kcat/KM in the range
of 3300 M1s1, which is only ten times lower than that of a
natural catechol oxidase [39]. Interestingly, the natural dizinc aminopeptidase produced a 4  1010-fold acceleration
of phosphodiester hydrolysis in addition to its natural
peptide hydrolytic activity [40]. Thus, this protein scaffold
has been shown to perform reactions that pass through
totally different transition states: hydrolysis of peptides
and phosphodiesters with the natural di-zinc enzyme, and
oxidation with the artificial di-copper enzyme. All reactions are catalyzed with efficiencies of the same magnitude
as their natural counterparts.
Induced catalytic promiscuity in pyridoxal-phosphatedependent enzymes
Pyridoxal-phosphate (PLP) is a versatile cofactor used by
more than 140 different enzymes from five out of the six
main enzyme EC classes [41]. These enzymes mainly
accept a-amino acids and catalyze reactions such as aracemization, transamination, a- or b-decarboxylation,
and retroaldol reactions. Their active site is similar, and
the cofactor is covalently bound through an imine linkage

to a lysine residue. The common mechanistic feature is the

formation of an external imine formed by the amino group
of the substrate and the carbonyl of the cofactor [42,43].
Many of the PLP-dependent enzymes subsequently form a
quinonoid intermediate, from which the mechanism
diverges depending on the type of PLP-dependent enzyme
(Figure 3). Mechanistic details suggested by Toney
indicate the consequence of a fully developed quinonoid
intermediate for the discrimination between racemization
and transamination, and it is clear that small changes are
needed in one enzyme to alter the activity into that of
another PLP enzyme [44]. Examples of such induced catalytic promiscuity have, for instance, been published by
Esaki et al. [45].
Seebeck and Hilvert describe another example of
induced catalytic promiscuity of a PLP-dependent enzyme.
They converted a racemase into an aldolase by a single
point mutation. The mutation, Tyr265Ala, in a racemase
from Geobacillus stearothermophilus resulted in a 3  103fold reduction in racemase activity and a 2  105-fold
increase in retro-aldol activity with the substrate D-phenylserine, compared with the wild-type enzyme. Replacement of Tyr265 with Ala was expected to promote the
nearby His166 to initiate a retro-aldol reaction of a bhydroxy-amino acid, similar to the retro-aldol reaction of
the non-related enzyme L-threonine aldolase (Figure 4).
Tyr265 is acting as a base in the active site and is necessary
for converting L- to D-alanine. Removal of Tyr265 was,
therefore, also expected to drastically lower the racemase

Figure 4. The retro-aldol reaction specificity of l-threonine aldolase was engineered into alanine racemase by a single-point active-site mutation [46]. Abbreviation: PLP,
pyridoxal-phosphate.
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activity. Interestingly, the retro-aldol reaction catalyzed by

the alanine racemase mutant was found to be highly
enantioselective because the conversion of racemic threob-phenylserine stopped at 50% conversion [46].
Induced catalytic promiscuity in glycoside synthesis
In 1898, Hill prepared maltose from glucose by
equilibrium-controlled synthesis using maltase as a catalyst
[1]. Owing to the need for water to dissolve the substrate,

this methodology produces low yields. Kinetically controlled

synthesis of glycosides requires activated substrates, and
might reach yields of up to 40%. In 1998, Withers
and colleagues reported how the yield could be increased
considerably by introducing catalytic promiscuity to suppress hydrolysis [47]. This first paper has led to an abundance of extremely useful glycosynthases [48].
Inverting glycosidases have two carboxylates in
the active site: one acts as a nucleophile to form a

Figure 5. Induced catalytic promiscuity. Glycosidases were transformed into glycosynthases, thioglycoligases or thioglycosynthases by rerouting reaction intermediates
achieved by deletion of catalytic amino acid side chains. The different mutations are shown in bold.
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TRENDS in Biotechnology

glycosylenzyme intermediate, whereas the other one acts

as an acid/base catalyst. Removing the nucleophilic carboxylate produces an enzyme that is unable to hydrolyze
glycosidic bonds. However, the otherwise intact active site
can bind a suitable activated a-glycosyl fluoride and an
acceptor sugar in such a way that the remaining acid/base
carboxylate can catalyze the formation of a glycosidic bond.
Because the mutant enzyme is devoid of hydrolytic
activity, the product can accumulate without being hydrolyzed. A mutant in which the nucleophilic carboxylate was
changed not to an alanine but to serine is especially good as
glycosynthase (Figure 5) [49].
An alternative modification of the reaction mechanism
is to cripple the ability of the enzyme to hydrolyze the
glycosylenzyme intermediate by removing the acid/base
carboxylic acid. The glycosyl enzyme can still be formed
from substrates with good leaving groups, such as dinitrophenol or fluoride; however, water and hydroxyl-containing sugars are too weak nucleophiles to react at an
appreciable reaction rate without the acid/base catalytic
group. Instead, thiol-containing sugars, which are much
more reactive, have to be used. The thioglycosides produced are not hydrolyzed by the enzyme and can, therefore,
be prepared in good yields. Withers et al. named this
activity thioglycoligase (Figure 5) [50].
In the two examples above, either of the two carboxylates
in the active site was removed, but what would happen if
both were removed? The double mutant would not be able to
form a glycosylenzyme intermediate and, in addition, there
would not be an acid/base to facilitate glycoside formation.
Again, Withers group conducted the experiments and found
an enzyme that could form glycosidic bonds between activated sugars and thiosugars. Presumably, the protein scaffold provided a proximity effect by binding and orienting the
substrates in a favorable position for reaction (Figure 5) [51].
Conclusions
Enzyme promiscuity, a property shown by many enzymes,
is nowadays exploited in applied enzymology. In this
review we have discussed some examples of various types
of enzyme promiscuity and some applications based on it.
We propose that the term, enzyme promiscuity, can be
classified into enzyme condition promiscuity, substrate
promiscuity and catalytic promiscuity. The applied aspects
of condition promiscuity and substrate promiscuity have
been explored for a long time, resulting in many industrial
applications. Enzyme catalytic promiscuity, however, has
only recently been exploited for synthetic applications.
Catalytic promiscuity can be further divided into accidental catalytic promiscuity and induced catalytic promiscuity. It is evident that enzyme catalytic promiscuity, and
particularly induced enzyme catalytic promiscuity, offers
great opportunities in the design and development of new
catalytic functions in the scaffold of stable enzymes. This is
particularly promising because metagenomics has
increased the availability of enzymes from extremophiles,
providing new, stable templates for this technology.
Exploiting enzyme catalytic promiscuity might lead to
new, efficient and stable catalysts with, as yet, unprecedented activity for reactions where no practically useful
enzyme alternatives exist today.
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237

Acknowledgements
This work was supported by the Swedish Research Council.

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