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Starch in Food: Structure, Function and Applications

Starch in Food: Structure, Function and Applications

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Starch in Food: Structure, Function and Applications

1,793 pagine
22 ore
Nov 25, 2017


Starch in Food: Structure, Function and Applications, Second Edition, reviews starch structure, functionality and the growing range of starch ingredients used to improve the nutritional and sensory quality of food. The new edition is fully updated and brings new chapters on starch and health, isolation, processing and functional properties of starch.

Part One illustrates how plant starch can be analyzed and modified, with chapters on plant starch synthesis, starch bioengineering and starch-acting enzymes. Part Two examines the sources of starch, from wheat and potato, to rice, corn and tropical supplies. Part Three looks at starch as an ingredient and how it is used in the food industry, with chapters on modified starches and the stability of frozen foods, starch-lipid interactions and starch-based microencapsulation. Part Four covers starch as a functional food, investigating the impact of starch on physical and mental performance, detecting nutritional starch fractions and analyzing starch digestion.

The book is a standard reference for those working in the food industry, especially to starch scientists, food researchers, post-docs, practitioners in the starch area and students.

  • Completely revised and updated with an overview of the latest developments in isolation, processing, functional properties and health attributes of starch
  • Reviews starch structure and functionality
  • Extensive coverage of the growing range of starch ingredients
  • Examines how starch ingredients are used to improve the nutritional and sensory quality of food
Nov 25, 2017

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Anteprima del libro

Starch in Food - Elsevier Science

Starch in Food

Structure, Function and Applications

Second Edition


Malin Sjöö

Lars Nilsson

Table of Contents

Cover image

Title page

Related Titles


List of Contributors

Part One. Analysing and Modifying Starch

Chapter 1. Plant Starch Synthesis

1. Localization of Plant Starch Synthesis in Plants

2. Starch Synthesis: Enzyme Reactions in Plants and Algae and Glycogen Synthesis in Cyanobacteria

3. Properties of Plant α-1,4-Glucan-Synthesizing Enzymes

4. Characterization of ADP-Glc PPases From Different Plant Sources

5. Differences in Interaction Between 3PGA and Pi in Different ADP-Glc PPases

6. Identification of Important Amino Acid Residues Within the ADP-Glc PPases

7. Characterization of the Regulatory Domain

8. Starch Synthases

Chapter 2. Analyzing Starch Molecular Structure

1. Introduction: Characterizing Structures of Starch Components

2. Fractionation of Starch

3. Analysis of Amylose

4. Analysis of Amylopectin Structure

5. Analysis of Intermediate Materials

6. Future Trends

Chapter 3. Understanding Starch Structure and Functionality

1. Introduction

2. Components of Starch Granules

3. Structures of Starch Granules

4. Effects of Structures on Starch Properties

5. Effects of Crop Maturation

6. Conclusions and Future Trends

Chapter 4. Starch Bioengineering

1. Introduction: Improving Starch Functionality Directly in the Crop

2. Technologies for Starch Bioengineering

3. The Metabolic Reactions Influencing Starch Yield and Structure

4. Physical and Chemical Properties of Bioengineered Starches

5. Functionality and Uses in Food Processing of Bioengineered Starches

6. Toward Predictable Biotechnological Modification of Starch

7. Future Prospects

Chapter 5. Physical Modification of Starch

1. Introduction

2. Thermal Treatments

3. Nonthermal Treatments

4. Physical Treatments That Produce Chemical Changes

Chapter 6. Measuring Starch in Food

1. Introduction

2. Sample Preparation

3. Methods of Analysis

4. Recent Developments: Automation and Future Trends

5. Sources of Further Information and Advice

Chapter 7. Chemical Modification of Starch

1. Introduction

2. Starch Source

3. Common Types of Chemical Modifications

4. Physicochemical Properties and Functionality of Modified Starches

5. Applications in Foods

Part Two. Sources of Starch

Chapter 8. The Functionality of Wheat Starch

1. Introduction

2. Wheat Starch Production and Use for the Food Industry

3. Granular and Molecular Structure of Wheat Starch

4. Interaction of Starch With Minor Components

5. Hydrolysis of Wheat Starch

6. Improving the Functionality of Wheat Starch for Use in the Food Industry

7. Conclusions and Future Trend

Chapter 9. Potato Starch

1. Introduction

2. Granular and Molecular Structure of Potato Starches

3. Amylopectin Potato Starches

4. Functionality of Potato Starch and Derivatives for the Food Industry

5. Recent Developments

6. Outlook

Chapter 10. Rice Flour and Starch Functionality

1. Introduction

2. Rice Flour and Starch as a Food Ingredient

3. Constitutes of Rice Starch

4. Structure and Functionality of Rice Starch

5. Gelatinization and the Structure of Rice Starch

6. Retrogradation and Other Properties of Rice Starch

7. Improving Rice Starch Functionality for Food Processing Applications

8. Future Trends

9. Sources of Further Information and Advise

Chapter 11. Functionality of Tuber Starches

1. Introduction

2. Tuber Crop Starch Production

3. Minor Constituents

4. Structure and Functionality of Starches

5. Food Applications of Tuber Starches

6. Improving Tuber Starch Functionality for Food Applications: Modifying Tropical Starches for Use in the Food Industry

7. Future Trends

Chapter 12. The Functionality of Pseudocereal Starches

1. Introduction

2. Quinoa Starch

3. Canihua Starch

4. Amaranth Starch

5. Buckwheat Starch

6. Future Trend

Part Three. Applications

Chapter 13. Utilizing Starches in Product Development

1. Introduction

2. Components of Starch

3. Food Applications for Natural and Modified Starches

4. Methods of Starch Selection

5. Factors Affecting Starch in Food Products

6. Using the Functional Properties of Starch to Enhance Food Products

Chapter 14. Modified Starches and the Stability of Frozen Foods

1. Introduction

2. Deterioration Mechanism of Frozen Foods

3. Improving Frozen Food Quality Through Modified Starches

4. Conclusion and Future Perspective

Chapter 15. Starch in Baked Products

Part I: Starch and Its Interaction With Ingredients in Baked Products

2. Morphology and Ultrastructure of Starch Grain

3. Hydrothermic Transitions

4. V Amylose and Inclusion Complexes

Part 2: Cases of Study

6. Starch in the Case of Pan Bread

7. Starch in the Case of Cakes

8. Starch in the Case of Cookies

9. Starch in the Case of Biscuits

10. Starch at Surface of Bakery Products and Impact on Crust Properties

11. Conclusion

Chapter 16. Starch in Brewing Applications

1. Introduction

2. History of Brewing Beer

3. Starch-Degrading Enzymes

4. Important Factors for Mashing

5. Better Understanding of the Substrate–Enzyme System

6. Other Sources for Fermentable Sugars

7. Fermentation

8. Wild Yeasts and Specialty Beers

9. Fermentable Sugars

10. Starch Impact on Whiskey and Other Distilled Products

11. Starch Gelatinization

12. The Barley Grain and Starch Structure

13. Variation in Starch Structure in Other Brewing Cereals

14. Conclusion

Chapter 17. Starch-Based Microencapsulation

1. Introduction

2. The Nature of Starch

3. Starch Modifications

4. Modified Starches for Microencapsulation

5. Conclusion

Chapter 18. Starch Nanoparticles

1. Introduction

2. Preparation of Starch Nanoparticles

3. Characterization of Starch Nanoparticles

4. Surface Functionalization of Starch Nanoparticles

5. Interaction of Starch Nanoparticles With Protein

6. Application of Starch Nanoparticles

7. Conclusion and Prospectives

Chapter 19. Starch-Based Films

1. Introduction

2. Definitions

3. Global Production

4. Regulations Governing Food Contact Materials

5. Conversion Techniques

6. Impact of Coating Technique

7. Challenges in Full-Scale Processes

8. Material Properties

9. Future Materials and Concluding Remarks

Chapter 20. Starch Interactions With Native and Added Food Components

1. Introduction

2. Starch Composition and Structure

3. Starch Phase Transitions

4. Starch Interactions With Other Food Constituents

5. Future Trends

6. Conclusions

Part Four. Starch and Health

Chapter 21. Starch Digestion and Applications of Slowly Available Starch

1. Introduction

2. Prevention of Postprandial Hyperglycemia: the Role of Starch

3. Targets to Influence Postprandial Glycemia

4. Techniques for Monitoring Starch Digestion

5. Current (Nutritional) Strategies to Prevent Postprandial Hyperglycemia

6. Future Trends

Chapter 22. Development of Foods High in Slowly Digestible and Resistant Starch

1. Introduction

2. Molecular and Physicochemical Characteristics of Functional Starches

3. Slowly Digestible and Resistant Starch Production as Ingredient for Food Industry

4. Development of Foods With Functional Starch Ingredients

5. Future Trends

6. Conclusions

Chapter 23. Starch: Physical and Mental Performance, and Potential Health Problems

1. Introduction

2. Dietary Starches and Physical Performance

3. Dietary Starches and Mental Performance

4. Dietary Starches—Adverse Effects

5. Conclusions


Related Titles

Starch, AP (ISBN 978-0-12-746275-2)

Yeasts in Food, Woodhead (ISBN 978-1-85573-706-8)


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List of Contributors

Yongfeng Ai,     University of Saskatchewan, Saskatoon, SK, Canada

Rajamohanan J. Anish,     Kerala University, Trivandrum, India

Raquel Antezana-Gomez,     San Simon University, Cochabamba, Bolivia

Jinsong Bao,     Zhejiang University, Hangzhou, China

Luis A. Bello-Perez,     Instituto Politécnico Nacional, Yautepec, Mexico

James N. BeMiller,     Purdue University, West Lafayette, IN, United States

Christine J. Bergman,     University of Nevada Las Vegas, Las Vegas, NV, United States

Eric Bertoft,     Bertoft Solutions, Turku, Finland

Andreas Blennow,     University of Copenhagen, Frederiksberg, Denmark

Pieter L. Buwalda

Wageningen University and Research, Wageningen, The Netherlands

Coöperatie AVEBE U.A., Veendam, The Netherlands

Yu-Fang Chen,     Massey University, Palmerston North, New Zealand

Coby Eelderink,     University of Groningen, Groningen, The Netherlands

Glen Fox

The University of Queensland, St Lucia, QLD, Australia

Stellenbosch University, Stellenbosch, South Africa

Suzanne Hendrich,     Iowa State University, Ames, IA, United States

Nesrin Hesso,     ONIRIS, UMR GEPEA CNRS 6144, Nantes, France

Javier D. Hoyos-Leyva,     Instituto Politécnico Nacional, Yautepec, Mexico

Jay-lin Jane,     Iowa State University, Ames, IA, United States

Yangyang Jin,     Ingredion Incorporated, Bridgewater, NJ, United States

Lovedeep Kaur,     Massey University, Palmerston North, New Zealand

Kristine Koch,     Swedish University of Agricultural Sciences, Uppsala, Sweden

Aleksandra Komisarczyk,     Lodz University of Technology, Lodz, Poland

Izabella Kwaśniewska-Karolak,     Lodz University of Technology, Lodz, Poland

Patricia Le-Bail,     INRA, UMR BIA, Nantes, France

Alain Le-Bail,     ONIRIS, UMR GEPEA CNRS 6144, Nantes, France

Jason Z. Li,     Ingredion Incorporated, Bridgewater, NJ, United States

Thomas Luallen,     Starquest F.O.O.D. Consulting, Clinton, IL, United States

Amir Malaki Nik,     International Flavors & Fragrances Incorporated, Union Beach, NJ, United States

Subramoney N. Moorthy,     CTCRI, Trivandrum, India

Ewa Nebesny,     Lodz University of Technology, Lodz, Poland

Daysi Perez-Rea,     San Simon University, Cochabamba, Bolivia

Miguel Peris-Tortajada,     Universitat Politècnica de València, Valencia, Spain

Jack Preiss,     Michigan State University, East Lansing, MI, United States

Marion G. Priebe,     University of Groningen, Groningen, The Netherlands

Carmen C. Quiroga Ledezma,     Universidad Privada Boliviana – UPB, Cochabamba, Bolivia

Justyna Rosicka-Kaczmarek,     Lodz University of Technology, Lodz, Poland

Moothandassery S. Sajeev,     CTCRI, Trivandrum, India

Cindy Semeijn,     Coöperatie AVEBE U.A., Veendam, The Netherlands

Jaspreet Singh,     Massey University, Palmerston North, New Zealand

Qingjie Sun,     Qingdao Agricultural University, Qingdao, China

Roel J. Vonk,     University of Groningen, Groningen, The Netherlands

Renate E. Wachters-Hagedoorn,     University of Groningen, Groningen, The Netherlands

Pei Wang,     Nanjing Agricultural University, Nanjing, China

Xueming Xu,     Jiangnan University, Wuxi, People's Republic of China

Part One

Analysing and Modifying Starch


Chapter 1. Plant Starch Synthesis

Chapter 2. Analyzing Starch Molecular Structure

Chapter 3. Understanding Starch Structure and Functionality

Chapter 4. Starch Bioengineering

Chapter 5. Physical Modification of Starch

Chapter 6. Measuring Starch in Food

Chapter 7. Chemical Modification of Starch

Chapter 1

Plant Starch Synthesis

Jack Preiss     Michigan State University, East Lansing, MI, United States


This chapter reviews enzymic reactions involved in starch synthesis in higher plants and algae. Existing information on the properties of various starch biosynthetic enzymes in the plant, algal, and cyanobacterial systems will be described and compared. Alteration of starch structure due to the mutational effects on these biosynthetic enzymes allows postulation for some specific functions for starch synthases and branching enzymes. Finally regulation of starch synthesis at the enzymatic level will be discussed and in relation to this regulation, recent results indicating how starch content has been increased in certain plants will be indicated. A previous chapter (Shannon and Garwood, l984) in the second edition of Starch Chemistry and Technology discusses the various maize endosperm mutants or mutant combinations (26 of them) that shows an effect on the quantity or the nature of the starch formed and is still of interest. This information remains of interest and the reader is referred to that review.


3PGA; Amylose–lipid complex; Biosynthetic enzymes; Branching enzyme; Pyrophosphorylase; Starch synthases; Starch synthesis; α-Amylase

This chapter reviews enzymic reactions involved in starch synthesis in higher plants and algae. Existing information on the properties of various starch biosynthetic enzymes in the plant, algal, and cyanobacterial systems will be described and compared. Alteration of starch structure due to the mutational effects on these biosynthetic enzymes allows postulation for some specific functions for starch synthases (SSs) and branching enzymes (BEs). Finally regulation of starch synthesis at the enzymatic level will be discussed and in relation to this regulation, recent results indicating how starch content has been increased in certain plants will be indicated. A previous chapter (Shannon and Garwood, l984) in the second edition of Starch Chemistry and Technology discusses the various maize endosperm mutants or mutant combinations (26 of them) that shows an effect on the quantity or the nature of the starch formed and is still of interest. This information remains of interest and the reader is referred to that review.

This review will deal with mutants where the biochemical process affecting the mutation has been elucidated. Pérez and Bertoft (2010) have published a recent and detailed informative review on starch structure based on recent instrumentation applications. That article presents the current view on the general features of the starch granules, and amylose and amylopectin structures. There is a number of recently published review articles (Preiss, 2009; Zeeman et al., 2010; Kötting et al., 2010; Preiss, 2010; Geigenberger, 2011; Stitt and Zeeman, 2012; Schwarte et al., 2015; Streb and Zeeman, 2012; Nakamura, 2015) on starch biosynthesis covering many of the areas presented in this chapter.

1. Localization of Plant Starch Synthesis in Plants

1.1. Leaf Starch

Starch is deposited in granules in almost all green plants and in various types of plant tissues and organs, e.g., leaves, roots, shoots, fruits, grains, and stems. Illumination of the leaf in bright light causes the formation of starch granules in the chloroplast organelle (Pérez and Bertoft, 2010). Disappearance of starch occurs either by exposure of the observed by iodine staining of the tissue or by light or electron microscopy. Starch accumulates due to carbon fixation during photosynthesis and the starch formed in the light is degraded in the dark to products that are in most cases utilized for sucrose synthesis. Mutants of Arabidopsis thaliana unable to synthesize starch, grow at the same rate as the wild type (WT) in a continuous light regime because they are able to synthesize sucrose (Caspar et al., 1986), but their growth rate is drastically reduced if grown in a day–night regime. The reason for this is that the accumulated starch is required for sucrose synthesis at night; the sucrose is transported from the leaf to the sink tissues. Biosynthesis and degradation of starch in the leaf is therefore a dynamic process having diurnal level fluctuations in its storage (Stitt and Zeeman, 2012; Streb and Zeeman, 2012).

Starch also plays an important role in the operation of stomatal guard cells, where it is degraded during the day. In the late afternoon or evening while the stomata are open, the starch is resynthesized. Leaf starch is lower in amylose content than what is observed in storage tissues (Matheson, 1996). The amylose structure is also of a smaller molecular size.

1.2. Starch in Storage Tissues

In storage organs, fruit or seed, during the development and maturation of the tissue, synthesis of starch occurs. At the time of sprouting or germination of the seed or tuber, or ripening of the fruit, starch degradation in these tissues can occur and the derived metabolites are used as a source both for carbon and energy. The degradation and biosynthetic processes in the storage tissues can therefore be temporally separated. However, there is a possibility that during each phase of starch metabolism some turnover of the starch molecule can occur. The main site of starch synthesis and accumulation in the cereals is the endosperm, with starch granules that are located within the amyloplasts. Starch content in potato tuber, maize endosperm, and in roots of yam, cassava, and sweet potato can range between 65% and 90% of the total dry matter (Pérez and Bertoft, 2010). Patterns of starch accumulation during development of the tissue are specific to the species and are related to the unique pattern of differentiation of the organ (Pérez and Bertoft, 2010).

Starch granules in storage tissues can vary in shape, size, and composition (Pérez and Bertoft, 2010). They can be spherical, oval, polygonal, lenticular elongated, or kidney shaped. The dimensions of the starch granules can vary from 2 to 3  μm as seen in small wheat granules to up to 100  μm as observed in canna starch. Starch granules as isolated in plants such as amaranth or taro, however, can have dimensions in the submicron level. Thus the shape and size of the granules depends on the source. But in each tissue there is a range of sizes and shapes. The diameter of the starch granule changes during the development of the reserve tissue. There are also some fine features, characteristic of each species, e.g., the growth rings, spaced 4–7  μm apart, and the fibrillar organization seen in potato starch, that allows one to identify the botanical source of the starch by microscopic examination (Pérez and Bertoft, 2010).

Two polymers are distinguished in the starch granule. Amylose is essentially a linear polymer and amylopectin, a highly branched polymer. Amylose is mainly found as linear chains of about 840 to 22,000 units of α-D-glucopyranosyl residues linked by α-(1→4) bonds (molecular weight around 136,000 to 3.5  ×  l0⁶). The number of anhydroglucose units, however, varies quite widely with plant species and stage of development. Some of the amylose molecules are branched to a small extent (α-1→6-D glucopyranose; one per l70 to 500 glucosyl units). Amylopectin, in contrast, which usually comprises about 70% of the starch granule, is more highly branched with about 4%–5% of the glucosidic linkages being α-1→6. Amylopectin molecules are large flattened disks consisting of α-(1,4)-glucan chains joined by frequent α-(1,6)-branch points. The satisfactory models of amylopectin structure proposed fitting the experimental data available are those proposed by Robin et al. (1974), Manners and Matheson (1981), and by Hizukuri (1986). These are known as cluster models. The chemical and physical aspects of the starch granule and components amylose and amylopectin are discussed in reviews by Morrison and Karkalis (1990) and Hizukuri (1996) and recently by Pérez and Bertroft (2010). Fig. 1.1 shows the proposed cluster model of amylopectin.

Figure 1.1  Proposed basic structure of amylopectin ( Pérez and Bertoft, 2010 ). The circles are glucose residues and lines represent α-1   →   4 linkages. The bent arrows represent α-1   →   6 linkages. The reducing end is seen as ø.

2. Starch Synthesis: Enzyme Reactions in Plants and Algae and Glycogen Synthesis in Cyanobacteria

2.1. Enzyme Reactions of Starch Synthesis

The specific sugar nucleotide utilized for synthesis of the α-1,4-glucosidic linkages in amylose and amylopectin is adenosine diphosphate-glucose (ADP-Glc). ADP-Glc synthesis is catalyzed by ADP-Glc pyrophosphorylase (Reaction (1.i), E.C.; ATP: α-D-glucose-1-phosphate adenylyl-transferase).

ATP  +  α-glucose-1-P  ↔  ADP-glucose  +  PPi (1.i)

Reaction (1.ii) is catalyzed by SS (E.C.; ADP-glucose; 1,4-α-D-glucan 4-α-glucosyl-transferase). A similar reaction is noted for glycogen synthesis in cyanobacteria and other bacteria (Preiss and Romeo, 1989; Preiss, 1996, 2014; Ball and Morell, 2003; Ballicora et al., 2003) where the reaction is referred to as glycogen synthase (E.C.

ADP-glucose  +  α-1,4-glucan  →  α-1,4-glucosyl-α-1,4-glucan  +  ADP (1.ii)

Reaction (1.iii) shows catalysis by BE (E.C.; 1,4-α-D-glucan6-α-(1,4-α-glucano)-transferase).

α-1,4-oligosaccharide chain  →  α-1,4, α-1,6-branched chain (1.iii)

The branch chains in amylopectin are longer than in glycogen and are about 20–24 glucose units long. There is less branching in amylopectin. About 5% of the glucosidic linkages are α-1,6. In glycogen, the chain lengths (CLs) are about 10–13 glucose units long and 10% of linkages are α-1,6. Thus, the (SBEs) may have different properties with respect to the size of chain transferred, or placement of branch point, than the enzyme that branches glycogen. Alternatively, the interaction of the SBEs with the SSs may be different from the interaction of the bacterial BEs with their respective glycogen synthases. The chain-elongating properties of the SSs could be different from those observed for the bacterial glycogen synthases and may account for some of the differences observed in the amylopectin structure. The differences in the catalytic properties of the SSs and BEs isolated from different plant sources and their interactions may also account for the differences observed in the various plant starch structures (Pérez and Bertoft, 2010).

Isozymic forms of plant SSs and BEs have been reported and the different properties of these isozymes will be discussed. They seem to play different roles in the synthesis of the two polymers of starch, amylose and amylopectin, and are products from different genes. In many different plants as well as in Chlamydomonas reinhardtii, a granule-bound starch synthase (GBSS) involved in the catalysis of Reaction (1.ii) has been shown to be involved in the synthesis of amylose.

3. Properties of Plant α-1,4-Glucan-Synthesizing Enzymes

3.1. ADP-Glucose Pyrophosphorylase: Kinetic and Regulatory Properties of the Enzyme

In bacteria and in plants there is only one physiological function for ADP-Glc and that is to be a donor of glucose for synthesis of α-1,4-glucosyl linkages of plant or algal starch. It would therefore be advantageous to conserve the ATP utilized for synthesis of the sugar nucleotide by regulating α-1,4-glucan synthesis at the level of ADP-Glc formation.

Over 50 ADP-glucose pyrophosphorylases (ADP-Glc PPases), bacterial, algal, and plant, have been studied with respect to their regulatory properties (Preiss, 1996, 2009, 2010, 2014; Preiss and Romeo, 1989; Ball and Morell, 2003; Ballicora et al., 2003, 2004; Smith-White and Preiss, 1992). In almost all cases, glycolytic intermediates activate ADP-Glc synthesis, while AMP, ADP, and/or Pi are inhibitors (Preiss, 1996, 2010, 2014; Preiss and Romeo, 1989; Ball and Morell, 2003; Ballicora et al., 2003, 2004; Smith-White and Preiss, 1992).

Glycolytic intermediates in the cell may be considered as a signal of carbon excess; therefore, under conditions of limited growth with available excess carbon in the environment, accumulation of glycolytic intermediates would be the signal for activation of ADP-Glc synthesis. In plants and algae, with active CO2 fixation either via the Calvin or Hatch-Slack pathway and ATP formation during photosynthesis, the enzyme ADP-Glc PPase would be affected by the availability of ATP in the cell as well as the carbon dioxide fixation product, 3-phospho-glycerate (3PGA). The ADP-Glc PPase of higher plants, green algae, and the cyanobacteria are allosterically activated by 3PGA and inhibited by inorganic phosphate (Pi) (Preiss, 2009, 2010; Ballicora et al., 2004; Smith-White and Preiss, 1992). The 3PGA activation can be anywhere from 3- to 60-fold. Of importance is that increasing concentrations of Pi can reverse activation by 3PGA. These effects are important in the regulation of starch synthesis. Spinach leaf (Ghosh and Preiss, 1966; Preiss et al., 1967; Copeland and Preiss, 1981) and other plant leaves (Sanwal and Preiss, 1967), Chlorella (Sanwal et al., 1968), and potato tuber ADP-Glc PPases (Iglesias et al., 1993; Ballicora et al., 1995, 1998) have been studied in the most detail with respect to kinetic properties and structure. The results obtained with potato tuber and spinach leaf enzyme are summarized in Table 1.1. 3PGA activates the spinach leaf ADP-Glc PPase 20-fold and activates the potato tuber enzyme 30-fold. Pi can inhibit both enzymes about 50% at 40–45  μM. However, in the presence of 1  mM 3PGA, the enzymes are less sensitive to inhibition by Pi and 50% inhibition occurs at 0.97  mM for the spinach enzyme. In the presence 3  mM PGA, 0.63  mM Pi is required for 50% inhibition. Thus the synthetic rate of ADP-Glc synthesis will be dependent on the ratio of 3PGA/Pi concentrations. It is expected that during photosynthesis that Pi concentrations would be lower due to higher rate of ATP synthesis and greater rate of synthesis and accumulation of phosphorylated glycolytic intermediates including 3PGA. Of interest is that the apparent affinity of the substrates, ATP and glucose-1-P (Glc-1-P) for the ADP-Glc PPase is higher in the presence of the activator, 3PGA. The spinach leaf enzyme requires about four- to sixfold less substrate concentration for 50% maximal velocity and the constant is denoted as S0.5. 3PGA lowers the S0.5 of the substrates for the potato tuber enzyme about 3.5- to 6-fold.

Table 1.1

Kinetic Constants of ADP-Glucose Pyrophosphorylase From Spinach Leaf and Potato Tuber

A0.5 is the activation constant; the concentration of activator required for 50% of maximal velocity. I0.5 is the inhibitor concentration required for 50% inhibition in the presence or absence of the activator, 3PGA. S0.5 is the substrate concentration required for 50% of maximal activity. NR stands for not reported.

The kinetic and regulatory properties of the ADP-Glc PPases from the leaf extracts of spinach, barley, butter lettuce, kidney bean, maize, peanut, rice, sorghum, sugar beet, tobacco, and tomato have been studied in detail and are quite similar (Sanwal et al., l968).

3.2. ADP-Glucose Pyrophosphorylase: Structure–Function Relationships: Quaternary Structure

Bacterial ADP-Glc PPases are homotetrameric in structure (Preiss, 1996, 2014; Ballicora et al., 2003). The catalytic and allosteric sites are on the same and each subunit. In plants and in green algae, however, the ADP-Glc PPases have been shown to be heterotetramers with two homologous subunits, α2β2 (Preiss, 2009, 2010; Ballicora et al., 2004; Smith-White and Preiss, 1992) having different molecular sizes. The small subunit is about 50–54  kDa and has catalytic activity. The large subunit, about 51–60  kDa, is generally the regulatory subunit. The large subunit modulates the sensitivity of the small subunit toward allosteric effectors, via large subunit/small subunit interactions (Preiss, 2009, 2010; Ballicora et al., 2004). However, recent results indicate that some large subunits, particularly those in leaf (Ventriglia et al., 2008), also have catalytic activity.

ADP-Glc PPase from potato tuber is composed of two different subunits, 50 and 51  kDa, with a α2β2 heterotetrameric subunit structure (Ballicora et al., 1995, 1998, 2004; Iglesias et al., 1993). The small subunit of many higher plant ADP-Glc PPases is highly conserved among plants with 85%–95% identity (Smith-White and Preiss, 1992; Ballicora et al., 1995, 1998). The homotetrameric potato enzyme composed exclusively of small subunits has a lower apparent affinity (A0.5  =  2.4  mM) for the activator 3PGA than the heterotetramer (A0.5  =  0.16  mM) and is more sensitive to the inhibitor Pi (I0.5  =  0.08  mM in the presence of 3  mM 3PGA) as compared with the heterotetramer (I0.5  =  0.63  mM) (Ballicora et al., 1995, 1998). The potato large subunit greatly increases the affinity of the small (catalytic) subunit for 3PGA and lowers the affinity for the inhibitor Pi (Ballicora et al., 1995, 1998).

In plants there may be only one conserved small (catalytic) subunit and several large (regulatory) subunits that are distributed in different parts of the plant (Ventriglia et al., 2008; Crevillén et al., 2003, 2005). This is of physiological significance as expression of different large subunits in different plant tissues may confer distinct allosteric properties to the ADP-Glc PPase needed for the different parts of the plant's distinct need for starch (Crevillén et al., 2003, 2005).

It has been shown with Arabidopsis ADP-Glc PPase that coexpression of its small subunit, APS1, with the different Arabidopsis large subunits, ApL1, ApL2, ApL3, and ApL4, resulted in heterotetramers with different regulatory and kinetic properties (Tables 1.2 and 1.3). Homotetramer APS1 had low affinity for 3PGA while the heterotetramers of APS and the large subunits had much greater affinity for 3PGA (Crevillén et al., 2003; Table 1.2). The heterotetramer of the small subunit APS1 with ApL1, the predominant leaf large subunit (Crevillén et al., 2003), had the highest sensitivity to the allosteric effectors, 3PGA and Pi, as well as the highest apparent affinity for the substrates ATP and Glc-1-P (Crevillén et al., 2003). The heterotetrameric pairs of APS1 with either ApL3 or ApL4, large subunits, prevalent in sink or storage tissues (Crevillén et al., 2005), had intermediate sensitivity to the allosteric effectors and intermediate affinity for the substrates ATP and Glc-1-P (Table 1.3; Crevillén et al., 2003). ApL2 also present mainly in sink tissues had low affinity for either 3PGA or Pi (Ventriglia et al., 2008; Crevillén et al., 2003). Thus, differences on the regulatory properties conferred by the Arabidopsis large subunits were found in vitro. Differences noted for source and sink large subunit proteins strongly suggests that starch synthesis is modulated in a tissue-specific manner in response to 3PGA and Pi, as well as to the substrate levels. APS1 and ApL1 would be finely regulated in source tissues by both effectors and substrates while in sink tissues the heterotetramers of APS1 with ApL2, ApL3, or ApL4 with lower sensitivity to effectors and substrates would be controlled more by the supply of substrates.

Table 1.2

Kinetic Parameters for 3PGA of Arabidopsis thaliana Recombinant ADP-Glc PPase in the Synthesis Direction (Crevillén et al., 2003)

APS1 is the small subunit 1, ApL1, ApL2, ApL3, and ApL4 are large subunits 1, 2, 3, and 4, respectively, from A. thaliana. N.D. indicates not determined. The kinetic parameters were calculated without inhibitor Pi, or in the presence of inhibitor Pi at 0.2  mmol or at 2  mM. A0.5 is the activator concentration required for 50% of maximal activation.

Table 1.3

Kinetic Parameters for the Substrates of Arabidopsis thaliana Recombinant ADP-Glc PPase in the Synthesis Direction (Crevillén et al., 2003).

The activator 3PGA concentrations used for each enzyme tetramer subunit combination was the concentration required for maximal velocity. S0.5 is the concentration of substrate required for 50% of maximal activity.

Based on mRNA expression, APS1 is the main small subunit or catalytic isoform responsible for ADP-Glc PPase activity in all tissues of the plant. ApL1 is the main large subunit in source tissues, whereas ApL3 and ApL4 are the main isoforms present in sink tissues (Crevillén et al., 2005). It was also found that sugar regulation of ADP-Glc PPase genes was restricted to ApL3 and ApL4 in leaves (Crevillén et al., 2005). Sucrose induction of ApL3 and ApL4 transcription in leaves allowed formation of heterotetramers that are less sensitive to the allosteric effectors, resembling the situation in sink tissues.

3.3. Relationship Between the Small and Large Subunits

The amino acid sequence similarity between the small and large subunits (∼50%–60% identity) suggests a common origin (Ballicora et al., 1995, 1998). In both sink and source tissues the small subunit has catalytic activity while catalytic activity is only observed for the large subunits that may reside in the leaf and not in the sink large subunits (Ventriglia et al., 2008). Most probably gene duplication and divergence has led to different and functional roles catalytic and regulatory for the subunits. The ancestor of small and large subunits possibly is a bacterial subunit having both catalytic as well as regulatory function in the same subunit. This is supported by the similarity between the two plant subunits with many active bacterial ADP-Glc PPases (Ballicora et al., 2004; Smith-White and Preiss, 1992).

The large subunit from the potato (Solanum tuberosum L.) tuber ADP-Glc PPase was shown to bind substrates (Fu et al., 1998a). The plant heterotetramer therefore, as well as bacterial homotetramers, binds four ADP-[¹⁴C]glucose molecules (Fu et al., 1998a; Haugen and Preiss, 1979). It can be postulated that the large subunit maintained its structure needed for binding of substrate, but catalytic ability was eliminated by mutations of essential residues. To test this hypothesis, it was attempted to create a large subunit with significant catalytic activity by the mutation of amino acid residues involved in substrate binding as well as in catalysis (Frueauf et al., 2003).

Thus, sequence alignments of ADP-Glc PPase large and small subunits with reported activity were compared to identify critical missing residues for catalytic activity in the large subunit (Frueauf et al., 2003). The subset of the ones absent in the large subunit was of particular interest. Lys44 and Thr54 in the large subunit of potato tuber were selected as the best candidates to study because the homologous residues, Arg33 and Lys43 in the small (catalytic) subunit, were completely conserved in the active bacterial and plant catalytic subunits. Moreover, Lys44 and Thr54 are in a highly conserved region of both bacterial and plant ADP-Glc PPases (Table 1.4).

Table 1.4

Sequence Comparison of ADP-Glc PPase Subunits in a Conserved Region With Critical Amino Acids for Catalysis (Frueauf et al., 2003)

Sequence comparison of the catalytic site of ADP-Glc PPases from E. coli, Arabidopsis, and potato. Sequences and their accession numbers are E. coli, P00584; PSS, potato tuber small subunit CAA88449; APS1, P55228; ApL1, P55229; ApL2, P55230; ApL3, P55231; ApL4, Q9SIK1; PLS, potato tuber large subunit Q00081. The conserved arginine and lysine (or threonine) residues are indicated in bold.

The potato large subunits Lys44 and Thr54 were mutagenized to Arg44 and Lys54, respectively. The mutant, LargeK44R/T54K, expressed in the absence of the small subunit had no activity. Possibly the large subunit cannot form a stable tetramer in the absence of the small subunit as seen earlier with Arabidopsis enzyme (Ventriglia et al., 2008). Because WT small subunit has intrinsic activity, the activity of the large subunit mutants cannot be tested when coexpressed. Thus, the large subunit mutants were coexpressed with inactive small subunit D145N, in which the catalytic residue Asp145 was mutated (Frueauf et al., 2003) reducing by more than three orders of magnitude small subunit activity (Table 1.5). Coexpression of the large subunit double-mutant K44R/T54K with Small D145N generated an enzyme having 10% and 18% of the WT enzyme in the ADP-Glc synthetic direction, respectively (Table 1.5). Single mutations K44R or T54K generated enzymes with no significant activity. The combination of both mutations in the large subunit (Small D145N/LargeK44R/T54K) gave the most dramatic effect (Table 1.5). Therefore, it was concluded that the two residues Arg44 and Lys54 are needed for restoring catalytic activity to the large subunit. Replacement of the homologous two residues with Lys and Thr in the small subunit (by mutations R33K and K43T) decreased the activity one and two orders of magnitude, respectively, in either the ADP-Glc synthetic or pyrophosphorolytic directions, confirming the hypothesis (Table 1.5; Frueauf et al., 2003).

Table 1.5

Activity Catalytic (Small) and Regulatory (Large) Subunits of Potato Tuber ADP-Glc PPase Mutants

The enzymes activities of purified coexpressed small and large subunits were measured for ADP-glucose (ADP-Glc) synthetic activity. For ADP-Glc synthesis, 4  mM of 3PGA (activator), 2  mM of ATP, and 0.5  mM of Glc-1-P were used.

The mutant enzymes were still activated by 3PGA and inhibited by phosphate (Pi). The WT enzyme and Small D145N/LargeK44R/T54K had very similar kinetic properties indicating that the substrate site domain has been conserved. The apparent affinities for the substrates and the allosteric properties of small subunit D145N/LargeK44R/T54K resembled those of the WT enzyme (Ballicora et al., 2005). The new form has a similar sensitivity to Pi inhibition and the activator–inhibitor interactions were the same as WT enzyme. That the large subunit restored enzyme activity to the inactive small subunit heterotetramer due to only two mutations is evidence that the large and small subunits are derived from the same ancestor.

3.4. The Glucose-1-P-Binding Site in Plant ADP-Glucose Pyrophosphorylase

The heterotetramer Small D145N mutant and LargeK44R/T54K mutant was disrupted in each subunit at their Glc1P site and their kinetic properties compared. As indicated above, catalysis occurs in the large subunit of Small D145N/LargeK44R/T54K heterotetramer. As seen in Table 1.6, the Glc-1-P substrate-binding residue in the small subunit is Lys198 (Fu et al., 1998a). Substitution of Lys with Arg lowers the affinity of the enzyme for Glc-1-P about 150-fold and substitution of the Lys with Ala or Glu lowers the affinity 440- or 546-fold, respectively (Table 1.6). Amino acid substitution of the homologous residue, Lys213, in the large subunit does not have the same effect. In Small D145N/LargeK44R/T54K enzyme, the mutation, K213R of the large subunit severely decreased the apparent affinity for G1P, whereas mutation of K198R on the small subunit did not (Table 1.6). This indicated that the large subunit double mutant, and not Small D145N, was the catalytic subunit. In the WT enzyme, Lys213 does not seem to play any important role, but in Small D145N/LargeK44R/T54K it recovered its ancestral ability to confer to the enzyme a high-apparent affinity for G1P. Previous results showed that Asp145 in the small subunit of the WT is essential for catalysis (Frueauf et al., 2003), and in the WT enzyme, replacement of Lys198 in the small subunit of the WT enzyme, decreased the Glc-1P affinity (Fu et al., 1998a). Disruption of the homologous residue Lys213 in the large subunit has much less effect. In Small D145N/LargeK44R/T54K where the large subunit is now the catalytic subunit, the K213R mutation of the large subunit severely decreased the apparent affinity for Glc1P, whereas the K198R mutation of the small subunit did not indicate that the large subunit double mutant, and not Small D145N, was the catalytic subunit. In the WT enzyme, Lys213 does not seem to play any important role, but Small D145N/LargeK44R/T54K recovered its ancestral ability for the enzyme to have a low physiological Km for Glc1P.

Table 1.6

Apparent Affinity of Glc-1-P and ADP-Glc of Potato Tuber Wild-Type and Mutant ADP-Glc PPases

Previous results showed that in the WT ADP-Glc PPase, Asp145 of the small subunit is essential for catalysis, but homologous Asp160 in the large subunit is not 39. Also mutation of D160 to N or E in the active large subunit LK44R/T54K abolished activity. This confirms that catalysis of Small D145N/LargeK44R/T54K does occur in the large subunit.

A comparative model of LK44R/T54K shows the predicted role of Arg44 and Lys54 (Fig. 1.2). In the model, Asp160, which is homologous to the catalytic Asp145 in the small subunit and catalytic Asp142 in the Escherichia coli ADP-Glc PPase (Frueauf et al., 2001, 2003), interacts with Lys54. This type of interaction (Lys54–Asp160) has also been observed in crystal structures of enzymes catalyzing similar reactions, such as dTDP-glucose pyrophosphorylase (dTDP-Glc PPase) (Blankenfeldt et al., 2000) and UDP-N-acetyl-glucosamine pyrophosphorylase (UDP-GlcNAc PPase) (Brown et al., 1999), and postulated to be important for catalysis by correctly orienting the aspartate residue (Blankenfeldt et al., 2000; Brown et al., 1999; Sivaraman et al., 2002). Lys54 interacts with the oxygen bridging the α- and β-phosphates as it has been observed in the crystal structure of E. coli dTDP-Glc PPase (Sivaraman et al., 2002). The interaction may neutralize a negative charge density stabilizing the transition state and making PPi a better leaving group. Arg44 interacts in the model with the β- and γ-phosphates of ATP, which correspond to the PPi by-product (Fig. 1.2). Likewise, Arg15 in the E. coli dTDP-Glc PPase was postulated to contribute to the departure of PPi42 and kinetic data agreed with interaction of PPi with Arg44 in the model. A Lys44, in both the catalytic large subunit mutant and the small subunit, of the potato ADP-Glc PPase decreased the apparent affinity for PPi at least 20-fold (Ballicora et al., 2005). In WT large subunit, Lys44 and Thr54 cannot interact as Arg44 and Lys54 (Fig. 1.2).

Figure 1.2  Involvement of the large (regulatory) subunit of mutant K44R/T54K in enzyme catalysis. The WT and double-mutant large subunits were modeled based on the dTDP-Glc PPase and UDP-GlcNAc PPases as indicated. Portions of residues 31–73 and 131–136 are shown. The deoxyribose triphosphate portion common to dTTP and ATP is modeled with Mg ²+ as a blue sphere . The nitrogen atom of the adenylyl group attached to the ribosyl unit is also in blue. The dotted green lines depict hydrogen bonds.

3.5. Phylogenetic Analysis of the Large and Small Subunits

A phylogenetic tree of the ADP-Glc PPases present in photosynthetic eukaryotes may also shed information about the origin of the two subunits. The tree shows that plant small and large subunits can be divided into two and four distinct groups, respectively (Ballicora et al., 2005). The two main groups of S subunits are from dicot and monocot plants, whereas large subunit groups correlate better with their documented tissue expression. The first Large-subunit group, group I, is generally expressed in photosynthetic tissues (Ballicora et al., 2005) and comprises large subunits from dicots and monocots. These subunits recently have been shown to have catalytic activity and have in their sequences Arg and Lys in the equivalent residues of 102 and 112 of A. thaliana large subunit, ApL1. Group II displays a broader expression pattern, whereas groups III and IV are expressed in storage organs (roots, stems, tubers, seeds). Subunits from group III are only from dicot plants, whereas group IV are seed-specific subunits from monocots. These last two groups stem from the same branch of the phylogenetic tree and split before monocot and dicot separation. These subunits are probably inactive in catalytic activity as they are lacking Arg and Lys in the homologous residues seen in A. thaliana ApLI and ApL234.

3.6. Crystal Structure of Potato Tuber ADP-Glc PPase

The crystal structure of potato tuber homotetrameric small (catalytic) subunit ADP-Glc PPase has been determined to 2.1  Å resolution (Fig. 1.3(a); Jin et al., 2005). The structures of the enzyme in complex with ATP and ADP-Glc were determined to 2.6 and 2.2  Å resolution, respectively. Ammonium sulfate was used in the crystallization process and was found tightly bound to the crystalline enzyme. It was also shown that the small subunit homotetrameric potato tuber ADP-Glc PPase was also inhibited by inorganic sulfate with the I0.5 value of 2.8  mM in the presence of 6  mM 3PGA. Sulfate is considered as an analog of phosphate, the allosteric inhibitor of plant ADP-Glc PPases. Thus the atomic resolution structure of the ADP-Glc PPase probably presents a conformation of the allosteric enzyme in its inhibited state. The crystal structure of the potato tuber ADP-Glc PPase (Jin et al., 2005) allows one to determine the location of the activator and substrate sites in the three-dimensional structure and their relation to the catalytic residue, Asp145. The structure also provides insights into the mechanism of allosteric regulation and these aspects will be discussed later.

The overall fold of the potato tuber ADP-Glc PPase small subunit catalytic domain is quite similar to that of two other pyrophosphorylases, viz., N-acetylglucosamine 1-phosphate uridylyl-transferase (GlmU) from E. coli 43 (Fig. 1.3(b)) and S. pneumoniae (Sulzenbacher et al., 2001; Kostrewa et al., 2001) and glucose 1-phosphate thymidylyl-transferase (Rffh) from P. aeruginosa (Blankenfeldt et al., 2000) and E. coli (Sivaraman et al., 2002), although their primary sequences have only very low sequence similarities. The catalytic domain is composed of a seven-stranded β sheet covered by α helices, a fold reminiscent of the dinucleotide-binding Rossmann fold (Raetz and Roderick, 1995). At one of its ends, the central β-sheet is topped by a two-stranded β-sheet. The catalytic domain makes strong hydrophobic interactions with the C-terminal domain through an α-helix that encompasses residues 285–297 (Fig. 1.3(c)). The catalytic domain is connected to the C-terminal β-helix domain by a long loop containing residues 300–320. This loop makes numerous interactions with the equivalent region of another monomer.

The C-terminal domain comprises residues 321–451 and adopts a left-handed β-helix fold composed of six complete or partial coils with two insertions, one of that encompasses residues 368–390. The other encompasses residues 401–431. This type of left-handed β-helix domain fold has been found in the structures of bacterial acetyl-transferases, including E. coli UDP-N-acetylglucosamine 3-O-acyl-transferase (Raetz and Roderick, 1995), Methanosarcina thermophila carbonic anhydrase (Kisker et al., 1996), Mycobacterium bovis tetrahydrodipicolinate N-succinyl-transferase (Beaman et al., 1997) and GlmU (Sivaraman et al., 2002), and in other proteins such as T4 bacteriophage gp5 (Kanamaru et al., 2000). However, the β-helix domain seen in the other structures is an acetyl-transferase or succinyl-transferase domain. In the present structure of ADP-Glc PPase, the β-helix domain is involved in cooperative allosteric regulation with the N-terminal catalytic region and interactions with the N-terminal region within each monomer, and contributes to oligomerization.

Figure 1.3  (a) Crystal structure of potato tuber ADP-glucose pyrophosphorylase (ADP-Glc PPase) small (catalytic) subunit monomer. The catalytic domain is in yellow and the β-helix subunit is in pink. ADP-Glc is shown in atom type where the carbon atoms are in green, the oxygen atoms red, nitrogen atoms blue, the phosphorus atoms magenta, and the sulfate groups orange. (b) Overlay of ADP-Glc PPase (cyan, RmlA (r.m.s.d. 1.9   Å) (magenta)) and GlmU (gold) (r.m.s.d. 2.5   Å). (c) ADP-Glc PPase tetramer. The disulfide bond is boxed . (d) Interactions between the monomers in the tetramer.

3.7. The Homotetramer Catalytic Subunit Structure of the Potato Tuber ADP-Glucose Pyrophosphorylase (Jin et al., 2005)

The crystalline potato tuber ADPGlc PPase small subunit is a tetramer with approximate 222 symmetry and approximate dimensions of 80  ×  90  ×  110  Å³ (Fig. 1.3(c)). It can be viewed as a dimer of dimers, labeled A, A′, B, and B′. Monomers A and B interact predominantly by end-to-end stacking of their β-helix domains, although there is also a significant interface between the linker loop connecting the two domains (Fig. 1.3(d)). This interface buries 2544  Å² of surface area. The catalytic domains of A and B′ (and B and A′) also make an extensive interface. Several hydrogen bond and hydrophobic interactions stabilize the interface between A and B′, burying a surface area of 1400  Å. All residues defining dimerization interfaces are identical or similar in the large subunit. Fig. 1.3(d) delineates all oligomerization interactions seen within the tetramer in the asymmetric unit. Cys12 of monomer A and the equivalent cysteine residue of monomer A′ make a disulfide bond, as do equivalent cysteine residues of monomers B and B′. The intersubunit disulfide bond between the small (catalytic) subunit is preserved in the heterotetramer. However, there is no disulfide bond between the large (regulatory) subunits, as Cys12 is not conserved. This disulfide bond establishes the relative orientation of the small subunits in the heterotetramer to be like A and A′ in the α4-homotetramer structure. The disulfide bond is the only interaction made between A and A′ (or B and B′ subunits). Potato tuber ADP-Glc PPase is redox-regulated by reduction and oxidation of the intermolecular disulfide bond between the two small subunits (Fu et al., 1998b; Ballicora et al., 1999, 2000). This covalent regulatory modification is discussed later.

The N-terminal catalytic domain resembles a dinucleotide-binding Rossmann fold and the C-terminal domain comprising residues 321–451 adopts a left-handed parallel β helix that is involved in cooperative allosteric regulation and a unique oligomerization. The structures of the enzyme in an ATP and ADP-Glc complex are also observed. Communication between the regulator-binding sites and the active site involves several distinct regions of the enzyme including the N-terminus, the Glc-1-P-binding site, and the ATP-binding site are proposed. These structures provide insights into the mechanism for catalysis and allosteric regulation of the enzyme.

Sulfate is an inhibitor of potato tuber ADP-Glc PPase small subunit homotetramer with I0.5  =  2.8  mM in the presence of 6  mM 3PGA (Jin et al., 2005). The electron density map for potato tuber ADP-Glc PPase small subunit suggests that there are three sulfate ions tightly bound to the enzyme (Fig. 1.4). Most probably, this is due to the high-sulfate concentration (150  mM) in the crystallization solution. Two sulfate ions bind within 7.5  Å of each other in a crevice located between the N- and C-terminal domains of the enzyme (Jin et al., 2005). A third sulfate ion binds between the two subunits of the enzyme. The sulfate ions make numerous interactions with residues shown to be involved in the allosteric activator-binding site, as demonstrated by chemical modification (Morell et al., 1988; Greene et al., 1996a) and site-directed mutagenesis studies (Fu et al., 1998b). The structures contain 12 sulfate ions within a tetramer in the asymmetric unit (three per monomer) and are all, therefore, representative of the inhibited conformation of the enzyme.

Figure 1.4  ADP-Glc PPase monomer showing (a) the sulfate-binding region between the catalytic and beta-helix domains and (b) the amino acid residues interacting with sulfate. The sulfate residues are yellow and the interacting residues are green in one subunit. The neighboring subunit and its residues are purple.

Sulfate 1 makes hydrogen bond interactions with R41, R53, K404, and K441 (Fig. 1.4; Jin et al., 2005). The side-chain nitrogen atom of R41 makes hydrogen bond interactions with one of the sulfate ion oxygen atoms, and D403 makes a salt bridge interaction with R41 to facilitate the binding. D413 in the potato tuber enzyme large subunit (D403 in the small subunit) was identified as important for activation by 3PGA (Greene et al., 1996a; Ballicora et al., 1998). All these residues are conserved in virtually all plant ADP-Glc PPases, and four of the five (all but K441) are strongly conserved in bacterial ADP-Glc PPases (Fig. 1.4). Site-directed mutagenesis studies have identified residues K441 and K404 in the small subunit of potato tuber as important for 3PGA activation (Greene et al., 1996a; Ballicora et al., 1998). The enzyme's affinity for 3PGA was lowered and the inhibition by Pi diminished when mutations at these residues were Ala (neutral) or Glu (negative). The kinetic parameters for the substrates, ADP-Glc, PPi and the cofactor Mg²+ were not affected. Mutations on the homologous residues in the large subunit showed lesser or no effects on regulation of the enzyme (Ballicora et al., 1998). Therefore, it was concluded that K404 and K441 in potato tuber ADP-Glc PPase small subunits are important for the binding of 3PGA and Pi, and the main role of the large subunit is to interact with the small subunit and modulate its activation mechanism (Ballicora et al., 1998). These studies indicate that the activator 3PGA binds at or near the inhibitor-binding site defined in the structure by sulfate 1.

Sulfate 2 makes similar interactions with surrounding positively charged residues, R53, R83, H84, Q314, and R316 (Fig. 1.4). Site-directed mutagenesis studies have shown that H83 of the E. coli enzyme (H84 in the potato tuber enzyme small subunit) is involved in activator binding (Hill and Preiss, 1998). Chemical modification with phenylglyoxal has identified R294 in the Anabaena sp. enzyme (R316 in the potato tuber enzyme small subunit) as an important residue for inhibition by Pi, as mutations of this residue lowered the apparent affinity for Pi more than 100-fold (Sheng and Preiss, 1997). Mutations of this Arg residue to Ala, Gln, or Lys caused a change in inhibitor selectivity such that these mutants were inhibited by NADPH or FBP (Frueauf et al., 2002). Taken together, these studies confirm the importance of the sulfate ion-binding site in the allosteric regulation of the enzyme and indicate that 3PGA may also bind near the sulfate 2-binding site.

Sulfate 3 is located between two subunits, viz., A and B′. This sulfate ion interacts with R83 of one monomer and K69, H134, and T135 of the other subunit. K69 and R83 are conserved in both the small and large subunits of all plant ADP-Glc PPases. H134 is conserved in all small subunits, and residue T135 is conservatively replaced by Asn in other plant small subunits. The precise role of this location is not yet clear. Sulfate binding may be nonspecific or it may interfere with the dimerization of the subunits, thus causing the R  →  T equilibrium to be more toward the T (inhibited) state.

The current structural results strongly support previous data on the allosteric regulation of this enzyme and provide some insights on how the binding of allosteric effectors could affect catalysis.

3.8. ATP Binding

When ATP binds to the enzyme, both A and A′ monomers undergo almost identical conformational changes. Several regions move significantly, viz., a loop region from residue 27 to 34, another loop region from residue 106 to 119, and residues K40, R41, Q75, and F76 (Fig. 1.4 in reference Jin et al. (2005)). Both loop regions make direct interactions with the adenine portion of the nucleotide. Specifically, the main chain nitrogen atom of G28 makes a hydrogen bond with N3 of the adenine ring; several hydrophobic interactions are established between the adenine ring and L26 and G29; the side chain of Q118 makes a hydrogen bond with N6. These residues all undergo correlated conformational change upon ATP binding. Interactions of Q75 with both G30 and W116, and interactions of the K40 side chain with P111 couple the motions of the Q75, G30, and 106–119 regions (Jin et al., 2005).

Furthermore, ATP binding in the A and A′ subunits drives conformational change in the B and B′ subunits, as P111 of A and A′ is packed snugly against W129 in B′ and B, respectively (Jin et al., 2005). Motion of P111 accompanying ATP binding leads directly to motion of W129 and, in fact, the entire region from 165 to 231 in the B/B′ subunits.

3.9. ADP-Glucose Binding

Three of the four subunits (A, A′, and B), bind ADP-Glc in the ADP-Glc PPase/ADP-Glc complex (Fig. 1.3(a)). The B′ subunit binds neither ATP nor ADP-Glc, and is conformationally more rigid than the other three subunits. A and A′ bind ADP-Glc identically, and ADP-Glc binding produces conformational changes in A and A′ identical to that which occurs when ATP binds (described above). The adenyl and ribosyl units of ADP-Glc in A and A′ are positioned identically to the adenyl unit of ATP, and the interactions between the enzyme and ADP-Glc are also identical to those seen in the ATP complex (Jin et al., 2005). No electron density is seen for the glucosyl moiety of ADP-Glc in the A and A′ active sites, indicating it to be disordered. This indicates that the conformational changes seen in A and A′ on ATP or ADP-Glc binding are due almost exclusively to the adenyl and ribosyl moieties. Both phosphate groups are also ordered. In contrast, the entire ADP-Glc molecule is well ordered in the B subunit active site. The adenyl and ribosyl positions are very similar to those seen in the A and A′ subunits through the region 112–117, which undergoes conformational change upon ATP or ADP-Glc binding in A and A′ and is disordered in B and B′ both with and without ADP-Glc in the active site. The two phosphate groups and the glucosyl units are very well ordered in the B active site and adopt positions and conformations similar to that seen in other sugar nucleotide pyrophosphorylase complex structures (Fig. 1.5). There are several direct interactions between the enzyme and the glucosyl unit of ADP-Glc. These include hydrogen bonds between E197, S229, D280, and the glucosyl unit (Fig. 1.6). In addition K198 makes a salt bridge with the phosphate group attached to the glucosyl unit.

Figure 1.5  Sequence alignment of ADP-Glc PPase from different species. Secondary structure of the potato tuber enzyme is shown above the sequence. Cylinders , helices ; straight block arrows , beta strands; curved block arrows , turns in the beta-helix domain. Green stars are residues interacting with ADP-Glc and red stars are residues interacting with ATP. Residues that are identical are shaded in purple . Ana_glgC , Anabaena ADP-Glc PPase; Ath_S , APS1 , Arabidopsis thaliana small subunit; Atu_glgC , Agrobacterium tumefaciens ADP-Glc PPase; Bst_glgC , Bacillus stearothermophilus ADP-Glc PPase; Cre_S , Chlamydomonas reinhardtii small subunit; Eco_glgC , Escherichia coli ADP-Glc PPase; Hvu_S_endosp , Hydra vulgaris small subunit; Rsp_glgC , Rhodopseudomonas spheroides ADP-Glc PPase; Stu_L , potato tuber large subunit; Stu_s , potato tuber small subunit; Zma_S_maize endosp , maize small subunit.

The B subunit undergoes a very large subdomain movement in response to ADP-Glc binding. Two residues, E197 and K198, are critical for binding the glucosyl and phosphate moieties of ADP-Glc; K198 has been characterized as a Glc1P binding residue by site-directed mutagenesis (Fu et al., 1998a) and both are part of a motif present in many sugar nucleotide pyrophosphorylases. These two residues are shifted out of the binding pocket in the B subunit of the ATP-bound structure, while they are pulled more inward in the unbound B subunit and are pulled in significantly in the ADP-Glc-bound molecule (Fig. 1.6).

Figure 1.6  Hydrogen bond interactions between ADP-glucose (ADP-Glc) and the ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalytic subunit. Protein carbon bonds are green. ADP-Glc carbon bonds are yellow, oxygen atoms are red, nitrogen atoms are blue, and phosphate atoms are purple.

3.10. Implication for Catalysis

Detailed kinetic studies on ADP-Glc PPase have shown that a sequential bi–bi mechanism fits the data with ATP binding first (Haugen and Preiss, 1979; Kleczkowski et al., 1993a; Paule and Preiss, 1971). Structural data from two related enzymes, GlmU46 and Rffh44, indicate the presence of a metal ion in the active site. A Co²+ ion (in GlmU) and an Mg²+ ion (in GlmU and Rffh) are located in almost identical locations in the two distinct enzymes when the active sites are aligned. In GlmU, both the Mg²+ and the Co²+ are chelated to two conserved residues (Asp105 and Asn227) and to the two phosphate groups of the product (UDP-N-acetylglucosamine). In Rffh, the Mg²+ is also bound to two conserved carboxylate residues (Asp223 and Asp108) and to the α-phosphate group of TTP. When the ADP-Glc PPase active site is aligned with these active sites, two acidic residues, Asp145 and Asp280, are spatially close to the metal-chelating residues in Rffh and GlmU. Mutation of Asp145 residue to Asn in the potato tuber enzyme and the equivalent Asp142 in the E. coli enzyme results in a reduction in catalytic activity by four orders of magnitude (Frueauf et al., 2001; Jin et al., 2005). Taken together, it is concluded that the metal-mediated catalytic mechanism proposed for RffH and GlmU is also used by ADP-Glc PPase. Also concluded is that the metal ion is chelated by the residues equivalent to D145 and D280 in all ADP-Glc PPases and that the mutational sensitivity of D145 is due to the requirement for metal ion in the reaction. The absolute requirement for a metal ion has been biochemically demonstrated for ADP-Glc PPase from several organisms (Preiss, l996, 2014; Ballicora et al., 2003, 2004; Gomez-Casati et al., 2001). Structural data from GlmU, RmlA, and RffH have shed considerable light on the mechanism of sugar nucleotide pyrophosphorylases, and the similarity between the active site of α-4-ADP-Glc PPase and these enzymes indicates a similar mechanism for all of these enzymes. The structure of the RffH/dTTP complex is particularly informative, because it identifies the GXGXRL loop, which is strongly conserved in all of these enzymes, to be the site of the triphosphate moiety of ATP and shows that the residue equivalent to R33 in the α subunit of ADP-Glc PPase is critical for triphosphate binding. Conformational change of this loop will therefore have profound effects on the activity of the enzyme. In addition to R33, D145, D280, K43, E197, and K198 (potato tuber α4 numbering, Fig. 1.6) are also conserved in the other sugar nucleotide pyrophosphorylases of known structures, are in similar locations in the active sites, and make similar interactions with the sugar nucleotide. Based on results with other sugar nucleotide pyrophosphorylase structures (Brown et al., 1999; Sivaraman et al., 2002), the following is postulated for

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