E. Ann MacGregor

Relationship of sequence and structure to specificity in the α-amylase family of enzymes

The hydrolases and transferases that constitute the alpha-amylase family are multidomain proteins, but each has a catalytic domain in the form of a (beta/alpha)(8)-barrel, with the active site being at the C-terminal end of the barrel beta-strands. Although the enzymes are believed to share the same catalytic acids and a common mechanism of action, they have been assigned to three separate families - 13, 70 and 77 - in the classification scheme for glycoside hydrolases and transferases that is based on amino acid sequence similarities. Each enzyme has one glutamic acid and two aspartic acid residues necessary for activity, while most enzymes of the family also contain two histidine residues critical for transition state stabilisation. These five residues occur in four short sequences conserved throughout the family, and within such sequences some key amino acid residues are related to enzyme specificity. A table is given showing motifs distinctive for each specificity as extracted from 316 sequences, which should aid in identifying the enzyme from primary structure information. Where appropriate, existing problems with identification of some enzymes of the family are pointed out. For enzymes of known three-dimensional structure, action is discussed in terms of molecular architecture. The sequence-specificity and structure-specificity relationships described may provide useful pointers for rational protein engineering.

Starch- and glycogen-debranching and branching enzymes: Prediction of structural features of the catalytic (β/α)8-barrel domain and evolutionary relationship to other amylolytic enzymes

Sequence alignment and structure prediction are used to locate catalytic α-amylase-type (β/α)8-barrel domains and the positions of their β-strands and α-helices in isoamylase, pullulanase, neopullulanase, α-amylase-pullulanase, dextran glucosidase, branching enzyme, and glycogen branching enzymes—all enzymes involved in hydrolysis or synthesis of α-1,6-glucosidic linkages in starch and related polysaccharides. This has allowed identification of the transferase active site of the glycogen debranching enzyme and the locations of β ⇑ α loops making up the active sites of all enzymes studied. Activity and specificity of the enzymes are discussed in terms of conserved amino acid residues and loop variations. An evolutionary distance tree of 47 amylolytic and related enzymes is built on 37 residues representing the four best conserved β-strands of the barrel. It exhibits clusters of enzymes close in specificity, with the branching and glycogen debranching enzymes being the most distantly related.

A circularly permuted α-amylase-type α/β-barrel structure in glucan-synthesizing glucosyltransferases

A motif of amino acid residues, located at the active site and specific β‐strands in a‐amylases, is recognized in α‐1,3‐ and α‐1,6‐glucan‐synthesizing glucosyltransferases, leading to the conclusion that these enzymes contain an α/β‐barrel closely related to the (β/α)8‐fold of the α‐amylase superfamily. The secondary structure elements of the transferase barrel, however, are circularly permuted to start with an α‐helix equivalent to helix 3 in the α‐amylases. Thus, the transferase counterpart of the long third β → α connection — constituting a domain in the α‐amylases — is divided to precede and succeed the barrel. This architectural arrangement may be coupled to sucrose scission and glucosyl transfer. The involvement in the mechanism in glucosyltransferases of active site residues recurring in amylolytic enzymes is discussed.

α-Amylase: an enzyme specificity found in various families of glycoside hydrolases

Abstractα-Amylase (EC 3.2.1.1) represents the best known amylolytic enzyme. It catalyzes the hydrolysis of α-1,4-glucosidic bonds in starch and related α-glucans. In general, the α-amylase is an enzyme with a broad substrate preference and product specificity. In the sequence-based classification system of all carbohydrate-active enzymes, it is one of the most frequently occurring glycoside hydrolases (GH). α-Amylase is the main representative of family GH13, but it is probably also present in the families GH57 and GH119, and possibly even in GH126. Family GH13, known generally as the main α-amylase family, forms clan GH-H together with families GH70 and GH77 that, however, contain no α-amylase. Within the family GH13, the α-amylase specificity is currently present in several subfamilies, such as GH13_1, 5, 6, 7, 15, 24, 27, 28, 36, 37, and, possibly in a few more that are not yet defined. The α-amylases classified in family GH13 employ a reaction mechanism giving retention of configuration, share 4–7 conserved sequence regions (CSRs) and catalytic machinery, and adopt the (β/α)8-barrel catalytic domain. Although the family GH57 α-amylases also employ the retaining reaction mechanism, they possess their own five CSRs and catalytic machinery, and adopt a (β/α)7-barrel fold. These family GH57 attributes are likely to be characteristic of α-amylases from the family GH119, too. With regard to family GH126, confirmation of the unambiguous presence of the α-amylase specificity may need more biochemical investigation because of an obvious, but unexpected, homology with inverting β-glucan-active hydrolases.

Structural and evolutionary aspects of two families of non-catalytic domains present in starch and glycogen binding proteins from microbes, plants and animals.

Starch-binding domains (SBDs) comprise distinct protein modules that bind starch, glycogen or related carbohydrates and have been classified into different families of carbohydrate-binding modules (CBMs). The present review focuses on SBDs of CBM20 and CBM48 found in amylolytic enzymes from several glycoside hydrolase (GH) families GH13, GH14, GH15, GH31, GH57 and GH77, as well as in a number of regulatory enzymes, e.g., phosphoglucan, water dikinase-3, genethonin-1, laforin, starch-excess protein-4, the β-subunit of AMP-activated protein kinase and its homologues from sucrose non-fermenting-1 protein kinase SNF1 complex, and an adaptor-regulator related to the SNF1/AMPK family, AKINβγ. CBM20s and CBM48s of amylolytic enzymes occur predominantly in the microbial world, whereas the non-amylolytic proteins containing these modules are mostly of plant and animal origin. Comparison of amino acid sequences and tertiary structures of CBM20 and CBM48 reveals the close relatedness of these SBDs and, in some cases, glycogen-binding domains (GBDs). The families CBM20 and CBM48 share both an ancestral form and the mode of starch/glycogen binding at one or two binding sites. Phylogenetic analyses demonstrate that they exhibit independent behaviour, i.e. each family forms its own part in an evolutionary tree, with enzyme specificity (protein function) being well represented within each family. The distinction between CBM20 and CBM48 families is not sharp since there are representatives in both CBM families that possess an intermediate character. These are, for example, CBM20s from hypothetical GH57 amylopullulanase (probably lacking the starch-binding site 2) and CBM48s from the GH13 pullulanase subfamily (probably lacking the starch/glycogen-binding site 1). The knowledge gained concerning the occurrence of these SBDs and GBDs through the range of taxonomy will support future experimental research.

A new clan of CBM families based on bioinformatics of starch-binding domains from families CBM20 and CBM21.

Approximately 10% of amylolytic enzymes are able to bind and degrade raw starch. Usually a distinct domain, the starch‐binding domain (SBD), is responsible for this property. These domains have been classified into families of carbohydrate‐binding modules (CBM). At present, there are six SBD families: CBM20, CBM21, CBM25, CBM26, CBM34, and CBM41. This work is concentrated on CBM20 and CBM21. The CBM20 module was believed to be located almost exclusively at the C‐terminal end of various amylases. The CBM21 module was known as the N‐terminally positioned SBD of Rhizopus glucoamylase. Nowadays many nonamylolytic proteins have been recognized as possessing sequence segments that exhibit similarities with the experimentally observed CBM20 and CBM21. These facts have stimulated interest in carrying out a rigorous bioinformatics analysis of the two CBM families. The present analysis showed that the original idea of the CBM20 module being at the C‐terminus and the CBM21 module at the N‐terminus of a protein should be modified. Although the CBM20 functionally important tryptophans were found to be substituted in several cases, these aromatics and the regions around them belong to the best conserved parts of the CBM20 module. They were therefore used as templates for revealing the corresponding regions in the CBM21 family. Secondary structure prediction together with fold recognition indicated that the CBM21 module structure should be similar to that of CBM20. The evolutionary tree based on a common alignment of sequences of both modules showed that the CBM21 SBDs from α‐amylases and glucoamylases are the closest relatives to the CBM20 counterparts, with the CBM20 modules from the glycoside hydrolase family GH13 amylopullulanases being possible candidates for the intermediate between the two CBM families.

The action of germinated barley alpha-amylases on linear maltodextrins

Abstract The actions of barley alpha-amylase isozymes 1 and 2 (EC 3.2.1.1) on malto-oligosaccharides and their p-nitrophenyl glycosides were similar, but not identical. For each isozyme, transglycosylation occurred with small substrates that were hydrolysed with difficulty, whereas the rates of hydrolysis increased with increase in the size of the substrate for both the malto-oligosaccharides and the p-nitrophenyl glycosides. A p-nitrophenyl group was found to mimic a glucose residue to a large extent. The differences in action of the isozymes are believed to be caused by differences at more than one subsite of the active site. A lysine-arginine substitution is postulated to account for some of the observed variations.

A remote but significant sequence homology between glycoside hydrolase clan GH-H and family GH31

Although both the α‐amylase super‐family, i.e. the glycoside hydrolase (GH) clan GH‐H (the GH families 13, 70 and 77), and family GH31 share some characteristics, their different catalytic machinery prevents classification of GH31 in clan GH‐H. A significant but remote evolutionary relatedness is, however, proposed for clan GH‐H with GH31. A sequence alignment, based on the idea that residues equivalent in the primordial catalytic GH‐H/GH31 (β/α)8‐barrel may not be found in the present‐day GH‐H and GH31 structures at strictly equivalent positions, shows remote sequence homologies covering β3, β4, β7 and β8 of the GH‐H and GH31 (β/α)8‐barrels. Structure comparison of GH13 α‐amylase and GH31 α‐xylosidase guided alignment of GH‐H and GH31 members for construction of evolutionary trees. The closest sequence relationship displayed by GH31 is to GH77 of clan GH‐H.

Possible Structure and Active Site Residues of Starch, Glycogen, and Sucrose Synthases

A group of enzymes that include muscle glycogen phosphorylase and sugar transferases involved in, for example, the glucosylation of DNA and the synthesis of peptidoglycan are known to possess the same basic three-dimensional fold. Here the possibility is examined that other monosaccharide transferases, those that catalyze synthesis of starch, glycogen, and the disaccharide sucrose, resemble the phosphorylase-type enzymes in structure. In particular, a clear relationship is shown, for the first time, between mammalian glycogen synthases and the phosphorylase structural group of proteins. Domain architecture and secondary structure are discussed, and the possible role of several conserved amino acids at the active site is explored.

Structure and activity of some starch-metabolising enzymes

Abstract Several enzymes that participate in the metabolism of starch are believed to share a common structural feature-their catalytic domain folds as a (β/α)8-barrel i.e. a cylinder of eight β-strands surrounded by eight α-helices. In each enzyme, the active site is made up of amino-acid residues situated on the β-strands or loops protruding from the C-terminal ends of the β-strands. α-Amylases are the most widely-studied enzymes in this “family”, but other enzymes belonging to the group can catalyse hydrolysis or synthesis of α-1,4- or α-1,6-glucosidic linkages or both bond types. In this review, a description is given of current ideas of the relationship between key structural variations and specificity differences amongst the enzymes of the α-amylase family.