Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology
ReviewRelationship of sequence and structure to specificity in the α-amylase family of enzymes
Introduction
Comparisons of amino acid sequences of glycoside hydrolases and transglycosylases have allowed a classification scheme to be established for these enzymes, based on structure rather than specificity [1], [2], [3], i.e., the enzymes have been grouped into more than 80 families, where the members of one family share a common three-dimensional structure and mechanism, and have from a few to many sequence similarities. Families 13, 70 and 77 in this classification contain structurally and functionally related enzymes catalysing hydrolysis or transglycosylation of α-linked glucans, with retention of anomeric configuration. Many of these enzymes act on starch, and one of the most important starch-degrading enzymes, α-amylase, is also the most widely-studied member of family 13. Hence this group of enzymes, here considered to consist of families 13, 70 and 77, is often known as the α-amylase family of enzymes, but is, in fact, comprised of enzymes with almost 30 different specificities. In some cases, specificities of different enzymes in the family overlap and this, coupled with amino acid sequence similarities, has led to confusion in identification, particularly of family 13 enzymes. In this review, we attempt to clarify the relationship between sequence and specificity, and discuss, where information is available, key structural features that contribute to specificity.
Section snippets
Domain architecture
The enzymes are multidomain proteins, but share a common catalytic domain in the form of a (β/α)8-barrel, i.e., a barrel of eight parallel β-strands surrounded by eight helices, the so-called domain A (Fig. 1). This structure has been demonstrated by X-ray crystallography in several enzymes of the α-amylase family (Table 1), although in one instance only seven of the eight helices in the barrel fold are present [16]. In addition, studies of amino acid sequence similarities have led to the
Catalytic mechanism and substrate binding at the active site
Throughout the family, the enzymes are believed to have a similar mechanism of action, and so the catalytic amino acid residues are thought to be common to all the enzymes [37]. Anomeric configuration is retained when the substrate is converted to product, i.e., the enzymes act on α-linkages in glucans or glucosides and yield α-linked products. The reaction is believed to proceed by a double displacement mechanism (Fig. 2). During the first displacement an acid group on the enzyme protonates
Conserved sequences and specificity
Different enzymes of the family have different specificities; some are active only on α-1,4 glycosidic bonds between glucose residues, others on α-1,6 bonds exclusively, some on both bond types, yet others can cleave sucrose, while there are related enzymes that can hydrolyse or form the inter-glucose link in trehalose (Fig. 5). For all of the enzymes, activity involves binding a glucose residue of the substrate at subsite −1, while the nature of the portion of the substrate binding at subsites
α-Amylases
α-Amylases are generally considered to be endo-acting enzymes. There are enzymes, however, that hydrolyse starch polysaccharides to products with the α-anomeric configuration and are believed to act preferentially at one end of a polysaccharide chain to give primarily one size of small oligosaccharide, i.e., are exo- rather than endo-acting hydrolases. Some of these enzymes now have Enzyme Commission numbers distinct from that of α-amylase, e.g., maltogenic ‘amylase’ (3.2.1.133),
Relevance to protein engineering
The large diversity of specificity and the different types of reaction catalysed by enzymes in glycoside hydrolase families 13, 70, and 77 – or clan GH-H – invite rational engineering of the enzyme specificity. Early mutational analyses investigated structure/function relationships [37] and protein engineering moreover addressed important industrial goals such as improvement of thermostability or changing the pH activity dependence [121], [122], [123]. Similarly, modification of the product
Conclusions and recommendations
Enzymes of the α-amylase family can bring about scission and synthesis of α-1,4-, α-1,6- and less commonly α-1,2- and α-1,3-glucosidic linkages, as well as act on sucrose and trehalose. The enzyme active sites may be considered to be composed of subsites, each capable of interacting with one monosaccharide residue. All enzymes of the family require an α-linked glucose residue in the substrate to interact with the glycone-binding subsite −1 adjacent to the catalytic acids. Enzymes of different
Acknowledgements
M.T. Jensen and J.G. Olsen are thanked for help with Fig. 1, Fig. 3, Fig. 6. E.A.MacG. wishes to thank the University of Manitoba for the provision of office space and computer facilities. S.J. thanks the Slovak Grant Agency for Science (VEGA Grant No. 2/6045/99), FEBS for a short-term fellowship, and the Slovak literary Fund for financial support. B.S. was supported by the EU 4th Framework Programme (BIO4-CT98-0022).
References (138)
- et al.
Crystal structure of calcium-depleted Bacillus licheniformis α-amylase at 2.2 Å resolution
J. Mol. Biol.
(1995) - et al.
Crystal structure of a catalytic-site mutant α-amylase from Bacillus subtilis complexed with maltopentaose
J. Mol. Biol.
(1998) - et al.
Crystal and molecular structure of barley α-amylase
J. Mol. Biol.
(1994) - et al.
Crystal structure of yellow meal worm α-amylase at 1.64 Å resolution
J. Mol. Biol.
(1998) - et al.
Structure and molecular model refinement of pig pancreatic α-amylase at 2.1 Å resolution
J. Mol. Biol.
(1993) - et al.
Crystal structure of a maltotetraose-forming exo-amylase from Pseudomonas stutzeri
J. Mol. Biol.
(1997) - et al.
Three-dimensional structure of Pseudomonas isoamylase at 2.2 Å resolution
J. Mol. Biol.
(1998) - et al.
Crystal structure of a maltogenic amylase provides insights into a catalytic versatility
J. Biol. Chem.
(1999) - et al.
Crystal structure of Thermoactinonyces vulgaris R-47 α-amylase II (TVAII) hydrolyzing cyclodextrins and pullulan at 2.6 Å resolution
J. Mol. Biol.
(1999) - et al.
Crystal structure of glycosyltrehalose trehalohydrolase from the hyperthermophilic archaeum Sulfolobus solfataricus
J. Mol. Biol.
(2000)