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

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Abstract

The hydrolases and transferases that constitute the α-amylase family are multidomain proteins, but each has a catalytic domain in the form of a (β/α)8-barrel, with the active site being at the C-terminal end of the barrel β-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.

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)

  • B.E Hofmann et al.

    Three-dimensional structure of cyclodextrin glucosyltransferase from Bacillus circulans at 3.4 Å resolution

    J. Mol. Biol.

    (1989)
  • C.L Lawson et al.

    Nucleotide sequence and X-ray structure of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 in a maltose-dependent crystal form

    J. Mol. Biol.

    (1994)
  • R.M.A Knegtel et al.

    Crystal structure at 2.3 Å resolution and revised nucleotide sequence of the thermostable cyclodextrin glycosyltransferase from Thermoanaerobacterium thermosulfurigenes EMl

    J. Mol. Biol.

    (1996)
  • I Przylas et al.

    Crystal structure of amylomaltase from Thermus aquaticus, a glycosyltransferase catalysing the production of large cyclic glucans

    J. Mol. Biol.

    (2000)
  • K.H Park et al.

    Structure, specificity and function of cyclomaltodextrinase, a multispecific enzyme of the α-amylase family

    Biochim. Biophys. Acta

    (2000)
  • S Janecek

    α-Amylase family: molecular biology and evolution

    Prog. Biophys. Mol. Biol.

    (1997)
  • E.A MacGregor et al.

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

    FEBS Lett.

    (1996)
  • J.C.M Uitdehaag et al.

    The cyclization mechanism of cyclodextrin glycosyltransferase (CGTase) as revealed by a γ-cyclodextrin-CGTase complex at 1.8 Å resolution

    J. Biol. Chem.

    (1999)
  • J Zhou et al.

    Nucleotide sequence of the maltotetrahydrolase gene from Pseudomonas saccharophila

    FEBS Lett.

    (1989)
  • T Yamamoto et al.

    Alteration of product specificity of cyclodextrin glucanotransferase from Thermococcus sp. B1001 by site-directed mutagenesis

    J. Biosci. Bioeng.

    (2000)
  • Y Yoshioka et al.

    Crystal structures of a mutant maltotetraose-forming exo-amylase cocrystallized with maltopentaose

    J. Mol. Biol.

    (1997)
  • A Kadziola et al.

    Molecular structure of a barley α-amylase-inhibitor complex: implications for starch binding and catalysis

    J. Mol. Biol.

    (1998)
  • I.C Kim et al.

    Catalytic properties of the cloned amylase from Bacillus licheniformis

    J. Biol. Chem.

    (1992)
  • T Tonozuka et al.

    Comparison of primary structures and subsite specificities of two pullulan-hydrolyzing α-amylases, TVA I and TVA II, from Thermoactinomyces vulgaris R-47

    Biochim. Biophys. Acta

    (1995)
  • J.F Robyt et al.

    The action pattern of porcine pancreatic α-amylase in relationship to the substrate binding site of the enzyme

    J. Biol. Chem.

    (1970)
  • A.W MacGregor et al.

    The action of germinated barley α-amylases on linear maltodextrins

    Carbohydr. Res.

    (1992)
  • J.F Robyt et al.

    Multiple attack hypothesis of α-amylase action: action of porcine pancreatic, human salivary, and Aspergillus oryzae α-amylases

    Arch. Biochem. Biophys.

    (1967)
  • M Machius et al.

    Activation of Bacillus licheniformis α-amylase through a disorder→order transition of the substrate-binding site mediated by a calcium-sodium-calcium metal triad

    Structure

    (1998)
  • B.A van der Veen et al.

    Rational design of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 to increase α-cyclodextrin production

    J. Mol. Biol.

    (2000)
  • D Penninga et al.

    The raw starch binding domain of cyclodextrin glycosyltransferase from Bacillus circulans strain 251

    J. Biol. Chem.

    (1996)
  • T Yamamoto et al.

    Alteration of product specificity of cyclodextrin glucanotransferase from Thermococcus sp. B1001 by site-directed mutagenesis

    J. Biosci. Bioeng.

    (2000)
  • T Kuriki et al.

    Construction of chimeric enzymes out of maize endosperm branching enzymes I and II

    J. Biol. Chem.

    (1997)
  • S Hong et al.

    Localization of C-terminal domains required for the maximal activity or for determination of substrate preference of maize branching enzymes

    Arch. Biochem. Biophys.

    (2000)
  • Y Hatada et al.

    Amino acid sequence and molecular structure of an alkaline amylopullulanase from Bacillus that hydrolyses α-1,4 and α-1,6 linkages in polysaccharides at different active sites

    J. Biol. Chem.

    (1996)
  • B Henrissat

    A classification of glycosyl hydrolases based on amino acid sequence similarities

    Biochem. J.

    (1991)
  • B Henrissat et al.

    New families in the classification of glyosyl hydrolases based on amino acid sequence similarities

    Biochem. J.

    (1993)
  • B Henrissat et al.

    Updating the sequence-based classification of glycosyl hydrolases

    Biochem. J.

    (1996)
  • N Aghajari et al.

    Crystal structures of the psychrophilic α-amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor

    Protein Sci.

    (1998)
  • R.L Brady et al.

    Solution of the structure of Aspergillus niger acid α-amylase by combined molecular replacement and multiple isomorphous replacement methods

    Acta Cryst.

    (1991)
  • Y Matsuura et al.

    Structure and possible catalytic residues of Taka amylase A

    J. Biochem.

    (1984)
  • G.D Brayer et al.

    The structure of human pancreatic α-amylase at 1.8 Å resolution and comparisons with related enzymes

    Protein Sci.

    (1995)
  • N Ramasubbu et al.

    Structure of human salivary α-amylase at 1.6 Å resolution: implications for its role in the oral cavity

    Acta Cryst.

    (1996)
  • H Kizaki et al.

    Polypeptide folding of Bacillus cereus ATCC7064 oligo-1,6-glucosidase revealed by 3 Å resolution X-ray analysis

    J. Biochem.

    (1993)
  • Z Dauter et al.

    X-ray structure of Novamyl, the five-domain ‘maltogenic’ α-amylase from Bacillus stearothermophilus: maltose and acarbose complexes at 1.7 Å resolution

    Biochemistry

    (1999)
  • L. Skov, O. Mirza, A. Henriksen, G. Potocki de Montalk, M. Remaud-Simeon, P. Sarbacal, R.-M. Willemot, P. Monsan, M....
  • Y. Matsuura, M. Kubota, Crystal structure of cyclodextrin glucanotransferase from Bacillus stearothermophilus and its...
  • K Harata et al.

    X-ray structure of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011. Comparison of two independent molecules at 1.8 Å resolution

    Acta Cryst.

    (1996)
  • E.A MacGregor et al.

    A super-secondary structure predicted to be common to several α-1,4-d-glucan-cleaving enzymes

    Biochem. J.

    (1989)
  • H.M Jespersen et al.

    Comparison of the domain-level organization of starch hydrolases and related enzymes

    Biochem. J.

    (1991)
  • H.M Jespersen et al.

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

    J. Protein Chem.

    (1993)
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