Protein engineering in the α-amylase family: catalytic mechanism, substrate specificity, and stability
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- Svensson, B. Plant Mol Biol (1994) 25: 141. doi:10.1007/BF00023233
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Most starch hydrolases and related enzymes belong to the α-amylase family which contains a characteristic catalytic (β/α)8-barrel domain. Currently known primary structures that have sequence similarities represent 18 different specificities, including starch branching enzyme. Crystal structures have been reported in three of these enzyme classes: the α-amylases, the cyclodextrin glucanotransferases, and the oligo-1,6-glucosidases. Throughout the α-amylase family, only eight amino acid residues are invariant, seven at the active site and a glycine in a short turn. However, comparison of three-dimensional models with a multiple sequence alignment suggests that the diversity in specificity arises by variation in substrate binding at the β→α loops. Designed mutations thus have enhanced transferase activity and altered the oligosaccharide product patterns of α-amylases, changed the distribution of α-, β- and γ-cyclodextrin production by cyclodextrin glucanotransferases, and shifted the relative α-1,4:α-1,6 dual-bond specificity of neopullulanase. Barley α-amylase isozyme hybrids and Bacillus α-amylases demonstrate the impact of a small domain B protruding from the (β/α)8-scaffold on the function and stability. Prospects for rational engineering in this family include important members of plant origin, such as α-amylase, starch branching and debranching enzymes, and amylomaltase.
starch binding domain
triose-phosphate isomerase. The mutations are described with the one-letter code, i.e. D164A is a mutant in which A in the mutant is substituted for D in the wild-type.