Protein engineering in the ?-amylase family: catalytic mechanism, substrate specificity, and stability

Abstract

Most starch hydrolases and related enzymes belong to the e-amylase family which contains a characteristic catalytic (fi/e)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 e-amylases, the cyclodextrin glucanotransferases, and the oligo-l,6-glucosidases. Throughout the e-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 fl--+ e loops. Designed mutations thus have enhanced transferase activity and altered the oligosaccharide product patterns of e-amylases, changed the distribution of e-,/3-and 7-cyclodextrin production by cyclodextrin glucanotransferases, and shifted the relative e-1,4: e-1,6 dual-bond specificity of neopullulanase. Barley e-amylase isozyme hybrids and Bacillus e-amylases demonstrate the impact of a small domain B protruding from the (/3/e)8-scaffold on the function and stability. Prospects for rational engineering in this family include important members of plant origin, such as e-amylase, starch branching and debranching enzymes, and amylomaltase.Abbreviations: CGTase, cyclodextrin glucanotransferase; SBD, starch binding domain; TAA, takaamylase A; TIM, triose-phosphate isomerase. The mutations are described with the one-letter code, i.e.

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“…However, when the intermediate b-glycosyl-enzyme bond is cleaved, such a correctly oriented orbital is not present, as pointed out in the text. letters the arguments in favor of an oxocarbenium intermediate in both a-and b-retaining mechanisms was the large distance between the nucleophilic residue and the substrate 8,20 . However, this distance was not much greater than the sum of the van der Waals radii of the atoms involved.…”

“…The a-retaining mechanism is used by enzymes from the a-amylase family (glycosyl hydrolase family 13; ref. 5), such as a-amylase, pullulanase, iso-amylase and cyclodextrin glycosyltransferase (CGTase), which all share a virtually identical catalytic site architecture 8,9 .…”

Inversion of configuration during hydrolysis of α‐1,4‐galacturonidic linkage by three Aspergillus polygalacturonases

“…However, this generalization seems to fail so far for exoglycanases. The two exo-acting e~-l,4-glucanases, amyloglucosidase and 13-amylase are inverting hydrolases, but a number of maltooligosaccharide-hydrolyzing exo-acting enzymes appear to be retaining glycanases [5]. Of the two cellobiohydrolases of Trichoderma reesei, considered as exo-acting enzymes, particularly when acting on cellulose or cellooligosaccharides, one is a retaining and the other is an inverting glycanase [6,7].…”

“…Practically all retaining glycanases tested under conditions of oversaturation with substrate catalyzed the reverse reaction leading to the synthesis of new glycosidic linkages [1,[8][9][10]. There is no report of glycosyl transfer reactions by inverting glycanases although the situation is complicated by the ability of some enzymes, like amyloglucosidase, to catalyze condensation reactions [5].…”

Close evolutionary relatedness among functionally distantly related members of the (α/β)8‐barrel glycosyl hydrolases suggested by the similarity of their fifth conserved sequence region

“…To demonstrate the evolutionary importance of the fifth conserved sequence region, the parts of the amino acid sequences comprising their eventual domains B (from the third t-strand to the fourth t-strand) of the s-amylases from Bacillus subtilis and Butyrivibriofibrisolvens (40% identity [10]) and of the functionally distantly related potato amylomaltase (its sequence exhibits substantial variability also in the four wellknown conserved regions [8]), were aligned using the program CLUS-TAL V [13].…”