Specificity modulation of barley alpha-amylase through biased random mutagenesis involving a conserved tripeptide in beta -> alpha loop 7 of the catalytic (beta/alpha)(8)-barrel domain

The relative specificity and bond cleavage pattern of barley alpha -amylase 1 (AMY1) were dramatically changed by mutation in (FVD)-V-286 that connect ed beta -strand 7 of the catalytic (beta/alpha)(8)-barrel, to a succeeding 3(10)-helix. This conserved tripeptide of the otherwise variable beta --> a lpha segment 7 lacked direct ligand contact, but the nearby residues His290 and Asp291 participated in transition-state stabilization and catalysis. O n the basis of sequences of glycoside hydrolase family 13, a biased random mutagenesis protocol was designed which encoded 174 putative (FVD)-V-286 va riants of C95A-AMY1, chosen as the parent enzyme to avoid inactivating glut athionylation by the yeast host. The FVG, FGG, YVD, LLD, and FLE mutants sh owed 12-380 and 1.8-33% catalytic efficiency (k(cat)/K-m) toward 2-chloro-4 -nitrophenyl beta -D-maltoheptaoside and amylose DP17, respectively, and 0. 5-50% activity for insoluble starch compared to that of C95A-AMY1. K-m and k(cat) were decreased 2-9- and 1.3-83-fold, respectively, for the soluble s ubstrates. The starch:oligosaccharide, and amylose:oligosaccharide specific ity ratios were 13-172 and 2.4-14 for mutants and 520 and 27 for C95A-AMY1, respectively. The FVG mutant released 4-nitrophenyl alpha -D-maltotrioside (PNPG(3)) from PNPG(5), whereas C95A-AMY1 produced PNPG and PNPG(2). The m utation thus favored interaction with the substrate aglycon part, while pro ducts from PNPG(6) reflected the fact that the mutation restored binding at subsite -6 which was lost in C95A-AMY1. The outcome of this combined irrat ional and rational protein engineering approach was evaluated considering s tructural accommodation of mutant side chains. FVG and FGG, present in the most active variants, represented novel sequences. This emphasized the wort h of random mutagenesis and launched flexibility as a goal for beta --> alp ha loop 7 engineering in family 13.