Though the three-dimensional structures of barley alpha-amylase isozymes AMY1 and AMY2 are very similar, they differ remarkably from each other in their affinity for Ca(2+) and when interacting with substrate analogs. A surface site recognizing maltooligosaccharides, not earlier reported for other alpha-amylases and probably associated with the different activity of AMY1 and AMY2 toward starch granules, has been identified. It is located in the C-terminal part of the enzyme and, thus, highlights a potential role of domain C. In order to scrutinize the possible biological significance of this domain in alpha-amylases, a thorough comparison of their three-dimensional structures was conducted. An additional role for an earlier-identified starch granule binding surface site is proposed, and a new calcium ion is reported.

Enzymatic properties of barley a-amylase 1 (AMY1) are altered as a result of amino acid substitutions at subsites 25/26 (Cys95 ! Ala/Thr) and þ1/þ 2 (Met298 ! Ala/ Asn/Ser) as well as in the double mutants, Cys95 ! Ala/ Met298 ! Ala/Asn/Ser. Cys95 ! Ala shows 176% activity towards insoluble Blue Starch compared to wild-type AMY1, k cat of 142 and 211% towards amylose DP17 and 2-chloro-4-nitrophenyl b-D-maltoheptaoside (Cl-PNPG 7 ), respectively, but fivefold to 20-fold higher K m . The Cys95 ! Thr-AMY1 AMY2 isozyme mimic exhibits the intermediary behaviour of Cys95 ! Ala and wild-type. Met298 ! Ala/Asn/Ser have slightly higher to slightly lower activity for starch and amylose, whereas k cat and k cat /K m for Cl-PNPG 7 are # 30% and # 10% of wild-type, respectively. The activity of Cys95 ! Ala/Met298 ! Ala/ Asn/Ser is 100-180% towards starch, and the k cat /K m is 15-30%, and 0.4-1.1% towards amylose and Cl-PNPG 7 , respectively, emphasizing the strong impact of the Cys95 ! Ala mutation on activity. The mutants therefore prefer the longer substrates and the specificity ratios of starch/Cl-PNPG 7 and amylose/Cl-PNPG 7 are 2.8-to 270-fold and 1.2-to 60-fold larger, respectively, than of wild-type. Bond cleavage analyses show that Cys95 and Met298 mutations weaken malto-oligosaccharide binding near subsites 25 and þ2, respectively. In the crystal structure Met298 CE and SD (i.e., the side chain methyl group and sulfur atom) are near C(6) and O(6) of the rings of the inhibitor acarbose at subsites þ1 and þ2, respectively, and Met298 mutants prefer amylose for glycogen, which is hydrolysed with a slightly lower activity than by wild-type. Met298 AMY1 mutants and wild-type release glucose from the nonreducing end of the main-chain of 6 000 -maltotriosyl-maltohexaose thus covering subsites 2 1 to þ5, while productive binding of unbranched substrate involves subsites 2 3 to þ3.Keywords: glycoside hydrolase family 13; (b/a) 8 barrel; site-directed mutagenesis; substrate specificity; branched malto-oligosaccharide.a-Amylase (a-1,4-D-glucan glucanohydrolase, EC 3.2.1.1) catalyzes hydrolysis of internal a-1,4-glucosidic linkages in starch and related saccharides [1]. a-Amylases belong to glycoside hydrolase family 13, which includes enzymes exhibiting 26 different specificities, e.g. a-glucosidase, cyclomaltodextrinase, pullulanase, cyclodextrin glucanotransferase, branching enzyme, and amylosucrase and showing very low sequence similarity [2,3]. In spite of a considerable amount of structural information available, there is a strong need to understand the impact of enzyme -substrate interactions at the level of specific binding subsites for the ultimate exploitation in rational design of enzymes with desired properties. The specificity of a family 13 member has been engineered into another enzyme class in only a few cases [4].Three-dimensional structures have been solved for several family members and have been shown to share a catalytic (b/a) 8 barrel (domain A), a domain that protrudes between the third b strand and a he...

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The relative specificity and bond cleavage pattern of barley alpha-amylase 1 (AMY1) were dramatically changed by mutation in F(286)VD that connected beta-strand 7 of the catalytic (beta/alpha)(8)-barrel to a succeeding 3(10)-helix. This conserved tripeptide of the otherwise variable beta --> alpha segment 7 lacked direct ligand contact, but the nearby residues His290 and Asp291 participated in transition-state stabilization and catalysis. On the basis of sequences of glycoside hydrolase family 13, a biased random mutagenesis protocol was designed which encoded 174 putative F(286)VD variants of C95A-AMY1, chosen as the parent enzyme to avoid inactivating glutathionylation by the yeast host. The FVG, FGG, YVD, LLD, and FLE mutants showed 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 substrates. The starch:oligosaccharide and amylose:oligosaccharide specificity 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 mutation thus favored interaction with the substrate aglycon part, while products 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 irrational and rational protein engineering approach was evaluated considering structural accommodation of mutant side chains. FVG and FGG, present in the most active variants, represented novel sequences. This emphasized the worth of random mutagenesis and launched flexibility as a goal for beta --> alpha loop 7 engineering in family 13.