1ven
Crystal Structure Analysis of Y164E/maltose of Bacilus cereus Beta-amylase at pH 4.6
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OverviewOverview
The optimum pH of Bacillus cereus beta-amylase (BCB, pH 6.7) differs from, that of soybean beta-amylase (SBA, pH 5.4) due to the substitution of a, few amino acid residues near the catalytic base residue (Glu 380 in SBA, and Glu 367 in BCB). To explore the mechanism for controlling the optimum, pH of beta-amylase, five mutants of BCB (Y164E, Y164F, Y164H, Y164Q, and, Y164Q/T47M/Y164E/T328N) were constructed and characterized with respect to, enzymatic properties and X-ray structural crystal analysis. The optimum pH, of the four single mutants shifted to 4.2-4.8, approximately 2 pH units, and approximately 1 pH unit lower than those of BCB and SBA, respectively, and their k(cat) values decreased to 41-3% of that of the wild-type, enzyme. The X-ray crystal analysis of the enzyme-maltose complexes showed, that Glu 367 of the wild type is surrounded by two water molecules (W1 and, W2) that are not found in SBA. W1 is hydrogen-bonded to both side chains, of Glu 367 and Tyr 164. The mutation of Tyr 164 to Glu and Phe resulted in, the disruption of the hydrogen bond between Tyr 164 Oeta and W1 and the, introduction of two additional water molecules near position 164. In, contrast, the triple mutant of BCB with a slightly decreased pH optimum at, pH 6.0 has no water molecules (W1 and W2) around Glu 367. These results, suggested that a water-mediated hydrogen bond network (Glu 367...W1...Tyr, 164...Thr 328) is the primary requisite for the increased pH optimum of, wild-type BCB. This strategy is completely different from that of SBA, in, which a hydrogen bond network (Glu 380...Thr 340...Glu 178) reduces the, optimum pH in a hydrophobic environment.
About this StructureAbout this Structure
1VEN is a Single protein structure of sequence from Bacillus cereus with GLC and CA as ligands. Active as Beta-amylase, with EC number 3.2.1.2 Full crystallographic information is available from OCA.
ReferenceReference
Engineering of the pH optimum of Bacillus cereus beta-amylase: conversion of the pH optimum from a bacterial type to a higher-plant type., Hirata A, Adachi M, Utsumi S, Mikami B, Biochemistry. 2004 Oct 5;43(39):12523-31. PMID:15449941
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