Graduate School of Agriculture, Hokkaido University, Sapporo, Japan.
FEBS J. 2012 Sep;279(17):3264-75. doi: 10.1111/j.1742-4658.2012.08713.x. Epub 2012 Aug 17.
Bacillus megaterium IAM 1030 (Bacillus sp. JCM 20016) possesses four d-glucose 1-dehydrogenase isozymes (BmGlcDH-I, -II, -III and -IV) that belong to the short-chain dehydrogenase/reductase superfamily. The BmGlcDHs are currently used for a clinical assay to examine blood glucose levels. Of these four isozymes, BmGlcDH-IV has relatively high thermostability and catalytic activity, but the disadvantage of its broad substrate specificity remains to be overcome. Here, we describe the crystal structures of BmGlcDH-IV in ligand-free, NADH-bound and β-D-glucose-bound forms to a resolution of 2.0 Å. No major conformational differences were found among these structures. The structure of BmGlcDH-IV in complex with β-D-glucose revealed that the carboxyl group at the C-terminus, derived from a neighboring subunit, is inserted into the active-site pocket and directly interacts with β-D-glucose. A site-directed mutagenic study showed that destabilization of the BmGlcDH-IV C-terminal region by substitution with more bulky and hydrophobic amino acid residues greatly affects the activity of the enzyme, as well as its thermostability and substrate specificity. Of the six mutants created, the G259A variant exhibited the narrowest substrate specificity, whilst retaining comparable catalytic activity and thermostability to the wild-type enzyme.
巨大芽孢杆菌 IAM 1030（芽孢杆菌 JCM 20016）拥有四种 d-葡萄糖 1-脱氢酶同工酶（BmGlcDH-I、-II、-III 和 -IV），它们属于短链脱氢酶/还原酶超家族。BmGlcDHs 目前用于临床检测血糖水平的分析。在这四种同工酶中，BmGlcDH-IV 具有相对较高的热稳定性和催化活性，但仍需要克服其广泛的底物特异性的缺点。在这里，我们描述了 BmGlcDH-IV 在无配体、NADH 结合和β-D-葡萄糖结合形式下的晶体结构，分辨率为 2.0 Å。这些结构之间没有发现明显的构象差异。BmGlcDH-IV 与β-D-葡萄糖复合物的结构表明，来自相邻亚基的 C 末端羧基基团插入活性口袋并与β-D-葡萄糖直接相互作用。定点突变研究表明，通过取代更大、更疏水的氨基酸残基使 BmGlcDH-IV C 末端区域不稳定，极大地影响了酶的活性以及其热稳定性和底物特异性。在所创建的六个突变体中，G259A 变体表现出最窄的底物特异性，同时保留与野生型酶相当的催化活性和热稳定性。
