Daniellou Richard, Zheng Hongyan, Langill David M, Sanders David A R, Palmer David R J
Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, Saskatchewan, Canada S7N 5C9.
Biochemistry. 2007 Jun 26;46(25):7469-77. doi: 10.1021/bi700281x. Epub 2007 Jun 1.
The active site of myo-inositol dehydrogenase (IDH, EC 1.1.1.18) from Bacillus subtilis recognizes a variety of mono- and disaccharides, as well as 1l-4-O-substituted inositol derivatives. It catalyzes the NAD+-dependent oxidation of the axial alcohol of these substrates with comparable kinetic constants. We have found that 4-O-p-toluenesulfonyl-myo-inositol does not act as a substrate for IDH, in contrast to structurally similar compounds such as those bearing substituted benzyl substituents in the same position. X-ray crystallographic analysis of 4-O-p-toluenesulfonyl-myo-inositol and 4-O-(2-naphthyl)methyl-myo-inositol, which is a substrate for IDH, shows a distinct difference in the preferred conformation of the aryl substituent. Conformational analysis of known substrates of IDH suggests that this conformational difference may account for the difference in reactivity of 4-O-p-toluenesulfonyl-myo-inositol in the presence of IDH. A sequence alignment of IDH with the homologous glucose-fructose oxidoreductase allowed the construction of an homology model of inositol dehydrogenase, to which NADH and 4-O-benzyl-scyllo-inosose were docked and the active site energy minimized. The active site model is consistent with all experimental results and suggests that a conserved tyrosine-glycine-tyrosine motif forms the hydrophobic pocket adjoining the site of inositol recognition. Y233F and Y235F retain activity, while Y233R and Y235R do not. A histidine-aspartate pair, H176 and D172, are proposed to act as a dyad in which H176 is the active site acid/base. The enzyme is inactivated by diethyl pyrocarbonate, and the mutants H176A and D172N show a marked loss of activity. Kinetic isotope effect experiments with D172N indicate that chemistry is rate-determining for this mutant.
来自枯草芽孢杆菌的肌醇脱氢酶（IDH，EC 1.1.1.18）的活性位点能够识别多种单糖和二糖，以及1l - 4 - O - 取代的肌醇衍生物。它以可比的动力学常数催化这些底物的轴向醇的NAD⁺依赖性氧化。我们发现，与结构相似的化合物（如在相同位置带有取代苄基取代基的化合物）相比，4 - O - 对甲苯磺酰基 - 肌醇不作为IDH的底物。对4 - O - 对甲苯磺酰基 - 肌醇和作为IDH底物的4 - O - （2 - 萘基）甲基 - 肌醇进行X射线晶体学分析，结果表明芳基取代基的优选构象存在明显差异。对IDH已知底物的构象分析表明，这种构象差异可能解释了4 - O - 对甲苯磺酰基 - 肌醇在IDH存在下反应性的差异。将IDH与同源的葡萄糖 - 果糖氧化还原酶进行序列比对，构建了肌醇脱氢酶的同源模型，将NADH和4 - O - 苄基 - 青蟹肌醇对接至该模型，并使活性位点能量最小化。活性位点模型与所有实验结果一致，表明保守的酪氨酸 - 甘氨酸 - 酪氨酸基序形成了与肌醇识别位点相邻的疏水口袋。Y233F和Y235F保留活性，而Y233R和Y235R则没有活性。一对组氨酸 - 天冬氨酸，H176和D（172），被认为是一个二元组，其中H176是活性位点的酸/碱。该酶被焦碳酸二乙酯灭活，突变体H176A和D172N表现出明显的活性丧失。对D172N进行的动力学同位素效应实验表明，化学过程是该突变体的速率决定因素。
