1.8 and 1.9 Å resolution structures of the Penicillium amagasakiense and Aspergillus niger glucose oxidases as a basis for modelling substrate complexes
1.8 and 1.9 Å resolution structures of the Penicillium amagasakiense and Aspergillus niger glucose oxidases as a basis for modelling substrate complexes
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DOI：
10.1107/s0907444999003431
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发表时间：
1999-05-01
影响因子：
2.2
通讯作者：
Hecht, HJ
中科院分区：
文献类型：
--
作者：
Wohlfahrt, G;Witt, S;Hecht, HJ
葡萄糖氧化酶是一种依赖于黄素的酶，它催化分子氧将β-D-葡萄糖氧化成β-葡萄糖内酯和过氧化氢，黑曲霉的该酶先前以2.3埃分辨率精制，其结构在1.9埃分辨率下被提纯为R值19.0%，而来自甘肃青霉的该酶的结构经分子置换确定，并在1.8埃分辨率下提纯为R值16.4%。部分脱糖基酶的结构具有R.M.S。用力场方法模拟了黑曲霉酶的底物复合体，主链原子偏离0.7埃，显示四个N-糖基化位点，糖链延伸到Asn89。所得到的模型与定点突变实验的结果一致，并表明β-D-葡萄糖分子位于葡萄糖氧化酶的活性部位，由12个氢键以及与邻近的三个芳香族残基和黄素腺嘌呤二核苷酸的疏水接触稳定。其他己糖，如α-D-葡萄糖、甘露糖和半乳糖，是酶的不良底物，以及2-脱氧-D-葡萄糖，与邻近氨基酸形成的键较少或不利于接触。对还原的酶和产物--葡萄糖内酯之间的复合体的模拟，为内酯没有抑制产物提供了一个解释。
Glucose oxidase is a flavin-dependent enzyme which catalyses the oxidation of beta-D-glucose by molecular oxygen to delta-gluconolactone and hydrogen peroxide, The structure of the enzyme from Aspergillus niger, previously refined at 2.3 Angstrom resolution, has been refined at 1.9 Angstrom resolution to an R value of 19.0%, and the structure of the enzyme from Penicillium amagasnkiense, which has 65% sequence identity, has been determined by molecular replacement and refined at 1.8 Angstrom resolution to an R value of 16.4%. The structures of the partially deglycosylated enzymes have an r.m.s. deviation of 0.7 Angstrom for main-chain atoms and show four N-glycosylation sites, with an extended carbohydrate moiety at Asn89, Substrate complexes of the enzyme from A. niger were modelled by force-field methods. The resulting model is consistent with results from site-directed mutagenesis experiments and shows the beta-D-glucose molecule in the active site of glucose oxidase, stabilized by 12 hydrogen bonds and by hydrophobic contacts to three neighbouring aromatic residues and to flavin adenine dinucleotide. Other hexoses, such as alpha-D-glucose, mannose and galactose, which are poor substrates for the enzyme, and 2-deoxy-D-glucose, form either fewer bonds or unfavourable contacts with neighbouring amino acids. Simulation of the complex between the reduced enzyme and the product, delta-gluconolactone, has provided an explanation for the lack of product inhibition by the lactone.