Role of glutamate 243 in the active site of 2-deoxy-scyllo-inosose synthase from Bacillus circulans
Role of glutamate 243 in the active site of 2-deoxy-scyllo-inosose synthase from Bacillus circulans
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DOI：
10.1016/j.bmc.2006.09.042
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发表时间：
2007-01-01
影响因子：
3.5
通讯作者：
Eguchi, Tadashi
中科院分区：
文献类型：
--
作者：
Hirayama, Toshifumi;Kudo, Fumitaka;Eguchi, Tadashi
2-脱氧鲨肌醇（DOI）合成酶参与含2-脱氧链霉胺的氨基糖苷类抗生素的生物合成，并催化D-葡萄糖-6-磷酸（G-6-P）形成DOI。反应机理被认为与莽草酸途径中脱氢奎尼酸（DHQ）合酶类似，包括C-4的氧化、磷酸的β-消除、C-4的还原、开环和分子内羟醛环化。为了研究DOI合酶的反应机制，对来自丁酰菌肽生产菌环状芽孢杆菌（BtrC）的DOI合酶的三个推测的催化重要氨基酸进行了定点突变分析。稳态和预稳态动力学分析表明BtrC的E243催化参与磷酸盐消除步骤。使用底物类似物葡萄糖-6-膦酸盐对BtrC的突变体E243 Q的进一步分析清楚地证实了E243负责在G-6-P中的C-5处提取质子并引发磷酸盐消除。该谷氨酸残基在迄今为止鉴定的所有DOI脱氢酶中是完全保守的，并且DHQ合酶的相应氨基酸作为天冬酰胺是完全保守的。因此，DOI合酶的这一特征性谷氨酸残基是区分DOI合酶和DHQ合酶反应机制以及一级序列的关键决定因素。(c)2006爱思唯尔有限公司保留所有权利。
2-Deoxy-scyllo-inosose (DOI) synthase is involved in the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics and catalyzes the carbocyclic formation from D-glucose-6-phosphate (G-6-P) into DOI. The reaction mechanism is proposed to be similar to that of dehydroquinate (DHQ) synthase in the shikimate pathway, and includes oxidation of C-4, beta-elimination of phosphate, reduction of C-4, ring opening, and intramolecular aldol cyclization. To investigate the reaction mechanism of DOI synthase, site-directed mutational analysis of three presumable catalytically important amino acids of DOI synthase derived from the butirosin producer Bacillus circulans (BtrC) was carried out. Steady state and pre-steady state kinetic analysis suggested that E243 of BtrC is catalytically involved in the phosphate elimination step. Further analysis of the mutant E243Q of BtrC using substrate analogue, glucose-6-phosphonate, clearly confirmed that E243 was responsible to abstract a proton at C-5 in G-6-P and set off phosphate elimination. This glutamate residue is completely conserved in all DOI synthases identified so far and the corresponding amino acid of DHQ synthase is completely conserved as asparagine. Therefore, this characteristic glutamate residue of DOI synthase is a key determinant to distinguish the reaction mechanism between DOI synthase and DHQ synthase as well as primary sequence. (c) 2006 Elsevier Ltd. All rights reserved.