OVEREXPRESSION, PURIFICATION, AND MECHANISTIC STUDY OF UDP-N-ACETYLENOLPYRUVYLGLUCOSAMINE REDUCTASE
OVEREXPRESSION, PURIFICATION, AND MECHANISTIC STUDY OF UDP-N-ACETYLENOLPYRUVYLGLUCOSAMINE REDUCTASE
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DOI：
10.1021/bi00059a019
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发表时间：
1993-03-02
期刊：
影响因子：
2.9
通讯作者：
WALSH, CT
中科院分区：
文献类型：
--
作者：
BENSON, TE;MARQUARDT, JL;WALSH, CT
最近分离的大肠杆菌 murB 基因（Pucci 等人，1992）已被克隆到表达载体中，编码的 UDP-N-乙酰烯醇丙酮酰葡萄糖胺还原酶（EC 1.1.1.158）过量产生约 10% 的可溶性细胞蛋白。编码的 38-kDa 蛋白质已纯化至接近均质。结果发现，它是一种单体，含有化学计量的结合 FAD，其催化转化率可降低。该酶利用 NADPH 的 4-pro-S 氢将 UDP-N-乙酰葡糖胺烯醇丙酮酸盐的烯醇丙酮基还原为 UDP-N-乙酰胞壁酸中的乳酰基醚。对 (H2O)-H-2 和 4S-[H-2]NADPH 孵育产物的 NMR 分析表明，NADPH 的氢化物通过 E.FADH2 转移至 3-O-乳基部分的 β-甲基，质子从溶剂转移至 UDP-N-乙酰胞壁酸乳酰部分的 α-碳。提出了细菌细胞壁组装中这种不寻常的烯醇醚减少的机制。
The recently isolated Escherichia coli murB gene (Pucci et al., 1992) has been cloned into an expression vector and the encoded UDP-N-acetylenolpyruvylglucosamine reductase (EC 1.1.1.158) was overproduced to about 10% of soluble cell protein. The encoded 38-kDa protein has been purified to near homogeneity. It was found,to be a monomer and to contain stoichiometric amounts of bound FAD which is reducible in catalytic turnover. The enzyme utilizes the 4-pro-S hydrogen of NADPH to reduce the enolpyruvyl group of UDP-N-acetylglucosamine enolpyruvate to the lactyl ether in UDP-N-acetylmuramic acid. NMR analysis of products from (H2O)-H-2 and 4S-[H-2]NADPH incubations establishes that a hydride from NADPH via E.FADH2 is transferred to the beta-methyl of the 3-O-lactyl moiety and a proton from solvent to the alpha-carbon of the lactyl moiety of UDP-N-acetylmuramic acid. A mechanism for this unusual enolether reduction in bacterial cell wall assembly is proposed.