Benson T E, Marquardt J L, Marquardt A C, Etzkorn F A, Walsh C T
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Biochemistry. 1993 Mar 2;32(8):2024-30. doi: 10.1021/bi00059a019.
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 2H2O and 4S-[2H]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.
最近分离得到的大肠杆菌murB基因（Pucci等人，1992年）已被克隆到一个表达载体中，编码的UDP-N-乙酰烯醇丙酮酸葡萄糖胺还原酶（EC 1.1.1.158）过量表达，达到可溶性细胞蛋白的约10%。编码的38 kDa蛋白已被纯化至接近均一。发现它是一种单体，含有化学计量的结合FAD，在催化周转中可被还原。该酶利用NADPH的4-pro-S氢将UDP-N-乙酰葡萄糖胺烯醇丙酮酸的烯醇丙酮酸基团还原为UDP-N-乙酰胞壁酸中的乳酰醚。对2H2O和4S-[2H]NADPH孵育产物的NMR分析表明，来自NADPH经E.FADH2的氢化物转移到3-O-乳酰部分的β-甲基，质子从溶剂转移到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醚还原机制。
