Strych U, Penland R L, Jimenez M, Krause K L, Benedik M J
Department of Biology and Biochemistry, University of Houston, Houston, TX 77204-5513, USA.
FEMS Microbiol Lett. 2001 Mar 15;196(2):93-8. doi: 10.1111/j.1574-6968.2001.tb10547.x.
D-Alanine is a necessary precursor in the biosynthesis of the bacterial peptidoglycan. The naturally occurring L-alanine isomer is racemized to its D-form through the action of a class of enzymes called alanine racemases. These enzymes are ubiquitous among prokaryotes, and with very few exceptions are absent in eukaryotes, making them a logical target for the development of novel antibiotics. The alanine racemase gene from both Mycobacterium tuberculosis and M. avium was amplified by PCR and cloned in Escherichia coli. Overexpression of the proteins in the E. coli BL21 system, both as native and as His-tagged recombinant products, has been achieved. The proteins have been purified to electrophoretic homogeneity and analyzed biochemically. A D-alanine requiring double knock-out mutant of E. coli (alr, dadX) was constructed and the cloned genes were able to complement its deficiencies.
D-丙氨酸是细菌肽聚糖生物合成中的必需前体。天然存在的L-丙氨酸异构体通过一类称为丙氨酸消旋酶的酶的作用外消旋化为其D-形式。这些酶在原核生物中普遍存在，并且在真核生物中除极少数例外均不存在，这使得它们成为新型抗生素开发的合理靶点。通过PCR扩增结核分枝杆菌和鸟分枝杆菌的丙氨酸消旋酶基因，并将其克隆到大肠杆菌中。已在大肠杆菌BL21系统中实现了该蛋白作为天然产物和His标签重组产物的过表达。这些蛋白已纯化至电泳纯，并进行了生化分析。构建了一株需要D-丙氨酸的大肠杆菌双敲除突变体（alr，dadX），并且克隆的基因能够弥补其缺陷。
