He Ming, Nie Yan-Fang, Xu Peilin
Biotechnology Research Center, The Key Laboratory of Gene Engineering of Education Ministry, Zhongshan University, Guangzhou 510275, The People's Republic of China.
Biosci Biotechnol Biochem. 2003 Jun;67(6):1405-9. doi: 10.1271/bbb.67.1405.
Mutants of class I enolpyruvylshikimate 3-phosphate synthase (EPSPS) with resistance to glyphosate were produced in a previous study using the staggered extension process with aroA genes from S. typhimurium and E. coli. Two of these mutants shared a common amino acid substitution, T42M, near the hinge region between the large globular domains of EPSPS. Using site-directed mutagenisis, we produced the T42M mutants without the other amino acid changes of the original mutants. The T42M substitution alone produced enzymes with a 9- to 25-fold decreased K(m)[PEP] and a 21- to 26-fold increased K(i)[glyphosate] compared to the wild-type enzymes. These results provide more testimony for the powerful approach for protein engineering by the combination of directed evolution and rational design.
在之前的一项研究中，利用交错延伸过程，采用来自鼠伤寒沙门氏菌和大肠杆菌的aroA基因，产生了对草甘膦具有抗性的I类5-烯醇丙酮酸莽草酸-3-磷酸合酶（EPSPS）突变体。这些突变体中的两个在EPSPS大球状结构域之间的铰链区附近有一个共同的氨基酸取代，即T42M。通过定点诱变，我们产生了没有原始突变体其他氨基酸变化的T42M突变体。与野生型酶相比，单独的T42M取代产生的酶的K(m)[PEP]降低了9至25倍，K(i)[草甘膦]增加了21至26倍。这些结果为通过定向进化和理性设计相结合的蛋白质工程强大方法提供了更多证据。
