Oh Bora, Kim Myungsook, Yoon Jongchul, Chung Kyungwha, Shin Yongchul, Lee Dongsoon, Kim Youngsoo
Division of Molecular Genomic Medicine, College of Medicine, Seoul National University, Yongon-Dong, Seoul 110-799, Republic of Korea.
Biochem Biophys Res Commun. 2003 Oct 10;310(1):19-27. doi: 10.1016/j.bbrc.2003.08.110.
Semisynthetic cephalosporins are primarily synthesized from 7-aminocephalosporanic acid (7-ACA), mainly by environmentally toxic chemical deacylation of cephalosporin C (CPC). Thus, the enzymatic conversion of CPC to 7-ACA by cephalosporin acylase (CA) would be very interesting. However, CAs use glutaryl-7-ACA (GL-7-ACA) as a primary substrate and the enzymes have low turnover rates for CPC. The active-site residues of a CA were mutagenized to various residues to increase the deacylation activity of CPC, based on the active-site conformation of the CA structure. The aim was to generate sterically favored conformation of the active-site to accommodate the D-alpha-aminoadipyl moiety of CPC, the side-chain moiety that corresponds to the glutaryl moiety of GL-7-ACA. A triple mutant of the CA, Q50betaM/Y149alphaK/F177betaG, showed the greatest improvement of deacylation activity to CPC up to 790% of the wild-type. Our current study is an efficient method for improving the deacylation activity to CPC by employing the structure-based repetitive saturation mutagenesis.
半合成头孢菌素主要由7-氨基头孢烷酸（7-ACA）合成，主要通过头孢菌素C（CPC）的环境毒性化学脱酰化反应。因此，利用头孢菌素酰化酶（CA）将CPC酶促转化为7-ACA将非常有意义。然而，CA以戊二酰-7-ACA（GL-7-ACA）作为主要底物，且这些酶对CPC的周转率较低。基于CA结构的活性位点构象，将CA的活性位点残基突变为各种残基，以提高CPC的脱酰化活性。目的是产生空间上有利的活性位点构象，以容纳CPC的D-α-氨基己二酸部分，即与GL-7-ACA的戊二酰部分相对应的侧链部分。CA的一个三重突变体Q50βM/Y149αK/F177βG对CPC的脱酰化活性提高最为显著，达到野生型的790%。我们目前的研究是一种通过基于结构的重复饱和诱变提高对CPC脱酰化活性的有效方法。
