Effect of a Y265F mutant on the transamination-based cycloserine inactivation of alanine racemase
Effect of a Y265F mutant on the transamination-based cycloserine inactivation of alanine racemase
复制标题
DOI：
10.1021/bi047842l
复制
发表时间：
2005-04-12
期刊：
影响因子：
2.9
通讯作者：
Ringe, D
中科院分区：
文献类型：
--
作者：
Fenn, TD;Holyoak, T;Ringe, D
细菌细胞壁肽聚糖层对D-丙氨酸的需求部分由丙氨酸消旋酶(EC 5.1.1.1)完成，丙氨酸消旋酶是一种5‘-磷酸吡哆醛(PLP)辅助的酶。该酶利用两个与PLP环垂直的位于C位的反平行碱基来促进丙氨酸对映体的平衡。因此，了解如何利用和控制这种双碱基系统来产生反应特异性是设计抗生素的潜在手段。环丝氨酸是一种已知的丙氨酸外消旋酶自杀底物，尽管其失活机制是基于转氨酶化学。在这里，我们研究了嗜热脂肪芽孢杆菌丙氨酸消旋酶Y265F突变体(在L异构体中以Tyr265为催化碱基)对环丝氨酸失活的影响。Y265F突变体将外消旋活性降低1600倍[Watanabe，A.，Yoshimura，T.，Mikami，B.和Esaki，N.(1999)J.126,781-786]，并且与使用野生型酶获得的结果相反，在D-环丝氨酸的情况下仅导致异恶唑最终产物的形成(转氨酶途径的结果)。另一方面，L-环丝氨酸在Y265缺失的情况下利用了许多替代途径，强调了Y265在失活和外消旋途径中的重要性。结合失活动力学，这些结果表明了两个催化碱基在外消旋和失活中的作用，以及Y265在“引导”化学倾向于一条途径而不是另一条途径方面的重要性。
The requirement for D-alanine in the peptidoglycan layer of bacterial cell walls is fulfilled in part by alanine racemase (EC 5.1.1.1), a pyridoxal 5'-phosphate (PLP)-assisted enzyme. The enzyme utilizes two antiparallel bases focused at the C, position and oriented perpendicular to the PLP ring to facilitate the equilibration of alanine enantiomers. Understanding how this two-base system is utilized and controlled to yield reaction specificity is therefore a potential means for designing antibiotics. Cycloserine is a known alanine racemase suicide substrate, although its mechanism of inactivation is based on transaminase chemistry. Here we characterize the effects of a Y265F mutant (Tyr265 acts as the catalytic base in the L-isomer case) of Bacillus stearothermophilus alanine racemase on cycloserine inactivation. The Y265F mutant reduces racemization activity 1600-fold [Watanabe, A., Yoshimura, T., Mikami, B., and Esaki, N. (1999) J. Biochem. 126, 781-786] and only leads to formation of the isoxazole end product (the result of the transaminase pathway) in the case Of D-cycloserine, in contrast to results obtained using the wild-type enzyme. L-Cycloserine, on the other hand, utilizes a number of alternative pathways in the absence of Y265, emphasizing the importance of Y265 in both the inactivation and racemization pathway. In combination with the kinetics of inactivation, these results suggest roles for each of the two catalytic bases in racemization and inactivation, as well as the importance of Y265 in "steering" the chemistry to favor one pathway over another.