Kinetic and mechanistic characterization of recombinant Lactobacillus viridescens FemX (UDP-N-acetylmuramoyl pentapeptide-lysine N6-alanyltransferase)
Kinetic and mechanistic characterization of recombinant Lactobacillus viridescens FemX (UDP-N-acetylmuramoyl pentapeptide-lysine N6-alanyltransferase)
复制标题
DOI：
10.1074/jbc.m301565200
复制
发表时间：
2003-06-20
影响因子：
4.8
通讯作者：
Blanchard, JS
中科院分区：
文献类型：
--
作者：
Hegde, SS;Blanchard, JS
FemABX家族编码将L-氨基酸掺入革兰氏阳性细胞壁肽聚糖的链间肽桥的酶，并且是使用氨酰-tRNA作为氨基酸供体的新型非核糖体肽基转移酶。我们先前报道了来自绿色乳杆菌的femX基因的鉴定和活性FemX（LvFemX）的重组表达，所述活性FemX（LvFemX）催化L-Ala从Ala-tRNA（Ala）转移到UDP-MurNAc五肽的L-赖氨酸的ε-氨基（Hegde，S.美国，和Shrader，T. E.（2001）J.Biol.Chem.276，6998-7003）。重组LvFemX对UDP-MurNAc五肽和大肠杆菌Ala-tRNA（Ala）的Km值分别为42和15 μ M，并且k（cat）值为660 min（-1）。初始速度和抑制动力学研究支持有序的顺序机制的酶，我们建议，催化收益通过三元复合物。的pH值的依赖性的活动是钟形的，这取决于两组表现出明显的pK（a）值为5.5和9.3的电离状态。酶的化学修饰和失活动力学以及底物的保护表明羧基参与了酶的催化功能。定点突变鉴定了Asp（109）作为催化碱基的候选物，并且Glu（320）在酶的催化功能中起另外的重要作用。
The FemABX family encodes enzymes that incorporate L-amino acids into the interchain peptide bridge of Gram-positive cell wall peptidoglycan and are novel nonribosomal peptidyl transferases that use aminoacyl-tRNA as the amino acid donor. We previously reported the identification of the femX gene from Lactobacillus viridescens and recombinant expression of active FemX (LvFemX) that catalyzes the transfer of L-Ala from Ala-tRNA(Ala) to the epsilon-amino group of L-lysine of UDP-MurNAc pentapeptide (Hegde, S. S., and Shrader, T. E. (2001) J. Biol. Chem. 276, 6998-7003). Recombinant LvFemX exhibits K-m values of 42 and 15 muM for UDP-MurNAc pentapeptide and Escherichia coli Ala-tRNA(Ala), respectively, and exhibited a k(cat) value of 660 min(-1). Initial velocity and inhibition kinetic studies support an ordered sequential mechanism for the enzyme, and we propose that catalysis proceeds via a ternary complex. The pH dependence of the activity was bell-shaped, depending on the ionization state of two groups exhibiting apparent pK(a) values of 5.5 and 9.3. Chemical modification of the enzyme and the kinetics of inactivation, and protection by substrate, indicated the involvement of carboxyl groups in the catalytic function of the enzyme. Site-directed mutagenesis identified Asp(109) as a candidate for the catalytic base and Glu(320) plays an additional important role in the catalytic function of the enzyme.