Kinetic and mechanistic characterization of recombinant Lactobacillus viridescens FemX (UDP-N-acetylmuramoyl pentapeptide-lysine N6-alanyltransferase)

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.