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-tRNAAla 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 Km values of 42 and 15 microm for UDP-MurNAc pentapeptide and Escherichia coli Ala-tRNAAla, respectively, and exhibited a kcat 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 pKa 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 Asp109 as a candidate for the catalytic base and Glu320 plays an additional important role in the catalytic function of the enzyme.
Hegde
Subray S
SS
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Blanchard
John S
JS
D019943:Amino Acid Substitution; D001483:Base Sequence; D017931:DNA Primers; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019884:Nitrogenous Group Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D011994:Recombinant Proteins; D019884:Nitrogenous Group Transferases; C476453:UDP-N-acetylmuramoyl pentapeptide-lysine N(6)-alanyltransferase