title: Characterization of tRNA-dependent peptide bond formation by MurM in the synthesis of Streptococcus pneumoniae peptidoglycan.
abstract: MurM is an aminoacyl ligase that adds l-serine or l-alanine as the first amino acid of a dipeptide branch to the stem peptide lysine of the pneumococcal peptidoglycan. MurM activity is essential for clinical pneumococcal penicillin resistance. Analysis of peptidoglycan from the highly penicillin-resistant Streptococcus pneumoniae strain 159 revealed that in vivo and in vitro, in the presence of the appropriate acyl-tRNA, MurM(159) alanylated the peptidoglycan epsilon-amino group of the stem peptide lysine in preference to its serylation. However, in contrast, identical analyses of the penicillin-susceptible strain Pn16 revealed that MurM(Pn16) activity supported serylation more than alanylation both in vivo and in vitro. Interestingly, both MurM(Pn16) acylation activities were far lower than the alanylation activity of MurM(159). The resulting differing stem peptide structures of 159 and Pn16 were caused by the profoundly greater catalytic efficiency of MurM(159) compared with MurM(Pn16) bought about by sequence variation between these enzymes and, to a lesser extent, differences in the in vivo tRNA(Ala):tRNA(Ser) ratio in 159 and Pn16. Kinetic analysis revealed that MurM(159) acted during the lipid-linked stages of peptidoglycan synthesis, that the d-alanyl-d-alanine of the stem peptide and the lipid II N-acetylglucosaminyl group were not essential for substrate recognition, that epsilon-carboxylation of the lysine of the stem peptide was not tolerated, and that lipid II-alanine was a substrate, suggesting an evolutionary link to staphylococcal homologues of MurM such as FemA. Kinetic analysis also revealed that MurM recognized the acceptor stem and/or the TPsiC loop stem of the tRNA(Ala). It is anticipated that definition of the minimal structural features of MurM substrates will allow development of novel resistance inhibitors that will restore the efficacy of beta-lactams for treatment of pneumococcal infection.
authors: Departments of Biological Sciences and Chemistry, University of Warwick, Gibbet Hill Road, Coventry, United Kingdom. adrian.lloyd@warwick.ac.uk
authors: Adrian J
authors: AJ
authors: Lloyd
authors: Andrea M
authors: AM
authors: Gilbey
authors: Anne M
authors: AM
authors: Blewett
authors: Gianfranco
authors: G
authors: De Pascale
authors: Ahmed
authors: A
authors: El Zoeiby
authors: Roger C
authors: RC
authors: Levesque
authors: Anita C
authors: AC
authors: Catherwood
authors: Alexander
authors: A
authors: Tomasz
authors: Timothy D H
authors: TD
authors: Bugg
authors: David I
authors: DI
authors: Roper
authors: Christopher G
authors: CG
authors: Dowson
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D004151:Dipeptides / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000187:drug effects / Q000235:genetics; D007700:Kinetics; D010406:Penicillins / Q000737:chemistry / Q000494:pharmacology; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010457:Peptidoglycan / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011008:Pneumococcal Infections / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics; D011499:Protein Processing, Post-Translational / Q000187:drug effects / Q000502:physiology*; D017433:Protein Structure, Secondary / Q000235:genetics; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013296:Streptococcus pneumoniae / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D014538:Uridine Diphosphate N-Acetylmuramic Acid / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004151:Dipeptides; C096686:FemA protein, Bacteria; D010406:Penicillins; D010457:Peptidoglycan; D012346:RNA, Transfer, Amino Acyl; D014538:Uridine Diphosphate N-Acetylmuramic Acid; C069249:muramyl-NAc-(pentapeptide)pyrophosphoryl-undecaprenol; C408066:MurM protein, Streptococcus pneumoniae; D010453:Peptide Synthases