RESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Peptídeo Sintases/metabolismo , Peptidoglicano/biossíntese , Processamento de Proteína Pós-Traducional/fisiologia , Aminoacil-RNA de Transferência/metabolismo , Streptococcus pneumoniae/enzimologia , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Catálise , Dipeptídeos/biossíntese , Dipeptídeos/química , Dipeptídeos/genética , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Cinética , Penicilinas/química , Penicilinas/farmacologia , Peptídeo Sintases/química , Peptídeo Sintases/genética , Peptidoglicano/química , Peptidoglicano/genética , Infecções Pneumocócicas/tratamento farmacológico , Infecções Pneumocócicas/enzimologia , Infecções Pneumocócicas/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estrutura Secundária de Proteína/genética , Aminoacil-RNA de Transferência/química , Aminoacil-RNA de Transferência/genética , Homologia de Sequência de Aminoácidos , Streptococcus pneumoniae/genética , Especificidade por Substrato/genética , Uridina Difosfato Ácido N-Acetilmurâmico/genética , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismoRESUMO
An ORF designated sp1530 (murE) in the Streptococcus pneumoniae TIGR4 genome sequence, identified as uridine 5'-diphospho-N-acetylmuramoyl-L-alanyl-D-glutamate:L-lysine ligase (MurE; EC 6.3.2.7), was cloned into the high-expression plasmid pET21b and overexpressed in Escherichia coli BL21 (DE3) Star. The enzyme was purified in three steps to 99% purity. Crystals were obtained by the hanging-drop vapour-diffusion method at 291 K from solutions containing 25%(w/v) polyethylene glycol 2000 monomethylether, 0.2 M potassium thiocyanate, 0.1 M MES pH 6.5 in the presence of uridine 5'-diphospho-N-acetylmuramoyl alanyl glutamate (UDP-MurNAc-L-Ala-D-Glu) with and without 5'-adenylyl imidophosphate (AMP-PNP), a non-hydrolysable analogue of ATP. Diffraction data to 1.5 and 2.7 A, respectively, were collected at the European Synchrotron Radiation Facility (ESRF). Crystals grown in the presence of two ligands belong to space group P1, with unit-cell parameters a = 68.4, b = 71.4, c = 74.8 A, alpha = 73.4, beta = 80.5, gamma = 72.3 degrees. Crystals grown in the presence of UDP-MurNAc-L-Ala-D-Glu alone belong to space group P2(1), with unit-cell parameters a = 71.1, b = 129.4, c = 74.6 A, beta = 106.3 degrees.