Structure of the low-affinity penicillin-binding protein 5 PBP5fm in wild-type and highly penicillin-resistant strains of Enterococcus faecium.
Among its penicillin-binding proteins (PBPs), Enterococcus faecium possesses a low-affinity PBP5, PBP5fm, which is the main target involved in beta-lactam resistance. A 7.7-kb EcoRI chromosomal fragment of E. faecium D63r containing the pbp5fm gene was cloned and sequenced. Two open reading frames (ORFs) were found. A 2,037-bp ORF encoded the deduced 73.8-kDa PBP5fm, the amino acid sequences of which were, respectively, 99.8, 78.5, and 62% homologous to those of the low-affinity plasmid-encoded PBP3r of Enterococcus hirae S185r and the chromosome-encoded PBP5 of E. hirae R40 and Enterococcus faecalis 56R. A second 597-bp ORF, designated psrfm, was found 2.3 kb upstream of pbp5fm. It appeared to be 285 bp shorter than and 74% homologous with the regulatory gene psr of E. hirae ATCC 9790. Different clinical isolates of E. faecium, for which a wide range of benzylpenicillin MICs were observed, showed that the increases in MICs were related to two mechanisms. For some strains of intermediate resistance (MICs of 16 to 64 micrograms/ml), the increased level of resistance could be explained by the presence of larger quantities of PBP5fm which had an affinity for benzylpenicillin (second-order rate constant of protein acylation [k+2/K] values of 17 to 25 M(-1) s(-1)) that remained unchanged. For the two most highly resistant strains, EFM-1 (MIC, 90 micrograms/ml) and H80721 (MIC, 512 micrograms/ml), the resistance was related to different amino acid substitutions yielding very-low-affinity PBP5fm variants (k+2/K < or = 1.5 M(-1) s(-1)) which were synthesized in small quantities. More specifically, it appeared, with a three-dimensional model of the C-terminal domain of PBP5fm, that the substitutions of Met-485, located in the third position after the conserved SDN triad, by Thr in EFM-1 and by Ala in H80721 were the most likely cause of the decreasing affinity of PBP5fm observed in these strains.
Zorzi
W
W
Centre d'Ingénierie des Protéines, Université de Liège, Sart Tilman, Belgium.
Zhou
X Y
XY
Dardenne
O
O
Lamotte
J
J
Raze
D
D
Pierre
J
J
Gutmann
L
L
Coyette
J
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D002876:Chromosomes, Bacterial; D017931:DNA Primers; D016983:Enterococcus / Q000235:genetics; D016984:Enterococcus faecium / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D005798:Genes, Bacterial; D005809:Genes, Regulator; D006602:Hexosyltransferases; D006801:Humans; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D002267:Muramoylpentapeptide Carboxypeptidase / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D010403:Penicillin Resistance; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000378:metabolism; D010458:Peptidyl Transferases; D010957:Plasmids; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D001426:Bacterial Proteins; D002352:Carrier Proteins; D017931:DNA Primers; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase