Site-directed mutagenesis of the mecA gene from a methicillin-resistant strain of Staphylococcus aureus.
The mecA-27r gene from Staphylococcus aureus 27r encodes penicillin-binding protein 2a (PBP2a-27r), which causes this strain to be methicillin resistant. Removal or replacement of the N-terminal transmembrane domain had no effect on binding of penicillin, but removal of portions of the putative transglycosylase domain (144, 245, or 341 amino acids after the transmembrane region) destroyed penicillin-binding activity. The SXXK, SXN, and KSG motifs, present in all penicillin-interacting enzymes, were found in the expected linear spatial arrangement within the putative transpeptidase region of PBP2a-27r. Alterations of amino acids in all three of these motifs resulted in elimination of penicillin-binding activity, confirming their roles in the interaction with penicillin.
Wu
C Y
CY
Infectious Disease Research, Eli Lilly and Company, Indianapolis, Indiana 46285.
Alborn
W E
WE
Flokowitsch
J E
JE
Hoskins
J
J
Unal
S
S
Blaszczak
L C
LC
Preston
D A
DA
Skatrud
P L
PL
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000235:genetics*; D002435:Cefamandole / Q000378:metabolism; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D006031:Glycosylation; D006602:Hexosyltransferases; D008565:Membrane Proteins / Q000235:genetics; D016106:Methicillin Resistance / Q000235:genetics*; D008969:Molecular Sequence Data; D002267:Muramoylpentapeptide Carboxypeptidase / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000378:metabolism; D010458:Peptidyl Transferases; D017434:Protein Structure, Tertiary; D013211:Staphylococcus aureus / Q000235:genetics*
D001426:Bacterial Proteins; D002352:Carrier Proteins; D008565:Membrane Proteins; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D002435:Cefamandole; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase