The ybxI gene of Bacillus subtilis 168 encodes a class D beta-lactamase of low activity.
The ybxI gene of Bacillus subtilis 168 encodes a preprotein of 267 amino acid residues, including a putative signal peptide of 23 residues. The YbxI primary structure exhibits high similarity scores with two members of the superfamily of the serine penicillin-recognizing enzymes: the class D beta-lactamases and the hydrophilic carboxy-terminal domains of the BlaR and MecR penicillin receptors. To determine the function and the activity of this putative penicillin-recognizing enzyme, we have subcloned the ybxI gene in the pET-26b expression vector. Transformation of Escherichia coli BL21(DE3) by the recombinant plasmid pCIP51 resulted in the export of the mature YbxI in the periplasm as a water-soluble protein. The recombinant protein was purified to 95% homogeneity. YbxI interacts with several beta-lactam antibiotics and can hydrolyze some of them. YbxI is not inactivated by clavulanic acid. The YbxI function and its enzymatic activity in B. subtilis remain unknown. The acyl-enzyme obtained after incubation of YbxI with a fluorescent derivative of ampicillin can be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, confirming that YbxI can be acylated by beta-lactam antibiotics. YbxI does not hydrolyze some of the standard substrates of D-alanyl-D-alanine peptidases, the targets of penicillin. YbxI belongs to the penicillin-recognizing enzyme family but has an activity intermediate between those of a penicillin-binding protein and a beta-lactamase.
Colombo
Maria-Luigi
ML
Centre d'Ingénierie des Protéines, Institut de Chimie B6a, Université de Liège, Sart Tilman, B-4000 Liège 1, Belgium.
Hanique
Sophie
S
Baurin
Stéphane L
SL
Bauvois
Cédric
C
De Vriendt
Kris
K
Van Beeumen
Jozef J
JJ
Frère
Jean-Marie
JM
Joris
Bernard
B
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D001426:Bacterial Proteins; D001639:Bicarbonates / Q000494:pharmacology; D002352:Carrier Proteins; D002511:Cephalosporins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial / Q000235:genetics*; D006602:Hexosyltransferases; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D002267:Muramoylpentapeptide Carboxypeptidase; D046915:Penicillin-Binding Proteins; D010457:Peptidoglycan / Q000378:metabolism; D010458:Peptidyl Transferases; D010957:Plasmids / Q000235:genetics; D012329:RNA, Bacterial / Q000096:biosynthesis; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000235:genetics*
D001426:Bacterial Proteins; D001639:Bicarbonates; D002352:Carrier Proteins; D002511:Cephalosporins; D046915:Penicillin-Binding Proteins; D010457:Peptidoglycan; D012329:RNA, Bacterial; D012333:RNA, Messenger; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D001618:beta-Lactamases; C021720:nitrocefin