The ybxI gene of Bacillus subtilis 168 encodes a class D beta-lactamase of low activity

Antimicrob Agents Chemother. 2004 Feb;48(2):484-90. doi: 10.1128/aac.48.2.484-490.2004.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacillus subtilis / drug effects
  • Bacillus subtilis / enzymology*
  • Bacillus subtilis / genetics*
  • Bacterial Proteins
  • Bicarbonates / pharmacology
  • Carrier Proteins
  • Cephalosporins / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Genes, Bacterial / genetics*
  • Hexosyltransferases
  • Hydrolysis
  • Kinetics
  • Molecular Sequence Data
  • Muramoylpentapeptide Carboxypeptidase
  • Penicillin-Binding Proteins
  • Peptidoglycan / metabolism
  • Peptidyl Transferases
  • Plasmids / genetics
  • RNA, Bacterial / biosynthesis
  • RNA, Messenger / biosynthesis
  • Reverse Transcriptase Polymerase Chain Reaction
  • beta-Lactamases / biosynthesis*
  • beta-Lactamases / genetics*

Substances

  • Bacterial Proteins
  • Bicarbonates
  • Carrier Proteins
  • Cephalosporins
  • Penicillin-Binding Proteins
  • Peptidoglycan
  • RNA, Bacterial
  • RNA, Messenger
  • Peptidyl Transferases
  • Hexosyltransferases
  • Muramoylpentapeptide Carboxypeptidase
  • beta-Lactamases
  • nitrocefin