MURPHY TA, SIMM AM, NIUMSUP P, WALSH TR; Interscience Conference on Antimicrobial Agents and Chemotherapy (43rd: 2003: Chicago, Ill.).
Abstr Intersci Conf Antimicrob Agents Chemother Intersci Conf Antimicrob Agents Chemother. 2003 Sep 14-17; 43: abstract no. C1-667.
BCARE, Bristol, United Kingdom.
BACKGROUND: C. crescentus possesses a metallo-beta-lactamase (MBL1b) with a high degree of homology (>35%) to the L1 metallo-beta-lactamase (MBL) of Stenotrophomonas maltophilia. MBL1b exhibits substrate inhibition kinetics, consistent with beta-lactam substrates binding in variable stoichiometries but still capable of enzyme-substrate complex formation and product release. Accordingly, other unusual MBLs from this genus were in biochemically analysed. METHODS: MBL1c was over expressed in E.coli and amplified using PCR and primers specific to MBL1b. MBL1c was sequenced using DuPont Automated systems and analysed using DNAstar. The MBL was purified using a periplasmic preparation followed by ion exchange chromatography and gel filtration. Purity was shown by SDS-PAGE. Kinetics and inhibition studies were determined for a range of beta-lactam antibiotics. RESULTS: PCR and sequencing showed C. crescentus 103472 contains a chromosomal gene of 710 bp which codes for a protein having 30% % identity with the L1-MBL of S. maltophilia the protein product of which is shown to be a monomeric zinc dependant metallo-b -lactamase. The primary sequence of the mature protein is very similar to the Caulobacter MBLs. MBL1a and MBL1b (from strain CB15 and 102123, respectively) with the exception of a 26 amino sequence at the N-terminus that is absent in 103472. Steady-state kinetics with a variety of beta-lactams show greatly increased K[m] values (e.g. 1200 micro-M for cefoxitin compared with 130 micro-M for strain10123). Substrate inhibition kinetics are consistent with a model in which conformations of the substrate bind reversibly but unproductively to enzyme. CONCLUSIONS: The substrate inhibition kinetics for MBL1a are significantly different from those of other known C. crecsentus enzymes. This difference is likely to be due to the 26 amino acid N-terminal region which influences the hydrolysis of beta-lactams and specifically the mechanism of inhibition with beta-lactams.
Publication Types:
Keywords:
- Amino Acids
- Base Sequence
- Caulobacter
- Caulobacter crescentus
- Cephalosporins
- Hydrolysis
- Kinetics
- Monobactams
- Stenotrophomonas maltophilia
- beta-Lactamases
- beta-Lactams
- beta-lactamase L1
- genetics
- pharmacokinetics
Other ID:
UI: 102264736
From Meeting Abstracts