Crystal structure of the Mycobacterium fortuitum class A beta-lactamase: structural basis for broad substrate specificity.

in Antimicrobial Agents and Chemotherapy (2006), 50(7), 2516-21

beta-Lactamases are the main cause of bacterial resistance to penicillins and cephalosporins. Class A beta-lactamases, the largest group of beta-lactamases, have been found in many bacterial strains ... [more ▼]

beta-Lactamases are the main cause of bacterial resistance to penicillins and cephalosporins. Class A beta-lactamases, the largest group of beta-lactamases, have been found in many bacterial strains, including mycobacteria, for which no beta-lactamase structure has been previously reported. The crystal structure of the class A beta-lactamase from Mycobacterium fortuitum (MFO) has been solved at 2.13-A resolution. The enzyme is a chromosomally encoded broad-spectrum beta-lactamase with low specific activity on cefotaxime. Specific features of the active site of the class A beta-lactamase from M. fortuitum are consistent with its specificity profile. Arg278 and Ser237 favor cephalosporinase activity and could explain its broad substrate activity. The MFO active site presents similarities with the CTX-M type extended-spectrum beta-lactamases but lacks a specific feature of these enzymes, the VNYN motif (residues 103 to 106), which confers on CTX-M-type extended-spectrum beta-lactamases a more efficient cefotaximase activity. [less ▲]

Catalytic mechanism of the Streptomyces K15 DD-transpeptidase/penicillin-binding protein probed by site-directed mutagenesis and structural analysis

in Biochemistry (2003), 42(10), 2895-2906

The Streptomyces K15 penicillin-binding DD-transpeptidase is presumed to be involved in peptide cross-linking during bacterial cell wall peptidoglycan assembly. To gain insight into the catalytic ... [more ▼]

The Streptomyces K15 penicillin-binding DD-transpeptidase is presumed to be involved in peptide cross-linking during bacterial cell wall peptidoglycan assembly. To gain insight into the catalytic mechanism, the roles of residues Lys38, Ser96, and Cys98, belonging to the structural elements defining the active site cleft, have been investigated by site-directed mutagenesis, biochemical studies, and X-ray diffraction analysis. The Lys38His and Ser96Ala mutations almost completely abolished the penicillin binding and severely impaired the transpeptidase activities while the geometry of the active site was essentially the same as in the wild-type enzyme. It is proposed that Lys38 acts as the catalytic base that abstracts a proton from the active serine Ser35 during nucleophilic attack and that Ser96 is a key intermediate in the proton transfer from the Ogamma of Ser35 to the substrate leaving group nitrogen. The role of these two residues should be conserved among penicillin-binding proteins containing the Ser-Xaa-Asn/Cys sequence in motif 2. Conversion of Cys98 into Asn decreased the transpeptidase activity and increased hydrolysis of the thiolester substrate and the acylation rate with most beta-lactam antibiotics. Cys98 is proposed to play the same role as Asn in motif 2 of other penicilloyl serine transferases in properly positioning the substrate for the catalytic process. [less ▲]

Unexpected Influence of a C-Terminal-Fused His-Tag on the Processing of an Enzyme and on the Kinetic and Folding Parameters

in FEBS Letters (1997), 413(2), 194-6

The addition of a poly-His C-terminal extension, designed to facilitate the purification of the protein, to the beta-lactamase of a thermophilic Bacillus licheniformis strain modified the site of action ... [more ▼]

The addition of a poly-His C-terminal extension, designed to facilitate the purification of the protein, to the beta-lactamase of a thermophilic Bacillus licheniformis strain modified the site of action of the signal peptidase. This resulted in the secretion of a protein with a different N-terminus, showing that this type of protein engineering might not always be as 'neutral' as generally assumed. [less ▲]

Penicillin and Beyond: Evolution, Protein Fold, Multimodular Polypeptides, and Multiprotein Complexes

in Microbial Drug Resistance : Mechanism, Epidemiology, & Disease (1996), 2(2, Summer), 163-175

As the protein sequence and structure databases expand, the relationships between proteins, the notion of protein superfamily, and the driving forces of evolution are better understood. Key steps of the ... [more ▼]

As the protein sequence and structure databases expand, the relationships between proteins, the notion of protein superfamily, and the driving forces of evolution are better understood. Key steps of the synthesis of the bacterial cell wall peptidoglycan are revisited in light of these advances. The reactions through which the D-alanyl-D-alanine depeptide is formed, utilized, and hydrolyzed and the sites of action of the glycopeptide and beta-lactam antibiotics illustrate the concept according to which new enzyme functions evolve as a result of tinkering of existing proteins. This occurs by the acquisition of local structural changes, the fusion into multimodular polypeptides, and the association into multiprotein complexes. [less ▲]