Structural basis of the inhibition of class A beta-lactamases and penicillin-binding proteins by 6-beta-iodopenicillanate.

6-Beta-halogenopenicillanates are powerful, irreversible inhibitors of various beta-lactamases and penicillin-binding proteins. Upon acylation of these enzymes, the inhibitors are thought to undergo a structural rearrangement associated with the departure of the iodide and formation of a dihydrothiazine ring, but, to date, no structural evidence has proven this. 6-Beta-iodopenicillanic acid (BIP) is shown here to be an active antibiotic against various bacterial strains and an effective inhibitor of the class A beta-lactamase of Bacillus subtilis BS3 (BS3) and the D,D-peptidase of Actinomadura R39 (R39). Crystals of BS3 and of R39 were soaked with a solution of BIP and their structures solved at 1.65 and 2.2 A, respectively. The beta-lactam and the thiazolidine rings of BIP are indeed found to be fused into a dihydrothiazine ring that can adopt two stable conformations at these active sites. The rearranged BIP is observed in one conformation in the BS3 active site and in two monomers of the asymmetric unit of R39, and is observed in the other conformation in the other two monomers of the asymmetric unit of R39. The BS3 structure reveals a new mode of carboxylate interaction with a class A beta-lactamase active site that should be of interest in future inhibitor design.

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

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.

Crystal structure of the sensor domain of the BlaR penicillin receptor from Bacillus licheniformis.

As in several staphylococci, the synthesis of the Bacillus licheniformis 749/I beta-lactamase is an inducible phenomenon regulated by a signal-transducing membrane protein BlaR. The C-terminal domain of this multimodular protein is an extracellular domain which specifically recognizes beta-lactam antibiotics. When it binds a beta-lactam, a signal is transmitted by the transmembrane region to the intracellular loops. In response, the hydrolytic activity of the BlaR large cytoplasmic L3 loop is induced, and a cascade of reactions is generated, leading to the transcription of the beta-lactamase gene. Here, we describe the crystal structure of the extracellular penicillin-receptor domain of BlaR (residues 346-601) at 2.5 A resolution in order to understand why this domain, whose folding is very similar to that of class D beta-lactamases, behaves as a highly sensitive penicillin-binding protein rather than a beta-lactamase. Two residues of the BlaR C-terminal domain, Thr452 and Thr542, modify the hydrophobic characteristic of the class D beta-lactamase active site. Both residues seem to be in part responsible for the lack of beta-lactamase activity of the BlaR protein due to the stability of the acyl-enzyme. Although further experimental data are needed to fully understand the transmembrane induction process, the comparison of the BlaR sensor domain structure with those of class D beta-lactamase complexes and penicillin-binding proteins provides interesting elements to hypothesize on possible signal transmission mechanisms.

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

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.

Crystal structures of the Bacillus licheniformis BS3 class A beta-lactamase and of the acyl-enzyme adduct formed with cefoxitin.

The Bacillus licheniformis BS3 beta-lactamase catalyzes the hydrolysis of the beta-lactam ring of penicillins, cephalosporins, and related compounds. The production of beta-lactamases is the most common and thoroughly studied cause of antibiotic resistance. Although they escape the hydrolytic activity of the prototypical Staphylococcus aureus beta-lactamase, many cephems are good substrates for a large number of beta-lactamases. However, the introduction of a 7alpha-methoxy substituent, as in cefoxitin, extends their antibacterial spectrum to many cephalosporin-resistant Gram-negative bacteria. The 7alpha-methoxy group selectively reduces the hydrolytic action of many beta-lactamases without having a significant effect on the affinity for the target enzymes, the membrane penicillin-binding proteins. We report here the crystallographic structures of the BS3 enzyme and its acyl-enzyme adduct with cefoxitin at 1.7 A resolution. The comparison of the two structures reveals a covalent acyl-enzyme adduct with perturbed active site geometry, involving a different conformation of the omega-loop that bears the essential catalytic Glu166 residue. This deformation is induced by the cefoxitin side chain whose position is constrained by the presence of the alpha-methoxy group. The hydrolytic water molecule is also removed from the active site by the 7beta-carbonyl of the acyl intermediate. In light of the interactions and steric hindrances in the active site of the structure of the BS3-cefoxitin acyl-enzyme adduct, the crucial role of the conserved Asn132 residue is confirmed and a better understanding of the kinetic results emerges.