The catalytic properties of three class B beta-lactamases (from Pseudomonas maltophilia, Aeromonas hydrophila and Bacillus cereus) were studied and compared with those of the Bacteroides fragilis enzyme. The A. hydrophila beta-lactamase exhibited a unique specificity profile and could be considered as a rather specific 'carbapenemase'. No relationships were found between sequence similarities and catalytic properties. The problem of the repartition of class B beta-lactamases into sub-classes is discussed. Improved purification methods were devised for the P. maltophilia and A. hydrophila beta-lactamases including, for the latter enzyme, a very efficient affinity chromatography step on a Zn(2+)-chelate column.

The sequences of class A ,I-lactamases were compared. Four main groups of enzymes were distinguished: those from the gram-negative organisms and bacilli and two distinct groups of Streptomyces spp. The Staphylococcus aureus PC1 enzyme, although somewhat closer to the enzyme from the Bacillus group, did not belong to any of the groups of I-lactamases. The similarities between the secondary structure elements of these enzymes and those of the class C P-lactamases and of the Streptomyces sp. strain R61 DD-peptidase were also analyzed and tentatively extended to the class D ,I-lactamases. A unified nomenclature of secondary structure elements is proposed for all the penicillin-recognizing enzymes.In the last few years, many different 13-lactamase (ii) Class C ,B-lactamases. Class C r-lactamase sequences were those from Escherichia coli K-12 (26); Citrobacter freundii 0S60 (34); Enterobacter cloacae P99, Q908R, and MHN1 (16); Serratia marcescens SR50 (38); and Pseudomonas aeruginosa (35).(iii) Class D ,3-lactamases. The aligned sequences of class D ,3-lactamases were those from OXA-1 (41), OXA-2 (9), and PSE-2 (23) (2), but the C-and N-terminal portions were deleted so that all compared sequences extended from residues 30 to 285 (ABL consensus numbering scheme). The final score represents the number of matches divided by the length of the shorter sequence, excluding the gaps. In the

The catalytic pathway of class A ␤-lactamases involves an acyl-enzyme intermediate where the substrate is ester-linked to the Ser-70 residue. Glu-166 and Lys-73 have been proposed as candidates for the role of general base in the activation of the serine OH group. The replacement of Glu-166 by an asparagine in the TEM-1 and by a histidine in the Streptomyces albus G ␤-lactamases yielded enzymes forming stable acyl-enzymes with ␤-lactam antibiotics. Although acylation of the modified proteins by benzylpenicillin remained relatively fast, it was significantly impaired when compared to that observed with the wild-type enzyme. Moreover, the E166N substitution resulted in a spectacular modification of the substrate profile much larger than that described for other mutations of ⍀-loop residues. Molecular modeling studies indicate that the displacement of the catalytic water molecule can be related to this observation. These results confirm the crucial roles of Glu-166 and of the "catalytic" water molecule in both the acylation and the deacylation processes.DD-peptidases and most ␤-lactamases, belong to the superfamily of active-site serine penicillin-recognizing enzymes (1). The interaction between these proteins and ␤-lactams involves the formation of an acyl-enzyme (E-S * ) intermediate where the antibiotic is covalently bound to the active-site serine residue,In contrast to DD-peptidases, ␤-lactamases efficiently catalyze the deacylation step (high k 3 value) which regenerates the active enzyme and releases a biologically inactive product (P). Serine ␤-lactamases are divided into three classes A, C, and D on the basis of their primary structures. Tertiary structures of various enzymes belonging to classes A and C have been solved by x-ray crystallography underlining similarities in the folds of all these proteins (2-6). Moreover, several conserved residues were identified, some of which appear to be essential for catalysis (7). The mechanism by which serine ␤-lactamases hydrolyze penicillins and cephalosporins has received a lot of attention and, for the class A enzymes the identity of the residue involved in the activation of the active serine (Ser-70 in the ABL numbering system (8)) has been subject of controversy. Both Lys-73 (9) and Glu-166 (10) have been proposed as potential candidates for this essential role. By contrast, the function of Glu-166 in activating the hydrolytic water molecule during the deacylation step is unanimously recognized. According to Adachi et al. (11) and Strynadka et al. (9), accumulation of an acyl-enzyme during the interaction between the TEM-1 Glu-166 3 Asn mutant (E166N) and benzylpenicillin suggested that the mutation affected only the deacylation step in a severe manner. In contrast, kinetic studies of the Glu-166 3 Ala mutant of the Bacillus licheniformis ␤-lactamase (12) and of the Glu-166 3 Asp mutant of the Bacillus cereus I ␤-lactamase (13) showed that accumulation of the acyl-enzyme could result from simultaneous but different decreases of the kinetic parameters characte...

In recent years, various mass spectrometry procedures have been developed for bacterial identification. The accuracy and speed with which data can be obtained by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS) could make this a powerful tool for environmental monitoring. However, minor variations in the sample preparation can influence the mass spectra significantly. Therefore, the first objectives of this study were the adjustment and the optimization of experimental parameters allowing a rapid identification of whole bacterial cells without laborious sample preparation. The tested experimental parameters were matrix, extraction solvent, salt content, deposition method, culture medium and incubation time. This standardized protocol was applied to identify reference and environmental bacterial strains of Escherichia coli, Salmonella and Acinetobacter. The environmental bacterial strains were isolated from sewage sludge using an original microextraction procedure based on repeated sonications and enzymatic treatments. The bacterial identification was realized by the observation of the respective genus‐, species‐ and strain‐specific biomarkers. This bacterial taxonomy could be completed within one hour, with minimal sample preparation, provided that sufficient bacteria had been collected prior to MALDI‐TOF analysis. Copyright © 2004 John Wiley & Sons, Ltd.

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