Is it necessary to change the classification of ß-lactamases?

Peer reviewe

The PECACE domain: a new family of enzymes with potential peptidoglycan cleavage activity in Gram-positive bacteria

BACKGROUND: The metabolism of bacterial peptidoglycan is a dynamic process, synthases and cleavage enzymes are functionally coordinated. Lytic Transglycosylase enzymes (LT) are part of multienzyme complexes which regulate bacterial division and elongation. LTs are also involved in peptidoglycan turnover and in macromolecular transport systems. Despite their central importance, no LTs have been identified in the human pathogen Streptococcus pneumoniae. We report the identification of the first putative LT enzyme in S. pneumoniae and discuss its role in pneumococcal peptidoglycan metabolism. RESULTS: Homology searches of the pneumococcal genome allowed the identification of a new domain putatively involved in peptidoglycan cleavage (PECACE, PEptidoglycan CArbohydrate Cleavage Enzyme). This sequence has been found exclusively in Gram-positive bacteria and gene clusters containing pecace are conserved among Streptococcal species. The PECACE domain is, in some instances, found in association with other domains known to catalyze peptidoglycan hydrolysis. CONCLUSIONS: A new domain, PECACE, putatively involved in peptidoglycan hydrolysis has been identified in S. pneumoniae. The probable enzymatic activity deduced from the detailed analysis of the amino acid sequence suggests that the PECACE domain may proceed through a LT-type or goose lyzosyme-type cleavage mechanism. The PECACE function may differ largely from the other hydrolases already identified in the pneumococcus: LytA, LytB, LytC, CBPD and PcsB. The multimodular architecture of proteins containing the PECACE domain is another example of the many activities harbored by peptidoglycan hydrolases, which is probably required for the regulation of peptidoglycan metabolism. The release of new bacterial genomes sequences will probably add new members to the five groups identified so far in this work, and new groups could also emerge. Conversely, the functional characterization of the unknown domains mentioned in this work can now become easier, since bacterial peptidoglycan is proposed to be the substrate

Mechanism of acyl transfer by the class A serine β-lactamase of Streptomyces albus G

Optimization by energy minimization of stable complexes occurring along the pathway of hydrolysis of benzylpenicillin and cephalosporin C by the Streptomyces albus G beta-lactamase has highlighted a proton shuttle that may explain the catalytic mechanism of the beta-lactamases of class A. Five residues, S70, S130, N132, T235 and A237, are involved in ligand binding. The gamma-OH group of T235 and, in the case of benzylpenicillin, the gamma-OH group of S130 interact with the carboxylate group, on one side of the ligand molecule. The side-chain NH2 group of N132 and the carbonyl backbone of A237 interact with the exocyclic CONH amide bond, on the other side of the ligand. The backbone NH groups of S70 and A237 polarize the carbonyl group of the scissile beta-lactam amide bond. Four residues, S70, K73, S130 and E166, and two water molecules, W1 and W2, perform hydrolysis of the bound beta-lactam compound. E166, via W1, abstracts the proton from the gamma-OH group of S70. While losing its proton, the O-gamma atom of S70 attacks the carbonyl carbon atom of the beta-lactam ring and, concomitantly, the proton is delivered back to the adjacent nitrogen atom via W2, K73 and S130, thus achieving formation of the acyl-enzyme. Subsequently, E166 abstracts a proton from W1. While losing its proton, W1 attacks the carbonyl carbon atom of the S70 ester-linked acyl-enzyme and, concomitantly, re-entry of a water molecule W'1 replacing W1 allows E166 to deliver the proton back to the same carbonyl carbon atom, thus achieving hydrolysis of the beta-lactam compound and enzyme recovery. The model well explains the differences found in the kcat. values for hydrolysis of benzylpenicillin and cephalosporin C by the Streptomyces albus G beta-lactamase. It also explains the effects caused by site-directed mutagenesis of the Bacillus cereus beta-lactamase I [Gibson, ChristensenPeer reviewe

Correction: Primary and predicted secondary structure of the actinomadura R39 extracellular DD-peptidase, a penicillin-binding protein (PBP) related to the escherichia coli PBP4 (Biochemical Journal(1992)282(786))

peer reviewe

Caractérisation structurale et fonctionnelle de PBP1b de Streptococcus pneumoniae et son implication dans la découverte de nouveaux inhibiteurs

Streptococcus pneumoniae, pathogène majeur de la sphère oro-pharyngée, résiste fréquemment aux antibiotiques de la famille des ?-lactamines généralement administrés dans les infections associées à ce pathogène. Les cibles de ces antibiotiques sont des enzymes responsables de la biosynthèse du peptidoglycane bactérien, les Penicillin-Binding Proteins (PBP) dont la structure du site actif est modifiée dans le cas de souches résistantes aux antibiotiques. Dans un premier temps, la résolution de la structure à haute résolution par cristallographie des rayons X de PBP1b, une des trois PBP bifonctionnelles du pneumocoque, a mis en évidence un phénomène de réorganisation structurale du site actif de la molécule en fonction de son interaction avec un pseudo-substrat de la réaction. Ce résultat nous a permis de proposer que l'ouverture du site actif joue un rôle clef lors du processus de division cellulaire. Dans un deuxième temps, nous avons résolu les structures de complexes entre PBP1b et de nouvelles molécules inhibitrices, ce qui permet d'ouvrir la voie de la mise au point rationnelle de nouveaux médicaments. Pour finir, nous avons caractérisé le domaine glycosyltransférase de PBP1b ,qui représente une nouvelle cible moléculaire pour le développement de nouveaux antibactériens, notamment par diffusion de rayons X aux petits angles (SAXS). Cette approche originale nous a permis de proposer un modèle d'organisation et de repliement de ce domaine au sein de ces protéines.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Crystal and molecular structure of akagerine,a new indole alkaloid , C20H24N2O2

Akagerine is a new alkaloid first isolated by one of us (LA) from roots of Strychnos usambarensis. The crystal structure was determined by direct methods from three-dimensional diffractometer data. The crystals are tetragonal. The absolute configuration was not determined. This is the first report of a seven-membered ring with an N1-C17 bond (conventional notation for indole alkaloid). The lone-pair of electrons on N4 is cis with respect to C(3)H and C(15)H.The cohesion of the structure is the result of one hydrogen bond OH...N (2.77O Angstroem) and van der Waals interactions

Crystal Structure and Absolute Configuration of Usambarensine Hydrobromide

Usambarensine is a bisindolinic alkaloid found in the bark of Strychnos usambarensis roots. The crystal and molecular structure of usambarensine.2HBr.2H2O has been solved by direct methods thanks to a Hilger-Watts four-circle diffractometer. The crystals are monoclinic; refinement of the parameters was carried out with 2459 reflexions. The absolute configuration of the molecule, determined by X-ray anomalous scattering, confirms the 3 alpha cis configuration ( 11alpha cis, IUPAC) assigned earlier by spectral, chromatographic and biogenetic data.This configuration seems dependent on the presence of an ethylidenic chain in the corynane part of the molecule

A metallo-beta-lactamase enzyme in action: Crystal structures of the monozinc carbapenemase CphA and its complex with biapenem

One strategy developed by bacteria to resist the action of beta-lactam antibiotics is the expression of metallo-beta-lactamases. CphA from Aeromonas hydrophila is a member of a clinically important subclass of metallo-beta-lactamases that have only one zinc ion in their active site and for which no structure is available. The crystal structures of wild-type CphA and its N220G mutant show the structural features of the active site of this enzyme, which is modeled specifically for carbapenem hydrolysis. The structure of CphA after reaction with a carbapenem substrate, biapenem, reveals that the enzyme traps a reaction intermediate in the active site. These three X-ray structures have allowed us to propose how the enzyme recognizes carbapenems and suggest a mechanistic pathway for hydrolysis of the beta-lactam. This will be relevant for the design of metallo-beta-lactamase inhibitors as well as of antibiotics that escape their hydrolytic activity. (C) 2004 Elsevier Ltd. All rights reserved