Carine Bebrone

Update of the Standard Numbering Scheme for Class B β-Lactamases

β-Lactamases represent the major cause of bacterial resistance against β-lactam antibiotics, and they have been divided into four classes (A to D) on the basis of their amino acid sequences (21). The class B enzymes have no sequence or structural similarity to the active-site serine enzymes of classes A, C, and D (6); require a bivalent metal ion (Zn2+) for activity; and constitute group 3 in the Bush-Jacoby-Medeiros functional classification (2). The identification of Zn-β-lactamase-producing pathogenic strains of Aeromonas, Bacteroides, Flavobacterium, Legionella, Serratia, and Stenotrophomonas has greatly increased interest in this class of enzymes (2). The fact that they hydrolyze almost all β-lactam antibiotics, including carbapenems, underlines their clinical relevance. In consequence, the potential spreading of these enzymes among pathogenic bacteria is a frightening possibility, which emphasizes the importance of understanding their properties. On the basis of the sequences, three subclasses of class B β-lactamases (B1 to B3) were identified, and a standard numbering scheme (BBL numbering) was proposed (13) by analogy to the ABL numbering scheme which has been widely used for class A β-lactamases. Due to the general low degree of identity between subclass sequences (<20%), classical alignment programs produce unreliable results. The proposed alignment (13) was facilitated by the availability of X-ray structures for B1 and B3 enzymes. Crystallographic structures have been described for several B1 enzymes: Bacillus cereus BcII (4, 11), Bacteroides fragilis CcrA (5, 8), Pseudomonas aeruginosa IMP-1 (7) and VIM-2 (unpublished data), and Chryseobacterium meningosepticum BlaB (14). Structural data are also available for two B3 enzymes: Stenotroptromonas maltophilia L1 (28) and Legionella gormanii FEZ-1 (15). Recently, we solved the first X-ray structure of a subclass B2 enzyme (CphA) produced by various species of Aeromonas (G. Garau, C. Bebrone, C. Anne, M. Galleni, J.-M. Frere, and O. Dideberg, unpublished data). Using all available three-dimensional structures, it is now possible to propose a bonafide structural alignment of the class B β-lactamases, and accordingly, to update the first proposed BBL scheme (Fig. ​(Fig.11). FIG. 1. Structural alignment of eight class B β-lactamases with known X-ray structures. The sequences are referred to by their familiar names. BCII, B. cereus 569H (16); IMP-1, P. aeruginosa 101/477 (17); CcrA, B. fragilis TAL3636 (25); VIM-2, P. aeruginosa ... For the three-dimensional structure comparison of the eight available structures, we used the program TOP (18) with the new option MAPS, allowing multiple alignments of protein structures. In addition, the program produces two ranking scores: the sequence identities of aligned residues and the structural diversity. The structural-diversity score was defined as RMS/(Nmatch/N0)3/2, where RMS is the root mean square deviation of the distances between matched Cα atoms, Nmatch is the number of matching residues, and (Nmatch/N0) is the matching fraction of two compared structures. N0 = (N1 + N2/2), where N1 and N2 are the numbers of amino acids in the two compared proteins. This score estimates the evolutionary distance between proteins. These two scores are shown in Table ​Table11 for all known X-ray structures. TABLE 1. Sequence identities of aligned residues and structure diversity among proteins Figure ​Figure11 displays the proposed alignment and numbering. Interestingly, the numbering of the important class B residues is conserved between old and new alignments. Improvements in the alignment concern mainly N and C termini and small shifts along the sequences. The main result of the new alignment is the identification of 14 sequence fragments of structurally conserved positions, which cover the entirety of all sequences (Fig. ​(Fig.1);1); they belong mainly to secondary-structure elements (α helices or β sheets). Notably, all Zn ligands are structurally aligned. The following comments can be made. (i) Only sequences of proteins of known structures are shown. (ii) For residues in lightface, the fact that they have the same number does not imply that they are structurally equivalent. (iii) For newly discovered enzymes, any insertion departing from the present numbering can be characterized by lowercase letters following the number of the last residue of the consensus sequence. Table ​Table22 shows the numbering of the putative zinc ligands. Not all proteins of known sequence are shown. Only enzymes with <50% sequence identity compared to the first reported sequence are included in the table. TABLE 2. Numbering of important class B residues In 1997, Neuwald et al. (23) detected a few proteins that have sequence similarities to (and may have given rise to) Zn-β-lactamases. They include enzymes with large variations in function (sulfatase; DNA cross-link repair enzyme) and which are encoded by yeast, plant, or bacterial open reading frames. Human glyoxalase II was also shown to belong to the superfamily. More recently, 17 groups with known functions were identified (9). In order to evaluate the structural diversity of the Zn-β-lactamase superfamily, human glyoxalase II (3) and rubredoxin oxygen-oxidoreductase from Desulfovibrio gigas (12) were also aligned using TOP, along with one member of each subclass. Table ​Table33 shows the sequence identities and structural diversity of the two proteins and BCII, CphA, and FEZ-1. As expected, low sequence identity corresponds to a high structural-diversity score. The structural-diversity scores for proteins belonging to a superfamily range from 1.4 to 2, in contrast to 3.5 to 4 for proteins with different folds (18). Interestingly and surprisingly, FEZ-1 is closer to glyoxalase II and rubredoxin oxygen-oxidoreductase than to BcII or CphA. TABLE 3. Sequence identities of aligned residues and structural diversity among proteins In the structural alignment, a large number of amino acid changes and insertions-deletions are observed. One hypothesis is that an ancient protein gave rise to the different subclasses of Zn-β-lactamases. A few candidates for the ancient protein are those related to essential biological functions within the cell, such as DNA or RNA processing or DNA repair (9). Nature used a limited number of scaffolds to generate a large variety of biological functions. Zn-β-lactamases are good examples of such a selection.

Current Challenges in Antimicrobial Chemotherapy

The use of the three classical β-lactamase inhibitors (clavulanic acid, tazobactam and sulbactam) in combination with β-lactam antibacterials is currently the most successful strategy to combat β-lactamase-mediated resistance. However, these inhibitors are efficient in inactivating only class A β-lactamases and the efficiency of the inhibitor/antibacterial combination can be compromised by several mechanisms, such as the production of naturally resistant class B or class D enzymes, the hyperproduction of AmpC or even the production of evolved inhibitor-resistant class A enzymes. Thus, there is an urgent need for the development of novel inhibitors. For serine active enzymes (classes A, C and D), derivatives of the β-lactam ring such as 6-β-halogenopenicillanates, β-lactam sulfones, penems and oxapenems, monobactams or trinems seem to be potential starting points to design efficient molecules (such as AM-112 and LK-157). Moreover, a promising non-β-lactam molecule, NXL-104, is now under clinical development. In contrast, an ideal inhibitor of metallo-β-lactamases (class B) remains to be found, despite the huge number of potential molecules already described (biphenyl tetrazoles, cysteinyl peptides, mercaptocarboxylates, succinic acid derivatives, etc.). The search for such an inhibitor is complicated by the absence of a covalent intermediate in their catalytic mechanisms and the fact that β-lactam derivatives often behave as substrates rather than as inhibitors. Currently, the most promising broad-spectrum inhibitors of class B enzymes are molecules presenting chelating groups (thiols, carboxylates, etc.) combined with an aromatic group.This review describes all the types of molecules already tested as potential β-lactamase inhibitors and thus constitutes an update of the current status in β-lactamase inhibitor discovery.

CENTA as a Chromogenic Substrate for Studying β-Lactamases

ABSTRACT CENTA, a chromogenic cephalosporin, is readily hydrolyzed by β-lactamases of all classes except for the Aeromonas hydrophila metalloenzyme. Although it cannot practically be used for the detection of β-lactamase-producing strains on agar plates, it should be quite useful for kinetic studies and the detection of the enzymes in crude extracts and chromatographic fractions.

Mercaptophosphonate Compounds as Broad-Spectrum Inhibitors of the Metallo-β-lactamases

Although commercialized inhibitors of active site serine beta-lactamases are currently used in coadministration with antibiotic therapy, no clinically useful inhibitors of metallo-beta-lactamases (MBLs) have yet been discovered. In this paper, we investigated the inhibitory effect of mercaptophosphonate derivatives against the three subclasses of MBLs (B1, B2, and B3). All 14 tested mercaptophosphonates, with the exception of 1a, behaved as competitive inhibitors for the three subclasses. Apart from 13 and 21, all the mercaptophosphonates tested exhibit a good inhibitory effect on the subclass B2 MBL CphA with low inhibition constants (K(i) < 15 muM). Interestingly, compound 18 turned out to be a potent broad spectrum MBL inhibitor. The crystallographic structures of the CphA-10a and CphA-18 complexes indicated that the sulfur atom of 10a and the phosphonato group of 18 interact with the Zn(2+) ion, respectively. Molecular modeling studies of the interactions between compounds 10a and 18 and the VIM-4 (B1), CphA (B2), and FEZ-1 (B3) enzymes brought to light different binding modes depending on the enzyme and the inhibitor, consistent with the crystallographic structures.

The Structure of the Dizinc Subclass B2 Metallo-β-Lactamase CphA Reveals that the Second Inhibitory Zinc Ion Binds in the Histidine Site

ABSTRACT Bacteria can defend themselves against β-lactam antibiotics through the expression of class B β-lactamases, which cleave the β-lactam amide bond and render the molecule harmless. There are three subclasses of class B β-lactamases (B1, B2, and B3), all of which require Zn2+ for activity and can bind either one or two zinc ions. Whereas the B1 and B3 metallo-β-lactamases are most active as dizinc enzymes, subclass B2 enzymes, such as Aeromonas hydrophila CphA, are inhibited by the binding of a second zinc ion. We crystallized A. hydrophila CphA in order to determine the binding site of the inhibitory zinc ion. X-ray data from zinc-saturated crystals allowed us to solve the crystal structures of the dizinc forms of the wild-type enzyme and N220G mutant. The first zinc ion binds in the cysteine site, as previously determined for the monozinc form of the enzyme. The second zinc ion occupies a slightly modified histidine site, where the conserved His118 and His196 residues act as metal ligands. This atypical coordination sphere probably explains the rather high dissociation constant for the second zinc ion compared to those observed with enzymes of subclasses B1 and B3. Inhibition by the second zinc ion results from immobilization of the catalytically important His118 and His196 residues, as well as the folding of the Gly232-Asn233 loop into a position that covers the active site.

Biochemical and Structural Characterization of the Subclass B1 Metallo-β-Lactamase VIM-4

ABSTRACT The metallo-β-lactamase VIM-4, mainly found in Pseudomonas aeruginosa or Acinetobacter baumannii, was produced in Escherichia coli and characterized by biochemical and X-ray techniques. A detailed kinetic study performed in the presence of Zn2+ at concentrations ranging from 0.4 to 100 μM showed that VIM-4 exhibits a kinetic profile similar to the profiles of VIM-2 and VIM-1. However, VIM-4 is more active than VIM-1 against benzylpenicillin, cephalothin, nitrocefin, and imipenem and is less active than VIM-2 against ampicillin and meropenem. The crystal structure of the dizinc form of VIM-4 was solved at 1.9 Å. The sole difference between VIM-4 and VIM-1 is found at residue 228, which is Ser in VIM-1 and Arg in VIM-4. This substitution has a major impact on the VIM-4 catalytic efficiency compared to that of VIM-1. In contrast, the differences between VIM-2 and VIM-4 seem to be due to a different position of the flapping loop and two substitutions in loop 2. Study of the thermal stability and the activity of the holo- and apo-VIM-4 enzymes revealed that Zn2+ ions have a pronounced stabilizing effect on the enzyme and are necessary for preserving the structure.

Mutational analysis of the two zinc-binding sites of the Bacillus cereus 569/H/9 metallo-β-lactamase

The metallo-beta-lactamase BcII from Bacillus cereus 569/H/9 possesses a binuclear zinc centre. The mono-zinc form of the enzyme displays an appreciably high activity, although full efficiency is observed for the di-zinc enzyme. In an attempt to assign the involvement of the different zinc ligands in the catalytic properties of BcII, individual substitutions of selected amino acids were generated. With the exception of His(116)-->Ser (H116S), C221A and C221S, the mono- and di-zinc forms of all the other mutants were poorly active. The activity of H116S decreases by a factor of 10 when compared with the wild type. The catalytic efficiency of C221A and C221S was zinc-dependent. The mono-zinc forms of these mutants exhibited a low activity, whereas the catalytic efficiency of their respective di-zinc forms was comparable with that of the wild type. Surprisingly, the zinc contents of the mutants and the wild-type BcII were similar. These data suggest that the affinity of the beta-lactamase for the metal was not affected by the substitution of the ligand. The pH-dependence of the H196S catalytic efficiency indicates that the zinc ions participate in the hydrolysis of the beta-lactam ring by acting as a Lewis acid. The zinc ions activate the catalytic water molecule, but also polarize the carbonyl bond of the beta-lactam ring and stabilize the development of a negative charge on the carbonyl oxygen of the tetrahedral reaction intermediate. Our studies also demonstrate that Asn(233) is not directly involved in the interaction with the substrates.

OXA-198, an acquired carbapenem-hydrolyzing class D beta-lactamase from Pseudomonas aeruginosa.

ABSTRACT A carbapenem-resistant Pseudomonas aeruginosa strain (PA41437) susceptible to expanded-spectrum cephalosporins was recovered from several consecutive lower-respiratory-tract specimens of a patient who developed a ventilator-associated pneumonia while hospitalized in an intensive care unit. Cloning experiments identified OXA-198, a new class D β-lactamase which was weakly related (less than 45% amino acid identity) to other class D β-lactamases. Expression in Escherichia coli TOP10 and in P. aeruginosa PAO1 led to transformants that were resistant to ticarcillin and showed reduced susceptibility to carbapenems and cefepime. The blaOXA-198 gene was harbored by a class 1 integron carried by a ca. 46-kb nontypeable plasmid. This study describes a novel class D β-lactamase involved in carbapenem resistance in P. aeruginosa.

Role of Cys221 and Asn116 in the zinc-binding sites of the Aeromonas hydrophila metallo-β-lactamase

The CphA metallo-β-lactamase produced by Aeromonas hydrophila exhibits two zinc-binding sites. Maximum activity is obtained upon binding of one zinc ion, whereas binding of the second zinc ion results in a drastic decrease in the hydrolytic activity. In this study, we analyzed the role of Asn116 and Cys221, two residues of the active site. These residues were replaced by site-directed mutagenesis and the different mutants were characterized. The C221S and C221A mutants were seriously impaired in their ability to bind the first, catalytic zinc ion and were nearly completely inactive, indicating a major role for Cys221 in the binding of the catalytic metal ion. By contrast, the binding of the second zinc ion was only slightly affected, at least for the C221S mutant. Mutation of Asn116 did not lead to a drastic decrease in the hydrolytic activity, indicating that this residue does not play a key role in the catalytic mechanism. However, the substitution of Asn116 by a Cys or His residue resulted in an approximately fivefold increase in the affinity for the second, inhibitory zinc ion. Together, these data suggested that the first zinc ion is located in the binding site involving the Cys221 and that the second zinc ion binds in the binding site involving Asn116 and, presumably, His118 and His196.

Discovery of novel lipophilic inhibitors of OXA-10 enzyme (class D beta-lactamase) by screening amino analogs and homologs of citrate and isocitrate.

Aminocitrate (and homolog) derivatives have been prepared by bis-alkylation of glycinate Schiff bases with bromoacetates (and ethyl acrylate), followed by N-acylation and esters (partial or complete) deprotection. Aminoisocitrate was similarly obtained by mono-alkylation with diethyl fumarate. Evaluation against representative beta-lactamases revealed that the free acid derivatives are modest inhibitors of class A enzymes, whilst their benzyl esters showed a good inhibition of OXA-10 (class D enzyme). A docking experiment featured hydrophobic interactions in the active site.