Active-site mutants of class B beta-lactamases: substrate binding and mechanistic study.

Increased resistance to beta-lactam antibiotics is mainly due to beta-lactamases. X-ray structures of zinc beta-lactamases unraveled the coordination of the metal ions, but their mode of action remains unclear. Recently, enzymes in which one of the zinc ligands was mutated have been characterized and their catalytic activity against several beta-lactam antibiotics measured. A molecular modeling study of these enzymes was performed here to explain the catalytic activity of the mutants. Coordination around the zinc ions influences the way the tetrahedral intermediate is bound; any modification influences the first recognition of the substrate by the enzyme. For all the studied mutants, at least one of the interactions fails, inducing a loss of catalytic efficiency compared to the wild type. The present studies show that the enzyme cavity is a structure of high plasticity both structurally and mechanistically and that local modifications may propagate its effects far from the mutated amino acid.

Quantitative analysis of the stabilization by substrate of Staphylococcus aureus PC1 beta-lactamase.

BACKGROUND: The stabilization of enzymes in the presence of substrates has been recognized for a long time. Quantitative information regarding this phenomenon is, however, rather scarce since the enzyme destroys the potential stabilizing agent during the course of the experiments. In this work, enzyme unfolding was followed by monitoring the progressive decrease of the rate of substrate utilization by the Staphylococcus aureus PC1 beta-lactamase, at temperatures above the melting point of the enzyme. RESULTS: Enzyme inactivation was directly followed by spectrophotometric measurements. In the presence of substrate concentrations above the K(m) values, significant stabilization was observed with all tested compounds. A combination of unfolding kinetic measurements and enzymatic studies, both under steady-state and non-steady-state regimes, allowed most of the parameters characteristic of the two concurrent phenomena (i.e. substrate hydrolysis and enzyme denaturation) to be evaluated. In addition, molecular modelling studies show a good correlation between the extent of stabilization, and the magnitude of the energies of interaction with the enzyme. CONCLUSIONS: Our analysis indicates that the enzyme is substantially stabilized towards heat-induced denaturation, independently of the relative proportions of non-covalent Henri-Michaelis complex (ES) and acyl-enzyme adduct (ES*). Thus, for those substrates with which the two catalytic intermediates are expected to be significantly populated, both species (ES and ES*) appear to be similarly stabilized. This analysis contributes a new quantitative approach to the problem.

Biochemical characterization of the FEZ-1 metallo-beta-lactamase of Legionella gormanii ATCC 33297T produced in Escherichia coli.

The bla(FEZ-1) gene coding for the metallo-beta-lactamase of Legionella (Fluoribacter) gormanii ATCC 33297T was overexpressed via a T7 expression system in Escherichia coli BL21(DE3)(pLysS). The product was purified to homogeneity in two steps with a yield of 53%. The FEZ-1 metallo-beta-lactamase exhibited a broad-spectrum activity profile, with a preference for cephalosporins such as cephalothin, cefuroxime, and cefotaxime. Monobactams were not hydrolyzed. The beta-lactamase was inhibited by metal chelators. FEZ-1 is a monomeric enzyme with a molecular mass of 29,440 Da which possesses two zinc-binding sites. Its zinc content did not vary in the pH range of 5 to 9, but the presence of zinc ions modified the catalytic efficiency of the enzyme. A model of the FEZ-1 three-dimensional structure was built.

Substrate binding and catalytic mechanism of class B beta-lactamases: a molecular modelling study.

Increased resistance to beta-lactam antibiotics is mainly due to beta-lactamases whose production by pathogenic bacteria makes their broad activity spectrum especially frightening. X-ray structures of several zinc beta-lactamases have revealed the coordination of the two metal ions, but their mode of action remains unclear. Geometry optimisation of stable complexes along the reaction pathway of benzylpenicillin hydrolysis highlighted a proton shuttle occurring from D120 of the Bacillus cereus beta-lactamase to the beta-lactam nitrogen via Zn2 which is central to the network. First, the Zn1 ion has a structural role maintaining Zn-bound waters, WAT1 and WAT2, either directly or through the Zn1 tetrahedrally coordinated histidine ligands. The Zn2 ion has a more catalytic role, stabilising the tetrahedral intermediate, accepting the beta-lactam nitrogen atom as a ligand. The role of Zn2 and the flexibility in the coordination geometry of both Zn ions is of crucial importance for catalysis.

Xylanase from the psychrophilic yeast Cryptococcus adeliae.

A xylanase belonging to family 10 is produced by Cryptococcus adeliae, an Antarctic yeast that exhibits optimal growth at low temperature. The mature glycosylated xylanase secreted by C. adeliae is composed of 338 amino acid residues and 26 +/- 3 osidic residues, and shares 84% identity with its mesophilic counterpart from C. albidus. The xylanase from C. adeliae is less thermostable than its mesophilic homologue when the residual activities are compared, and this difference was confirmed by differential scanning calorimetry experiments. In the range 0 degrees-20 degrees C, the cold-adapted xylanase displays a lower activation energy and a higher catalytic efficiency. All these observations suggest a less compact, more flexible molecular structure. Analysis of computerized molecular models built up for both psychrophilic and mesophilic xylanases indicates that the adaptation to cold consists of discrete changes in the tridimensional structure: of 53 substitutions, 22 are presumably involved in the adaptation process. These changes lead mainly to a less compact hydrophobic packing, to the loss of one salt bridge, and to a destabilization of the macrodipoles of the helices.

pK(a) calculations for class C beta-lactamases: the role of Tyr-150.

The Poisson-Boltzmann method was used to compute the pK(a) values of titratable residues in a set of class C beta-lactamases. In these calculations, the pK(a) of the phenolic group of residue Tyr150 is the only one to stand out with an abnormally low value of 8.3, more than one pK(a) unit lower than the measured reference value for tyrosine in solution. Other important residues of the catalytic pocket, such as the conserved Lys67, Lys315, His314, and Glu272 (hydrogen-bonded to the ammonium group of Lys315), display normal protonation states at neutral pH. pK(a) values were also computed in catalytically impaired beta-lactamase mutants. Comparisons between the relative k(cat) values and the Tyr150 pK(a) value in these mutants revealed a striking correlation. In active enzymes, this pK(a) value is always lower than the solution reference value while it is close to normal in inactive enzymes. These results thus support the hypothesis that the phenolate form of Tyr150 is responsible for the activation of the nucleophilic serine. The possible roles of Lys67 and Lys315 during catalysis are also discussed.

An additional aromatic interaction improves the thermostability and thermophilicity of a mesophilic family 11 xylanase: structural basis and molecular study.

In a general approach to the understanding of protein adaptation to high temperature, molecular models of the closely related mesophilic Streptomyces sp. S38 Xyl1 and thermophilic Thermomonospora fusca TfxA family 11 xylanases were built and compared with the three-dimensional (3D) structures of homologous enzymes. Some of the structural features identified as potential contributors to the higher thermostability of TfxA were introduced in Xyl1 by site-directed mutagenesis in an attempt to improve its thermostability and thermophilicity. A new Y11-Y16 aromatic interaction, similar to that present in TfxA and created in Xyl1 by the T11Y mutation, improved both the thermophilicity and thermostability. Indeed, the optimum activity temperature (70 vs. 60 degrees C) and the apparent Tm were increased by about 9 degrees C, and the mutant was sixfold more stable at 57 degrees C. The combined mutations A82R/F168H/N169D/delta170 potentially creating a R82-D169 salt bridge homologous to that present in TfxA improved the thermostability but not the thermophilicity. Mutations R82/D170 and S33P seemed to be slightly destabilizing and devoid of influence on the optimal activity temperature of Xyl1. Structural analysis revealed that residues Y11 and Y16 were located on beta-strands B1 and B2, respectively. This interaction should increase the stability of the N-terminal part of Xyl1. Moreover, Y11 and Y16 seem to form an aromatic continuum with five other residues forming putative subsites involved in the binding of xylan (+3, +2, +1, -1, -2). Y11 and Y16 might represent two additional binding subsites (-3, -4) and the T11Y mutation could thus improve substrate binding to the enzyme at higher temperature and thus the thermophilicity of Xyl1.

Structural, kinetic, and calorimetric characterization of the cold-active phosphoglycerate kinase from the antarctic Pseudomonas sp. TACII18.

The gene encoding the phosphoglycerate kinase (PGK) from the Antarctic Pseudomonas sp. TACII18 has been cloned and found to be inserted between the genes encoding for glyceraldhyde-3-phosphate dehydrogenase and fructose aldolase. The His-tagged and the native recombinant PGK from the psychrophilic Pseudomonas were expressed in Escherichia coli. The wild-type and the native recombinant enzymes displayed identical properties, such as a decreased thermostability and a 2-fold higher catalytic efficiency at 25 degrees C when compared with the mesophilic PGK from yeast. These properties, which reflect typical features of cold-adapted enzymes, were strongly altered in the His-tagged recombinant PGK. The structural model of the psychrophilic PGK indicated that a key determinant of its low stability is the reduced number of salt bridges, surface charges, and aromatic interactions when compared with mesophilic and thermophilic PGK. Differential scanning calorimetry of the psychrophilic PGK revealed unusual variations in its conformational stability for the free and substrate-bound forms. In the free form, a heat-labile and a thermostable domain unfold independently. It is proposed that the heat-labile domain acts as a destabilizing domain, providing the required flexibility around the active site for catalysis at low temperatures.

Sequence, overproduction and purification of the family 11 endo-beta-1,4-xylanase encoded by the xyl1 gene of Streptomyces sp. S38.

The xyl1 gene encoding the Xyl1 xylanase of Streptomyces sp. strain S38 was cloned by screening an enriched DNA library with a specific DNA probe and sequenced. Three short 5 bp -CGAAA- sequences are located upstream of the Streptomyces sp. S38 xyl1 gene 105, 115 and 250 bp before the start codon. These sequences, named boxes 1, 2 and 3, are conserved upstream of the Actinomycetales xylanase genes and are specifically recognized by a DNA-binding protein (Giannotta et al., 1994. FEMS Microbiol. Lett. 142, 91-97) and could be probably involved in the regulation of xylanase production. The Xyl1 ORF encodes a 228 residue polypeptide and the Xyl1 preprotein contains a 38 residue signal peptide whose cleavage yields a 190 residue mature protein of calculated M(r) = 20,585 and basic pI value of 9.12. The molecular mass of the produced and purified mature protein determined by mass spectrometry (20,586 +/- 1 Da) and its pI (9.8) agree with these calculated values. Its N-terminal amino-acid sequence confirmed the proposed cleavage site between the signal peptide and the mature protein. Comparisons between Xyl1 and the 62 other xylanases belonging to family 11 allowed the construction of a phylogenetic tree and revealed its close relationship with Actinomycetales enzymes. Moreover, nine residues were found to be strictly conserved among the 63 xylanases.

pKa calculations for class A beta-lactamases: influence of substrate binding.

Beta-Lactamases are responsible for bacterial resistance to beta-lactams and are thus of major clinical importance. However, the identity of the general base involved in their mechanism of action is still unclear. Two candidate residues, Glu166 and Lys73, have been proposed to fulfill this role. Previous studies support the proposal that Glu166 acts during the deacylation, but there is no consensus on the possible role of this residue in the acylation step. Recent experimental data and theoretical considerations indicate that Lys73 is protonated in the free beta-lactamases, showing that this residue is unlikely to act as a proton abstractor. On the other hand, it has been proposed that the pKa of Lys73 would be dramatically reduced upon substrate binding and would thus be able to act as a base. To check this hypothesis, we performed continuum electrostatic calculations for five wild-type and three beta-lactamase mutants to estimate the pKa of Lys73 in the presence of substrates, both in the Henri-Michaelis complex and in the tetrahedral intermediate. In all cases, the pKa of Lys73 was computed to be above 10, showing that it is unlikely to act as a proton abstractor, even when a beta-lactam substrate is bound in the enzyme active site. The pKa of Lys234 is also raised in the tetrahedral intermediate, thus confirming a probable role of this residue in the stabilization of the tetrahedral intermediate. The influence of the beta-lactam carboxylate on the pKa values of the active-site lysines is also discussed.

The catalytic mechanism of DD-peptidases: unexpected importance of tyrosine 280 in the transpeptidation reaction catalysed by the Streptomyces R61 DD-peptidase.

The study of the interactions between the Tyr280Phe mutant of the Streptomyces R61 DD-peptidase, various substrates and beta-lactam antibiotics shows that Tyr280 is involved not only in the formation of the acylenzyme with the peptide substrate and beta-lactam antibiotics, but also and specifically in the catalysis of the transpeptidation reaction. Surprisingly, this residue does not belong to the conserved structural and functional elements which characterise the penicillin-recognising enzymes.

Catalytic properties of class A beta-lactamases: efficiency and diversity.

beta-Lactamases are the main cause of bacterial resistance to penicillins, cephalosporins and related beta-lactam compounds. These enzymes inactivate the antibiotics by hydrolysing the amide bond of the beta-lactam ring. Class A beta-lactamases are the most widespread enzymes and are responsible for numerous failures in the treatment of infectious diseases. The introduction of new beta-lactam compounds, which are meant to be 'beta-lactamase-stable' or beta-lactamase inhibitors, is thus continuously challenged either by point mutations in the ubiquitous TEM and SHV plasmid-borne beta-lactamase genes or by the acquisition of new genes coding for beta-lactamases with different catalytic properties. On the basis of the X-ray crystallography structures of several class A beta-lactamases, including that of the clinically relevant TEM-1 enzyme, it has become possible to analyse how particular structural changes in the enzyme structures might modify their catalytic properties. However, despite the many available kinetic, structural and mutagenesis data, the factors explaining the diversity of the specificity profiles of class A beta-lactamases and their amazing catalytic efficiency have not been thoroughly elucidated. The detailed understanding of these phenomena constitutes the cornerstone for the design of future generations of antibiotics.

pKa calculations for class A beta-lactamases: methodological and mechanistic implications.

Beta-lactamases are responsible for resistance to penicillins and related beta-lactam compounds. Despite numerous studies, the identity of the general base involved in the acylation step is still unclear. It has been proposed, on the basis of a previous pKa calculation and analysis of structural data, that the unprotonated Lys73 in the active site could act as the general base. Using a continuum electrostatic model with an improved treatment of the multiple titration site problem, we calculated the pKa values of all titratable residues in the substrate-free TEM-1 and Bacillus licheniformis class A beta-lactamases. The pKa of Lys73 in both enzymes was computed to be above 10, in good agreement with recent experimental data on the TEM-1 beta-lactamase, but inconsistent with the proposal that Lys73 acts as the general base. Even when the closest titratable residue, Glu166, is mutated to a neutral residue, the predicted downward shift of the pKa of Lys73 shows that it is unlikely to act as a proton abstractor in either enzyme. These results support a mechanism in which the proton of the active Ser70 is transferred to the carboxylate group of Glu166.

Unexpected influence of a C-terminal-fused His-tag on the processing of an enzyme and on the kinetic and folding parameters.

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.

Cold adaptation parameters derived from cDNA sequencing and molecular modelling of elastase from Antarctic fish Notothenia neglecta.

The primary structure of an elastase from the Antarctic fish Notothenia neglecta (NE) was elucidated by molecular cloning and cDNA sequence analysis. The cDNA of interest was isolated from a cDNA library obtained from Notothenia's pyloric caeca. The amino acid sequence identity with mammalian elastases ranges between 53 and 64%, but interestingly reaches 79% with one isoform (CEB) of two recently isolated cod elastases. The most interesting changes distinguishing the model of NE, predicted from the three dimentional structure of the native porcine elastase (PE), concern the catalytic crevice located in the inter-domains region. These features might be involved in the adaptation to cold of the Antarctic elastase.

Phylogenetic classification of the mitochondrial carrier family of Saccharomyces cerevisiae.

The screening of the open reading frames identified in the whole yeast genome has allowed us to discover 34 proteins belonging to the mitochondrial carrier family. By phylogenetic study, they can be divided into 27 subfamilies including ADP/ATP, phosphate and citrate carriers, putative oxoglutarate and GDC carriers and 22 new subfamilies. Topology predictions using the 'positive inside rule' approach have shown that the yeast carriers are similarly oriented with both extremities exposed to the cytosol. In each subfamily, a strict conservation of the charged residues in the six transmembrane alpha-helices is observed, suggesting a functional role for these residues and the existence of 27 functionally distinct carriers.

Site-directed mutagenesis of glutamate 166 in two beta-lactamases. Kinetic and molecular modeling studies.

The catalytic pathway of class A beta-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 beta-lactamases yielded enzymes forming stable acyl-enzymes with beta-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 Omega-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.

Enzymes from cold-adapted microorganisms. The class C beta-lactamase from the antarctic psychrophile Psychrobacter immobilis A5.

A heat-labile beta-lactamase has been purified from culture supernatants of Psychrobacter immobilis A5 grown at 4 degrees C and the corresponding chromosomal ampC gene has been cloned and sequenced. All structural and kinetic properties clearly relate this enzyme to class C beta-lactamases. The kinetic parameters of P. immobilis beta-lactamase for the hydrolysis of some beta-lactam antibiotics are in the same range as the values recorded for the highly specialized cephalosporinases from pathogenic mesophilic bacteria. By contrast, the enzyme displays a low apparent optimum temperature of activity and a reduced thermal stability. Structural factors responsible for the latter property were analysed from the three-dimensional structure built by homology modelling. The deletion of proline residues in loops, the low number of arginine-mediated H-bonds and aromatic-aromatic interactions, the lower global hydrophobicity and the improved solvent interactions through additional surface acidic residues appear to be the main determinants of the enzyme flexibility.

A disulfide bridge near the active site of carbapenem-hydrolyzing class A beta-lactamases might explain their unusual substrate profile.

Bacterial resistance to beta-lactam antibiotics, a clinically worrying and recurrent problem, is often due to the production of beta-lactamases, enzymes that efficiently hydrolyze the amide bond of the beta-lactam nucleus. Imipenem and other carbapenems escape the activity of most active site serine beta-lactamases and have therefore become very popular drugs for antibacterial chemotherapy in the hospital environment. Their usefulness is, however, threatened by the appearance of new beta-lactamases that efficiently hydrolyze them. This study is focused on the structure and properties of two recently described class A carbapenemases, produced by Serratia marcescens and Enterobacter cloacae strains and leads to a better understanding of the specificity of beta-lactamases. In turn, this will contribute to the design of better antibacterial drugs. Three-dimensional models of the two class A carbapenemases were constructed by homology modeling. They suggested the presence, near the active site of the enzymes, of a disulfide bridge (C69-C238) whose existence was experimentally confirmed. Kinetic parameters were measured with the purified Sme-1 carbapenemase, and an attempt was made to explain its specific substrate profile by analyzing the structures of minimized Henri-Michaelis complexes and comparing them to those obtained for the "classical" TEM-1 beta-lactamase. The peculiar substrate profile of the carbapenemases appears to be strongly correlated with the presence of the disulfide bridge between C69 and C238.