Colette Duez

The 3-D structure of a zinc metallo-beta-lactamase from Bacillus cereus reveals a new type of protein fold.

The 3-D structure of Bacillus cereus (569/H/9) beta-lactamase (EC 3.5.2.6), which catalyses the hydrolysis of nearly all beta-lactams, has been solved at 2.5 A resolution by the multiple isomorphous replacement method, with density modification and phase combination, from crystals of the native protein and of a specially designed mutant (T97C). The current model includes 212 of the 227 amino acid residues, the zinc ion and 10 water molecules. The protein is folded into a beta beta sandwich with helices on each external face. To our knowledge, this fold has never been observed. An approximate internal molecular symmetry is found, with a 2-fold axis passing roughly through the zinc ion and suggesting a possible gene duplication. The active site is located at one edge of the beta beta sandwich and near the N-terminal end of a helix. The zinc ion is coordinated by three histidine residues (86, 88 and 149) and a water molecule. A sequence comparison of the relevant metallo-beta-lactamases, based on this protein structure, highlights a few well-conserved amino acid residues. The structure shows that most of these residues are in the active site. Among these, aspartic acid 90 and histidine 210 participate in a proposed catalytic mechanism for beta-lactam hydrolysis.

Isolation and characterization of the human prolactin gene

Prolactin (PRL) and growth hormone (GH) genes derive from a common ancestor and still share some sequence homologies. Their expression in the pituitary gland is regulated in opposite directions by most of the many hormones acting on them. This provides an interesting system to study sequences involved in gene expression. Using a human PRL cDNA clone as a probe, we screened a human genomic DNA library in lambda phage and isolated a single recombinant comprising the whole hPRL gene. It was characterized by restriction endonuclease mapping and cDNA hybridization, by DNA heteroduplex analysis and by nucleotide sequencing. The hPRL gene is present as a single copy per haploid genome, is approximately 10 kb long and contains four introns, three of which interrupt the coding sequence at the same locations as in the known GH and PRL genes. The origin of transcription was determined by S1 mapping on prolactinoma mRNAs. The search for direct and inverted repeats, as well as dyad symmetries was carried out in the 900‐bp sequenced in the 5′‐flanking region. Sequence homologies between hPRL, hGH and rPRL were derived from computer drawn matrices for these upstream regions.

Transcription and expression analysis, using lacZ and phoA gene fusions, of Mycobacterium fortuitum beta-lactamase genes cloned from a natural isolate and a high-level beta-lactamase producer.

The gene encoding a class A β‐lactamase was cloned from a natural isolate of Mycobacterium fortuitum (blaF) and from a high‐level amoxicillin‐resistant mutant that produces large amounts of β‐lactamase (blaF). The nucleotide sequences of the two genes differ at 11 positions, including two in the region upstream from the coding sequence. Gene fusions to Escherichia coli lacZ and transcription and expression analysis of the cloned genes in Mycobacterium smegmatis indicated that high‐level production of the β‐lactamase in the mutant is mainly or wholly due to a single base pair difference in the promoter. These analyses also showed that transcription and translation start at the same position. A comparison of the amino acid sequence of BlaF, as predicted from the nucleotide sequence, with the determined N‐terminal amino acid sequence indicated the presence of a typical signal peptide. The fusion of blaF (or biaF) to the E. coli gene phoA resulted in the production of BlaF‐PhoA hybrid proteins that had alkaline phosphatase activity. These results demonstrate that phoA can be used as a reporter gene for studying protein export in mycobacteria.

Occurrence of a serine residue in the penicillin-binding site of the exocellular DD-carboxy-peptidase-transpeptidase from Streptomyces R61

According to the hypothesis proposed by Tipper and Strominger in 1965 for the mechanism of action of penicillin [ 11, the antibiotic, acting as a structural analogue of the substrate, would acylate an essential sulfhydryl group of an enzyme that catalyzes peptide crosslinking during wall peptidoglycan synthesis in bacteria. However, the experimental results presented in favour of the involvement of a sulfhydryl group in the transpeptidation reaction as well as in the binding of penicillin have never been very convincing. The exocellular DD-carboxypeptidase-transpeptidase that is excreted by Streptomyces R61 during growth, is very sensitive to penicillin [2] and appears to be a good model for the study of the interaction between the enzyme, substrates and antibiotic [3,4]. At 37°C the stoichiometric complex formed between benzylpenicillin and this enzyme decomposes with half-life of 80 min, yielding active enzyme, phenylacetylglycine and N-formyl-D-penicillamine [5,6] . More recently, however, it has been observed that when a solution of the [ 14C] benzylpeniciIlin-enzyme complex (in 10 mM phosphate buffer, pH 7.0) is boiled for 1 min, the radioactivity remains stably attached to the denatured protein. Moreover, whereas the native enzyme is very resistant to the action of various proteases, the denatured protein can be readily degrad-

X-ray analysis of the NMC-A beta-lactamase at 1.64-A resolution, a class A carbapenemase with broad substrate specificity

The treatment of infectious diseases by penicillin and cephalosporin antibiotics is continuously challenged by the emergence and the dissemination of the numerous TEM and SHV mutant β-lactamases with extended substrate profiles. These class A β-lactamases nevertheless remain inefficient against carbapenems, the most effective antibiotics against clinically relevant pathogens. A new member of this enzyme class, NMC-A, was recently reported to hydrolyze at high rates, and hence destroy, all known β-lactam antibiotics, including carbapenems and cephamycins. The crystal structure of NMC-A was solved to 1.64-Å resolution, and reveals modifications in the topology of the substrate-binding site. While preserving the geometry of the essential catalytic residues, the active site of the enzyme presents a disulfide bridge between residues 69 and 238, and certain other structural differences compared with the other β-lactamases. These unusual features in class A β-lactamases involve amino acids that participate in enzyme-substrate interactions, which suggested that these structural factors should be related to the very broad substrate specificity of this enzyme. The comparison of the NMC-A structure with those of other class A enzymes and enzyme-ligand complexes, indicated that the position of Asn-132 in NMC-A provides critical additional space in the region of the protein where the poorer substrates for class A β-lactamases, such as cephamycins and carbapenems, need to be accommodated.

The linker region plays a key role in the adaptation to cold of the cellulase from an Antarctic bacterium

The psychrophilic cellulase, Cel5G, from the Antarctic bacterium Pseudoalteromonas haloplanktis is composed of a catalytic module (CM) joined to a carbohydrate-binding module (CBM) by an unusually long, extended and flexible linker region (LR) containing three loops closed by three disulfide bridges. To evaluate the possible role of this region in cold adaptation, the LR was sequentially shortened by protein engineering, successively deleting one and two loops of this module, whereas the last disulfide bridge was also suppressed by replacing the last two cysteine residue by two alanine residues. The kinetic and thermodynamic properties of the mutants were compared with those of the full-length enzyme, and also with those of the cold-adapted CM alone and with those of the homologous mesophilic enzyme, Cel5A, from Erwinia chrysanthemi. The thermostability of the mutated enzymes as well as their relative flexibility were evaluated by differential scanning calorimetry and fluorescence quenching respectively. The topology of the structure of the shortest mutant was determined by SAXS (small-angle X-ray scattering). The data indicate that the sequential shortening of the LR induces a regular decrease of the specific activity towards macromolecular substrates, reduces the relative flexibility and concomitantly increases the thermostability of the shortened enzymes. This demonstrates that the long LR of the full-length enzyme favours the catalytic efficiency at low and moderate temperatures by rendering the structure not only less compact, but also less stable, and plays a crucial role in the adaptation to cold of this cellulolytic enzyme.

Cloning and nucleotide sequencing of the gene encoding the β-lactamase from Citrobacter diversus

The gene coding for the class A beta-lactamase of Citrobacter diversus has been cloned and sequenced. It contains the information for a 294-amino-acid precursor protein, including a 27-residue N-terminal signal peptide. The deduced sequence of the N-terminal portion of the mature protein is in excellent agreement with that determined by microsequencing of the protein and readily explains the pI differences observed between the naturally occurring forms I and II of the enzyme. The sequence of the mature protein exhibits a very high degree of similarity with that of the Klebsiella oxytoca class A beta-lactamase.

Cloning and amplified expression in Streptomyces lividans of a gene encoding extracellular β-lactamase from Streptomyces albus G

A 4.9-kb DNA fragment containing the bla gene for the extracellular beta-lactamase (BLA) of Streptomyces albus G was cloned in Streptomyces lividans using the conjugative, low-copy-number plasmid pIJ61 as vector. No expression of bla was observed when this DNA fragment was introduced into Escherichia coli HB101 on a plasmid vector. A 1.5-kb PstI-SstI fragment containing the bla gene was cloned in S. lividans on the nonconjugative, high-copy-number plasmid pIJ702. A tenfold higher yield of BLA was obtained from S. lividans carrying this plasmid than from S. albus G grown under optimal production conditions. The BLA from the clone reacts with beta-iodopenicillanate according to a branched pathway which is characteristic of the original S. albus G BLA enzyme.

Serine-Type D-Ala-D-Ala Peptidases and Penicillin-Binding Proteins

Publisher Summary This chapter describes serine-type D-Ala-D-Ala peptidases and penicillin-binding proteins. Penicillin is a suicide substrate. Because of the endocyclic nature of the scissile β-lactam amide bond, the leaving group of the enzyme acylation step remains part of the acyl enzyme. The first part only of the transfer cycle is achieved, leading to a long-lived; serine ester-linked acyl(penicilloyl)-enzyme and the enzyme behaves as a penicillin-binding protein (PBP). All bacteria possess an assortment of low and high molecular weight membrane-bound PBPs. The low molecular weight PBPs are single catalytic entities. The bulk of the protein is on the outer face of the plasma membrane and bears a carboxy-terminal extension, the end of which serves as membrane anchor. The low molecular weight PBPs helps to control the extent of wall peptidoglycan cross-linking throughout the life cycle of the cells. The high molecular weight PBPs involved in wall peptidoglycan assembly and cell morphogenesis are multimodule proteins. The bulk of the protein is on the outer face of the membrane and consists of an N-terminal module, fused to a C-terminal, penicillin-binding module.

The dppA gene of Bacillus subtilis encodes a new d‐aminopeptidase

Different strains of Bacillus were screened for their ability to hydrolyse d‐alanyl‐p‐nitroanilide. Activity was detected in Bacillus pumilus, Bacillus brevis, Bacillus licheniformis 749I and Bacillus subtilis 168. The last strain was the best producer and was selected for the production and purification of the enzyme. The determination of the N‐terminal sequence identified the enzyme as the product of the dppA gene (previously named dciAA) belonging to the dipeptide ABC transport (dpp) operon expressed early during sporulation. Open reading frames (ORFs) encoding putative related proteins were found in the genomes of a variety of Archaea and both sporulating and non‐sporulating bacteria. The enzyme behaves as a d‐aminopeptidase and represents the prototype of a new peptidase family. Among the tested substrates, the highest activities were found with d‐Ala‐d‐Ala and d‐Ala‐Gly‐Gly. The active enzyme behaves as an octamer of identical 30 kDa subunits. It exhibits a broad pH optimum, extending between pH 9 and 11. It is reversibly inhibited in the presence of Zn2+ chelators, and the sequence comparisons highlight the conservation of potential Zn‐binding residues. As it has been shown by others that null mutations in the dpp operon do not inhibit spore formation, the physiological role of DppA is probably an adaptation to nutrient deficiency.