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Cell-free expression-based screening is sometimes more suitable than cell-based assays for enzyme discovery. The advantage of cell-free systems for expression of toxic, poorly expressed, or insoluble proteins has already been well documented. Cell-free methods can advantageously replace cell-based ones when screening has to be performed on cell lysates prepared from harvested cells, for instance, when dealing with protein-ligand interactions particularly when the latter is hydrophobic. From our experience, cell-free extracts efficient in both transcription and translation can be prepared from potentially any microorganism. Here we present a general method for preparation of cell-free extracts from prokaryotic and eukaryotic cells, selection of the best systems, and optimized conditions for specific protein expression. The method allows to select proteins for their ability to bind a selected target, to identify the inhibitors of such binding, or to identify novel enzymatic activities.

Screening of prokaryotic genomes in order to identify enzymes with a desired catalytic activity can be performed in vivo in bacterial cells. We propose a strategy of in vitro expression screening of large prokaryotic genomic libraries based on Escherichia coli cell-free transcription-translation systems. Because cell-based expression may be limited by poor yield or protein misfolding, cell-free expression systems may be advantageous in permitting a more comprehensive screen under conditions optimized for the desired enzyme activity. However, monocistronic messages with an improved leader initiation context are typically used for protein production in vitro. Here, we describe successful use of a Pseudoalteromonas genomic DNA library for in vitro expression of DNA fragments carrying multiple open reading frames (ORFs) in the context of their authentic translation initiation sites and regulatory regions. We show that ORFs located far from the 5' and 3' ends of polycistronic transcripts can be expressed at a sufficient level in an in vitro transcription-translation system in order to allow functional screening. We demonstrate the overall cell-free functional screen strategy with the successful selection of an esterase from Pseudoalteromonas.

A PCR protocol was used to identify and sequence a gene encoding a DNA ligase from Thermococcus fumicolans (Tfu). The recombinant enzyme, expressed in Escherichia coli BL21(DE3) pLysS, was purified to homogeneity and characterized. The optimum temperature and pH of Tfu DNA ligase were 65 degrees C and 7.0, respectively. The optimum concentration of MgCl2, which is indispensable for the enzyme activity, was 2 mM. We showed that Tfu DNA ligase displayed nick joining and blunt-end ligation activity using either ATP or NAD+, as a cofactor. In addition, our results would suggest that Tfu DNA ligase is likely to use the same catalytic residues with the two cofactors. The ability for DNA ligases, to use either ATP or NAD+, as a cofactor, appears to be specific of DNA ligases from Thermococcales, an order of hyperthermophilic microorganisms that belongs to the euryarchaeotal branch of the archaea domain.

The 5'-region of the TIF4631 gene of Saccharomyces cerevisiae (encoding the translation initiation factor eIF4G1) was reported earlier to harbor a very active internal ribosome entry site (IRES) allowing for internal initiation of translation of TIF4631 mRNA. Here, we report the presence of a promoter in the region -112 to -36 relative to the translation initiation codon of the TIF4631 gene. This promoter stimulates transcription from a start site at position -36 and generates an mRNA that is actively translated in vitro and able to sustain growth of yeast cells in vivo as the only source of eIF4G. The data show that the IRES activity reported earlier is due to this promoter. On the contrary, the presumed IRES represents a strongly inhibitory element for translation in vitro.

In this paper we report on searching for suitable reporters to monitor gene expression and protein secretion in the amylolytic yeast Schwanniomyces occidentalis. Several potential reporter and marker genes, formerly shown to be functional in other yeasts, were cloned downstream from the homologous invertase gene (INV) promoter and their activity was followed in conditions of repression and derepression of the INV promoter. However, neither beta-glucuronidase nor beta-lactamase nor phleomycin resistance-conferring gene, all originating from E. coli, were expressed in S. occidentalis cells to such a level to allow for monitoring of their activity. All the reporter genes tested have a higher percentage of GC (47-62%) in their DNA compared to the DNA composition of S. occidentalis genes that are more AT-rich (36% GC). The codon usage of all the reporter genes also varies from that of 16 so far sequenced S. occidentalis genes. This suggests that an appropriate composition of DNA and a codon usage similar to S. occidentalis genes might be very important parameters for an efficient expression of a heterologous gene in Schwanniomyces occidentalis. Indeed, two genes originating from Staphylococcus aureus, with an AT-content in their DNA similar to that of S. occidentalis, were functionally expressed in S. occidentalis cells. Both a phleomycin resistance-conferring gene and a chloramphenicol acetyltransferase-encoding gene thus represent suitable reporters of gene expression and protein secretion in S. occidentalis. Additionally, we show in this work that the transcription-regulating region and the signal peptide sequence of the S. occidentalis invertase gene were efficient to direct gene expression and subsequent protein secretion in Saccharomyces cerevisiae., (Copyright 2003 John Wiley & Sons, Ltd.)

Bleomycin (Bm), a 1.4 kDa glycopeptide excreted by Streptomyces verticillus, is a natural antibacterial compound used in therapy as antineoplastic drug. To counteract its biological activity, cells have developed several resistance mechanisms, one of these based on proteins able to tightly bind Bm. In this paper, the interaction of Zn(2+)-Bm with the Streptoalloteichus hindustanus Bm resistance protein (ShBle) has been investigated by solution state NMR. Sequential nOe and chemical shift index have shown that the fold of the protein (in absence or presence of Bm) is identical to the previously published X-ray structure. The dimeric nature of ShBle is confirmed by the diffusion tensor as determined by NMR relaxation data. Using isotope filtered nOe experiment, intermolecular nOes between Bm and ShBle have been observed as used for modeling. While the interaction of the Bm metal binding site with ShBle appears to be uniquely defined, several conformations of the bithiazole moieties are compatible with the NMR data. Binding of Bm also induces changes of the local dynamics (stretch N85-G91), as shown by (15)N relaxation data. These results are discussed in the context of several Bm analogues able to interact with ShBle and of the recently published X-rays structures.

PI-TfuI, an intein spliced from the DNA polymerase of Thermococcus fumicolans, is a highly specific endonuclease, whose cleavage efficiency and specificity depend on both the substrate topology and the divalent cation used as cofactor. An open circular intermediate was observed during the cleavage of supercoiled DNA by PI-TfuI, suggesting a two-step cleavage of the DNA. We characterized this nicked intermediate and, through the development of a method of analysis of the cleavage reaction based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we demonstrated that the cleavage of DNA by PI-TfuI indeed results from two cleavage events. One step results in the cleavage of the bottom strand, which is independent of the DNA conformation or choice of the metal ion cofactor. A second step, which is slower, leads to the cleavage of the top strand and governs the specific requirements of PI-TfuI concerning the essential cofactor and the DNA topology. These two steps were shown to be independent in optimal conditions of cleavage. These data give support to the existence of two distinct and independent active sites in the endonuclease domain of the archaeal intein.

Among the 14 inteins of the Pyrococcus abyssi genome, 10 harbour the LAGLIDADG motifs of dodecapeptide endonucleases. Four of these were cloned, expressed in Escherichia coli and purified to assay their potential endonuclease activity. PabRIR1-2 and PabRIR1-3 are specific endonucleases, named PI-PabI and PI-PabII, respectively, cleaving the sequence spanning their homing site. This is consistent with their size and with the relative positions and sequences of their endonuclease motifs. However, PI-PabI is 10-fold more active than PI-PabII and a discrepancy of the DNA recognition and cleavage mechanisms was observed between the two inteins. In particular, analysis of the DNA cleavage reactions by MALDI-TOF highlighted that while the cleavage of DNA by PI-PabI consists of two steps corresponding to the cleavage of each DNA strand, PI-PabII processes the two DNA strands simultaneously. Furthermore, the two inteins interact differently with DNA. In addition, we did not detect any endonuclease activity for PabLon and PabRIR1-1. Deletions in the intein sequences and mutations in the putative endonuclease motifs probably abolish this activity. Hence, inteins from the same archaebacteria, even if contained in the same host protein, did not evolve uniformly and are presumably at different stages of the invasion cycle.

We report here that PBP1a can dimerize but does not interact with PBP1b to form PBP1a/PBP1b heterodimers in Escherichia coli. These findings support the idea of a relevant involvement of dimerization of both PBP1a and PBP1b during murein synthesis and suggest the existence of different peptidoglycan synthesis complexes.

The worldwide recrudescence of tuberculosis and the widespread appearance of antibiotic resistance have strengthened the need for rapid and specific diagnostic tools. The prevailing microbiological identification of Mycobacterium tuberculosis, the causative agent of tuberculosis, which implies the use of in vitro cultures and acid-fast staining microscopy, is time-consuming. Detection of M. tuberculosis directly in clinical samples through PCR amplification of mycobacterium-specific genes, designed to shorten diagnostic delay, demonstrated reliability and high sensitivity. However, the quality of the diagnosis depends on the specificity of the target sequence for M. tuberculosis complex strains. In the present study, we demonstrated the specificity of recA and pps1 inteins for this complex and thus the feasibility of using intein-coding sequences as a new target for PCR diagnosis. Indeed, the recA and pps1 genes of 36 clinical isolates of M. tuberculosis and 10 field strains of M. bovis were found to be interrupted by an intein sequence at the RecA-a and Pps1-b sites, respectively, while a large number of nontuberculous mycobacterial species failed to demonstrate these insertions. Besides, the MtuPps1, which was cloned and expressed in Escherichia coli, was shown to possess an endonuclease activity. The intein cleaves the 40-bp sequence spanning the intein insertion site Pps1-b in the inteinless pps1 gene. In addition to the PCR amplification of recA and pps1 intein genes as a tool for diagnosis, the specific endonuclease activity could represent a new molecular approach to identify M. tuberculosis.

To gain further insights into the understanding of the intein invasion process in mycobacteria, intein sequences in the gyrA gene of 42 mycobacterial strains were searched and a new gyrA intein was found in Mycobacterium gastri (Mga). This 1260 bp intein, named MgaGyrA, inserted at the GyrA-a site, is highly homologous to the members of the Mycobacterium leprae GyrA allelic family. As the recA intein, MgaGyrA was detected in only one out of six Mga strains examined, while the pps1 intein was a constant character of Mga. This data supports the genomic heterogeneity of Mga towards intein invasion, a finding that may have phylogenetic implications.

A survey of a vast range of mycobacterial strains led us to discover a new Pps1 intein allele in Mycobacterium gastri which differs from those of Mycobacterium tuberculosis and Mycobacterium leprae in both its sequence and insertion site. While little is known about Pps1, except that it belongs to the YC24 family of ABC transporters, we show that, unlike the other inteins described so far from Eubacteria, the MgaPps1 intein possesses a specific endonuclease activity. The intein is the first eubacterial intein to be characterised as an endonuclease. Like other intein endonucleases, its minimal sequence for recognition and cleavage is quite large, with 22 bp spanning the Pps1-c site. The fact that an active endonuclease is found among the mycobacterial inteins supports the concept of a cyclical model of invasion by horizontal transfer of these genes, followed by degeneration and loss until a new invasion event, thus explaining their long-term persistence in closely related eubacterial species.

Penicillin-binding protein 1b (PBP1b) is the major high-molecular-weight PBP in Escherichia coli. Although it is coded by a single gene, it is usually found as a mixture of three isoforms which vary with regard to the length of their N-terminal cytoplasmic tail. We show here that although the cytoplasmic tail seems to play no role in the dimerization of PBP1b, as was originally suspected, only the full-length protein is able to protect the cells against lysis when both PBP1a and PBP3 are inhibited by antibiotics. This suggests a specific role for the full-length PBP1b in the multienzyme peptidoglycan-synthesizing complex that cannot be fulfilled by either PBP1a or the shorter PBP1b proteins. Moreover, we have shown by alanine-stretch-scanning mutagenesis that (i) residues R(11) to G(13) are major determinants for correct translocation and folding of PBP1b and that (ii) the specific interactions involving the full-length PBP1b can be ascribed to the first six residues at the N-terminal end of the cytoplasmic domain. These results are discussed in terms of the interactions with other components of the murein-synthesizing complex.

Biodiversity screening and directed evolution are two fruitful complementary approaches for the discovery and design of novel biocatalysts. A new technology for directed evolution, L-Shuffling, has been designed and patented by Proteus. L-Shuffling technology offers several competitive advantages over other technologies including (i) directed evolution of large genes: L-Shuffling" means "Large-Shuffling"; (ii) high fidelity recombination and (iii) Control over location and frequency of recombination. The thousands of new recombinants generated by L-Shuffling can be further screened for their biochemical characteristics using Phenomics. Phenomics is a proprietary functional HTS technology designed and patented by Proteus for the screening of natural biodiversity as well as biodiversity generated by combinatorial biology. Phenomics is a function to gene structure approach which provides an alternative to genomics and proteomics. The traditional limits of expression libraries are thereby circumvented especially those related to cytotoxic products in usual or specific surrogate hosts. The quality of the answer given by the screening is directed dependent on the quality of the question asked. Thanks to a new substrates synthesis technology named CLIPS-O, the company can design highly specific molecules simulating the chemical structure and energetic state of the industrial substrates. The whole process of novel biocatalysts discovery has been automated using commercially available high throughput robotics.

THY Pol-2 intein, from Thermococcus hydrothermalis, belongs to the same allelic family as TLI Pol-2 (PI-TLII), Tfu Pol-2 (PI-TFUII) and TspTY Pol-3 mini-intein, all inserted at the pol-c site of archaeal DNA polymerase genes. This new intein was cloned, expressed in Escherichia coli and purified. The intein is a specific endonuclease (PI-THY:I) which cleaves the inteinless sequence of the THY DNA pol gene. Moreover, PI-TLII, PI-TFUII and PI-THYI are very similar endonucleases which cleave DNA in the same optimal conditions at 70 degrees C yielding similar 3'-hydroxyl overhangs of 4 bp and the reaction is subject to product inhibition. The three enzymes are able to cleave the three DNA sequences spanning the pol-c site and a 24 bp consensus cleavage site was defined for the three isoschizomers. However, the exact size of the minimal cleavage site depends both on the substrate sequence and the endonuclease. The inability of the isoschizomers to cleave the inteinless DNA polymerase gene from Pyrococcus spp. KOD is due to point substitutions on the 5' side of the pol-c site, suggesting that the absence of inteins of this allelic family in DNA polymerase genes from Pyrococcus spp. can be linked to small differences in the target site sequence.

This study provides evidence that the differences in membrane composition found from one cell type to another can represent a limiting factor to recovering the functionality of transmembrane proteins when expressed in heterologous systems. Restoring the properties of the human mu-opioid receptor in yeast (Saccharomyces cerevisiae), similar to those observed in native cells, was achieved by replacing ergosterol from yeast by cholesterol, which is normally found in mammalian plasma membranes. The results suggest that these two sterols have opposite effects with respect to the ligand binding function of the receptor. Ergosterol was found to constrain the mu-opioid receptor in an inactive state in yeast plasma membranes and cannot replace cholesterol in activating it. These data differ from previous works dealing with the function of related G-protein-coupled receptors (GPCR) in ergosterol-enriched membranes. This suggests that structural requirements of GPCR with respect to their modulation by lipid components differ from one protein to another. As a consequence, we assume that the presence of appropriate lipids around transmembrane proteins determines their function. This highlights the functional significance of lateral heterogeneities of membrane components within biological membranes.

Five new inteins were discovered in a survey of 39 mycobacterial strains that was undertaken to clarify the role of RecA inteins in mycobacteria. They are all inserted at the RecA-b site of the recA gene of Mycobacterium chitae, 4. fallax, M. gastri, M. shimodei and M. thermoresistibile and belong to the MleRecA allelic family. Sequence analysis showed that although only M. tuberculosis harbours an intein at the RecA-a site the sequence of the RecA-b site is well conserved between species. Furthermore, the presence of inteins does not correlate with specific characteristics of the species such as pathogenicity or growth rate.

We have analyzed ARS elements linked to homologous and heterologous ADE2 loci functioning in Schwanniomyces occidentalis. We have identified a region of the ADE2 locus of S. occidentalis which promotes autonomous replication of plasmids in S. occidentalis cells. This region is within 385 bp preceding the ATG codon of the S. occidentalis ADE2 gene. It contains sequences similar to ARS core consensus sequences, ARS boxes, and a potential transcription activator binding site characterized in Saccharomyces cerevisiae. The ADE2 gene of S. cerevisiae was found to complement the ade2 mutation in S. occidentalis cells and the 5' UTR region of this gene is capable of supporting autonomous replication of plasmids in S. occidentalis. Furthermore, we confirmed that the origin of replication of the 2 microm plasmid and the ARS1 sequence of S. cerevisiae are also functional in S. occidentalis cells. Plasmids carrying either ARS, the SwARSA element of S. occidentalis, the ARS linked to the ADE2 gene of S. cerevisiae, and the ARS1 sequence or the 2 microm ori, were found to be maintained in S. occidentalis cells as episomal monomers or oligomers. However, their stability was low as already reported for the ARS in S. occidentalis.

The DNA polymerase gene of Thermococcus fumicolans harbors two intein genes. Both inteins have been produced in Escherichia coli and purified either as naturally spliced products from the expression of the complete DNA polymerase gene or directly from the cloned inteins genes. Both recombinant inteins exhibit endonuclease activity, with an optimal temperature of 70 degrees C. The Tfu pol-1 intein, which belongs to the Psp KOD pol-1 allelic family, recognizes and cleaves a minimal sequence of 16 base pairs (bp) on supercoiled DNA with either Mn(2+) or Mg(2+) as cofactor. It cleaves linear DNA only with Mn(2+) and requires a 19-bp minimal recognition sequence. The Tfu pol-2 intein, which belongs to the Tli pol-2 allelic family, is a highly active homing endonuclease using Mg(2+) as cofactor. Its minimal recognition and cleavage site is 21 bp long either on linear or circular DNA substrates. Its endonuclease activity is strongly inhibited by the 3' digestion product, which remains bound to the enzyme after the cleavage reaction. According to current nomenclature, these endonucleases were named PI-TfuI and PI-TfuII. These two inteins thus exhibit different requirements for metal cofactor and substrate topology as well as different mechanism of action.

PBP1b can be found as a dimer in Escherichia coli. Previous results suggested that dimerization involved the cysteine(s) in an intermolecular disulfide bond. We show that either deletion mutants or a mutant without cysteines is fully active and still binds penicillin and that the latter can also form dimers.

The human mu-opioid receptor was expressed in Saccharomyces cerevisiae. Binding of [3H]diprenorphine to yeast spheroplasts was specific and saturable (Kd = 1 nm, Bmax = 0.2-1 pmol x mg-1 of membrane proteins). Inhibition of [3H]diprenorphine binding by antagonists and agonists with varying opioid selectivities (mu, delta and kappa) occurred with the same order of potency as in mammalian tissues. Affinities of antagonists were the same with yeast spheroplasts as in reference tissues whereas those of agonists, except etorphine and buprenorphine, were 10-fold to 100-fold lower. Addition of heterotrimeric Gi,o-proteins purified from bovine brain shifted the mu-opioid receptor into a high-affinity state for agonists. Using individually purified Galpha-subunits re-associated with betagamma-dimers, we showed that alphao1, alphao2, alphai1, alphai2 and alphai3 reconstituted high-affinity agonist binding with equal efficiency. This suggests that the structural determinants of the mu-opioid receptor responsible for G-protein coupling are not able to confer a high degree of specificity towards any member of the Gi,o family. The selective effects of opioid observed in specialized tissues upon opioid stimulation may be a result of regulation of G-protein activity by cell-specific factors which should conveniently be analysed using the reconstitution assay described here.

Xenobiotic resistance is the major cause of failure in human therapies. Because of their serious clinical and economical consequences, resistance phenomena have been intensively studied in the case of antibacterial, anticancer, antipaludic and anti-human immunodeficiency virus-1 therapies. Beside pharmacological factors that can impede the action of the drugs, several cellular mechanisms of resistance have been described. Surprisingly, these mechanisms are conserved among bacteria, eucaryotic cells, parasites and viruses. The efficiency of drugs can be circumvented by alteration of the drug cellular concentration (altered influx, enhanced efflux or sequestration), detoxification, alteration of the drug target, nonactivation or inactivation of the drug, or by enhanced DNA repair.

Human vascular endothelial cells (EC) have been implicated in the dissemination of human immunodeficiency virus type-1 (HIV-1). HIV-1-tat, a viral gene product essential for HIV replication, has been shown to interact with different cell types, altering their growth and inducing gene expression. In the present report, we have examined the effect of HIV-tat on the expression of various adhesion molecules in human umbilical vein EC. Our results show that treatment of EC with HIV-tat induces the cell surface expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and endothelial leukocyte adhesion molecule-1 in a time- and dose-dependent manner. Cycloheximide abolished the HIV-tat-dependent induction of all the adhesion molecules, indicating that protein synthesis was required for induction. The effect of HIV-tat on expression of adhesion molecules was potentiated by tumor necrosis factor (TNF), a well-known inducer of adhesion molecules. Like TNF, HIV-tat also enhanced the adhesion of human promyelomonocytic HL-60 cells to EC, and this effect was abolished by treatment with antibodies either against HIV-tat or adhesion molecules. Our results thus indicate that the HIV-tat protein can activate human vascular EC to induce the expression of various adhesion molecules that may play a role in the extravasation of HIV-infected cells.

Penicillin-binding proteins (PBPs) are the targets of beta-lactam antibiotics. We have used a systematic five-alanine substitution method (called ASS [alanine stretch scanning] mutagenesis) to investigate the functional or structural role of various stretches of amino acids in the PBP1b of Escherichia coli. To probe the specific activity of each variant, the antibiotic discs assay was used with strain QCB1 (delta ponB) in the presence of cefaloridine, which totally inhibits the complementing action of PBP1a. This in vivo test has been combined with a quick and efficient in vitro test of the penicillin-binding activity of each of these variants with fluorescent penicillin. This approach has enabled us to show an unexpected role of the N-terminal and C-terminal tails of PBP1b. Moreover, we have established the correct position in PBP1b of the SMN motif that, with the SXXK and the KTG motifs, constitutes the signature of the penicilloyl serine transferases family. Finally, we have shown that the transglycosylase and the transpeptidase domains are separated by an inert linker region, where substitutions and insertions can be made without hindering the in vivo and in vitro activity of the protein.

The catELISA technique was modified and standardized for measuring HIV-1 aspartyl protease activity and evaluating the potency of synthetic peptide inhibitors. This immuno-quantified solid phase assay combines the use of an immobilized C-terminal biotinylated peptide as substrate, a crude enzyme preparation, and a highly specific antiserum elicited against the C-terminal product of the enzyme reaction. A standard curve of this C-terminal product was constructed to determine the enzyme activity. This assay, which requires less enzyme and substrate, is more sensitive than the conventional HPLC method. The amounts of C-terminal peptide produced in solution as determined from ELISA and HPLC standard curves were comparable. Analogues of peptidomimetics designed in our laboratory were assayed for their potency to inhibit the enzyme. One of them, H4, which is a hydroxyethylamine isostere of the Phe-Pro peptide bond, was a powerful inhibitor.

Background: Oligomerization is often necessary for protein activity or regulation and its efficiency is fundamental for the cell. The quaternary structure of a large number of oligomers consists of protomers tightly anchored to each other by exchanged arms or swapped domains. However, nothing is known about how the arms can be kept in a favourable conformation before such an oligomerization., Results: Upon examination of such quaternary structures, we observe an extremely frequent occurrence of proline residues at the point where the arm leaves the protomer. Sequence alignment and site-directed mutagenesis confirm the importance of these prolines. The conservation of these residues at the hinge regions can be explained by the constraints that they impose on polypeptide conformation and dynamics: by rigidifying the mainchain, prolines favour extended conformations of arms thus favouring oligomerization, and may prevent interaction of the arms with the core of the protomer., Conclusions: Hinge prolines can be considered as 'quaternary structure helpers'. The presence of a proline should be considered when searching for a determinant of oligomerization with arm exchange and could be used to engineer synthetic oligomers or to displace a monomers to oligomers equilibrium by mutation of this proline residue.

We have developed a foolproof method to substitute a stretch of residues by alanines. After the introduction of aPstI site by IPCR, thus creating two alanine codons, additional codons are introduced at this site through the use of an 'alanine-stretch cartridge'. These cartridges comprise an antibiotic resistance gene flanked on both sides by alanine codons. Excision of the resistance gene byPvuII then yields the correct insertion of codons. The method is both highly reliable and flexible and should be of general use.

The E166Y and the E166Y/R164S TEM-1 beta-lactamase mutant enzymes display extended spectrum substrate specificities. Electrospray mass spectrometry demonstrates that, with penicillin G as substrate, the rate-limiting step in catalysis is the hydrolysis of the E166Y acyl-enzyme complex. Comparison of the 1.8-A resolution x-ray structures of the wild-type and of the E166Y mutant enzymes shows that the binding of cephalosporin substrates is improved, in the mutant enzyme, by the enlargement of the substrate binding site. This enlargement is due to the rigid body displacement of 60 residues driven by the movement of the omega-loop. These structural observations strongly suggest that the link between the position of the omega-loop and that of helix H5, plays a central role in the structural events leading to extended spectrum TEM-related enzymes. The increased omega-loop flexibility caused by the R164S mutation, which is found in several natural mutant TEM enzymes, may lead to similar structural effects. Comparisons of the kinetic data of the E166Y, E166Y/R164S, and R164S mutant enzymes supports this hypothesis.

We have determined the nucleotide sequence of a 3.3 kb fragment containing the gene (ADE2) encoding phosphoribosylaminoimidazole carboxylase (AIRC) from the yeast Schwanniomyces occidentalis. Translation of a 1671 bp open reading frame predicts a protein of 557 amino acids which has significant homology to AIRC from Saccharomyces cerevisiae and Schizosaccharomyces pombe. The 5' untranslated region of the S. occidentalis gene contains a sequence corresponding to the consensus binding site of the S. cerevisiae transcription regulatory proteins GCN4, BAS1 and BAS2.

The G238S substitution found in extended-spectrum natural mutants of TEM-1 beta-lactamase induces a new capacity to hydrolyze cefotaxime and a large loss of activity against the good substrates of TEM-1. To understand this phenomenon at the molecular level, a method to determine the acylation and deacylation elementary rate constants has been developed by using electrospray mass spectrometry combined with UV spectrophotometry. The hydrolysis of penicillins and cefotaxime by TEM-1 and the G238S mutant shows that the behavior of penicillins and cefotaxime is very different. With both enzymes, the limiting step is deacylation for penicillin hydrolysis, but acylation for cefotaxime hydrolysis. Further analyses of the G238S mutant show that the loss of activity against penicillins is due to a large decrease in the deacylation rate and that the increase in catalytic efficiency against cefotaxime is the result of a better Km and an increased acylation rate. These modifications of the elementary rate constants and the hydrolytic capacity in the G238S mutant could be linked to structural effects on the omega-loop conformation in the active site.

TEM-35 (inhibitor resistant TEM (IRT)-4) and TEM-36 (IRT-7) clavulanic acid-resistant beta-lactamases have evolved from TEM-1 beta-lactamase by two substitutions: a methionine to a leucine or a valine at position 69 and an asparagine to an aspartic acid at position 276. The substitutions at position 69 have previously been shown to be responsible for the resistance to clavulanic acid, and they are the only mutations encountered in TEM-33 (IRT-5) and TEM-34 (IRT-6). However, the N276D substitution has never been found alone in inhibitor-resistant beta-lactamases, and its role in resistance to clavulanic acid was thus unclear. The N276D mutant was constructed, purified, and kinetically characterized. It was shown that the substitution has a direct effect on substrate affinities and leads to slightly decreased catalytic efficiencies and that clavulanic acid becomes a poor substrate of the enzyme. Electrospray mass spectrometry demonstrated the simultaneous presence of free and inhibited enzymes after incubation with clavulanic acid and showed that a cleaved moiety of clavulanic acid leads to the formation of the major inactive complex. The kinetic properties of the N276D mutant could be linked to a salt-bridge interaction of aspartic acid 276 with arginine 244 that alters the electrostatic properties in the substrate binding area.

Background: Escherichia coli TEM1 is a penicillinase and belongs to class A beta-lactamases. Its naturally occurring mutants are responsible for bacterial resistance to beta-lactamin-based antibiotics. X-ray structure determinations show that all class A beta-lactamases are similar, but, despite the numerous kinetic investigations, the reaction mechanism of these enzymes is still debated. We address the questions of what the molecular contexts during the acylation and deacylation steps are and how they contribute to the efficiency of these penicillinases., Results: Electrostatic analysis of the 1.8 A resolution refined X-ray structure of the wild-type enzyme, and of its modelled Michaelis and acyl-enzyme complexes, showed that substrate binding induces an upward shift in the pKa of the unprotonated Lys73 by 6.4 pH units. The amine group of Lys73 can then abstract the Ser70 hydroxyl group proton and promote acylation. In the acyl-enzyme complex, the deacylating water is situated between the carboxylate group of Glu166, within the enzyme, and the estercarbonyl carbon of the acyl-enzyme complex, in an electrostatic potential gradient amounting to 30 kTe-1 A-1. Other residues, not directly involved in catalysis, also contribute to the formation of this gradient. The deacylation rate is related to the magnitude of the gradient. The kinetic behavior of site-directed mutants that affect the protonation state of residue 73 cannot be explained on the basis of the wild-type enzyme mechanism., Conclusions: In the wild-type enzyme, the very high rates of acylation and deacylation of class A beta-lactamases arise from an optimal chemical setup in which the acylation reaction seems triggered by substrate binding that changes the general base property of Lys73. In site-directed mutants where Lys73 is protonated, acylation may proceed through activation of a water molecule by Glu166, and Lys73 contributes as a proton shuffle partner in this pathway.

Residue 104 is frequently mutated from a glutamic acid to a lysine in the extended-spectrum TEM beta-lactamases responsible for the resistance to third-generation cephalosporins in clinical Gram negative strains. Among class A beta-lactamases, it is the most variable residue within a highly conserved loop which delineates one side of the active site of the enzymes. To investigate the role of this residue in the extended-spectrum phenotype, it has been replaced by serine, threonine, lysine, arginine, tyrosine and proline. All these substitutions yield active enzymes, with no drastic changes in kinetic properties compared with the wild-type enzyme, except with cefaclor, but an overall improved affinity for second- and third-generation cephalosporins. Only mutant E104K exhibits a significant ability to hydrolyse cefotaxime. Molecular modelling shows that the substitutions have generally no impact on the conformation of the 101-111 loop as the side chains of residues at position 104 are all turned towards the solvent. Unexpectedly, the E104P mutant turns out to be the most efficient enzyme. All our results argue in favour of an indirect role for this residue 104 in the substrate specificity of the class A beta-lactamases. This residue contributes to the precise positioning of residues 130-132 which are involved in substrate binding and catalysis. Changing residue 104 could also modify slightly the local electrostatic potential in this part of the active site. The limited kinetic impact of the mutations at this position have to be analysed in the context of the microbiological problem of resistance to third-generation cephalosporins. Although mutation E104K improves the ability of the enzyme to hydrolyse these compounds, it is not sufficient to confer true resistance, and is always found in clinical isolates associated with at least one mutation at another part of the active site. It is the combined effect of the two mutations that synergistically enhances the hydrolytic capability of the enzyme towards third-generation cephalosporins.

A method has been developed for introducing heterologous DNA rapidly and efficiently by electropermeabilization into the yeast Schwanniomyces occidentalis. A transformation efficiency as high as 2 x 10(5) transformants/microgram of plasmid DNA was obtained with a square-wave electric pulse of 2.17 kV/cm during 18 ms. Small quantities of DNA (5 ng) can be used to transform 3 x 10(8) cells. The main parameters which have been optimized are: presence of adenine in the culture medium, pretreatment of the cells with dithiothreitol during the exponential growth phase of the cells, amount of cells treated, and pulse-field strength and duration. Competent cells can be stored to allow electrotransformation whenever needed.

Heterologous expression in Escherichia coli is often limited by yield and solubility of the foreign protein in the bacterial cytoplasm. In many cases, overexpression also results in growth inhibition. In order to produce retroviral proteins that are especially difficult to overexpress in E. coli, we designed a set of beta-galactosidase fusion cassettes. Fusions with beta-galactosidase increase significantly both yield and solubility of the foreign proteins, thus making purification much easier. These cassettes allow for N- or C-terminal fusions, and the retroviral proteins can be released from the fusion by automaturation in vivo for the HIV-1 protease or cleavage by thrombine for Tat. More generally, any synthetic sequence coding for a given cleavage site can be introduced 5' or 3' to the lacZ gene through a convenient set of unique restriction sites, making these fusion cassettes highly versatile.

The antibiotic bleomycin, a strong DNA cutting agent, is naturally produced by actinomycetes which have developed a resistance mechanism against such a lethal compound. The crystal structure, at 2.3 A resolution, of a bleomycin resistance protein of 14 kDa reveals a structure in two halves with the same alpha/beta fold despite no sequence similarity. The crystal packing shows compact dimers with a hydrophobic interface and involved in mutual chain exchange. Two independent solution studies (analytical centrifugation and light scattering) showed that this dimeric form is not a packing artefact but is indeed the functional one. Furthermore, light scattering also showed that one dimer binds two antibiotic molecules as expected. A crevice located at the dimer interface, as well as the results of a site-directed mutagenesis study, led to a model wherein two bleomycin molecules are completely sequestered by one dimer. This provides a novel insight into antibiotic resistance due to drug sequestering, and probably also into drug transport and excretion.

From sequence alignments, two groups can be defined for the carbenicillin-hydrolysing beta-lactamases (CARB enzymes). One group includes the Pseudomonas-specific enzymes PSE-1, PSE-4, CARB-3, CARB-4 and also the Proteus mirabilis GN79, for which the well-conserved residue Lys 234 in all class-A beta-lactamases is changed to an arginine residue. The second group includes the enzymes PSE-3 and AER-1 which have an arginine or a lysine residue at position 165. All these enzymes also have leucine at position 68, threonine at position 104 and glycine at position 240. We engineered these mutations into the TEM-1 beta-lactamase to study their potential role in defining the substrate profile of the CARB enzymes. The mutations K234R and E240G in TEM-1 noticeably increased the hydrolysis of carboxypenicillins relative to other penicillins by approximately sixfold and twofold, respectively. The variant E240G also demonstrated an improved rate of second-generation cephalosporin and cefotaxime hydrolysis. In contrast, the substitution of Trp165 by arginine does not extend the substrate profile to alpha-carboxypenicillins nor does it noticeably modify the kinetic behavior of the enzyme. The mutations M68L and E104T do not have a large effect on the hydrolysis rate but the mutation E104T enhances the affinity of the enzyme for third-generation cephalosporins. As the mutation K234R resulted in a severe decrease in the affinity for carboxypenicillins, the double mutant E240G/K234R was constructed in an attempt to enhance the CARB character of the enzyme. Contrary to what could be expected, the additional mutation E240G for the TEM-1 K234R enzyme increases neither the catalytic constant for the carboxypenicillins nor the affinity towards these substrates. Consequently, this study strongly suggests that the three-dimensional structures of the active site of the TEM-1 enzyme and PSE-3, PSE-4 or other related enzymes are significantly different. This probably explains the discrepancy of the substrate profile between the CARB enzymes and the TEM-1 protein variants.

The X-ray structure of Escherichia coli TEM1 beta-lactamase has been refined to a crystallographic R-factor of 16.4% for 22,510 reflections between 5.0 and 1.8 A resolution; 199 water molecules and 1 sulphate ion were included in refinement. Except for the tips of a few solvent-exposed side chains, all protein atoms have clear electron density and refined to an average atomic temperature factor of 11 A2. The estimated coordinates error is 0.17 A. The substrate binding site is located at the interface of the two domains of the protein and contains 4 water molecules and the sulphate anion. One of these solvent molecules is found at hydrogen bond distance from S70 and E166. S70 and S130 are hydrogen bonded to K73 and K234, respectively. It was found that the E. coli TEM1 and Staphylococcus aureus PC1 beta-lactamases crystal structures differ in the relative orientations of the two domains composing the enzymes, which result in a narrowed substrate binding cavity in the TEM1 enzyme. Local but significant differences in the vicinity of this site may explain the occurrence of TEM1 natural mutants with extended substrate specificities.

Arginine 244 is a highly conserved residue in Class A beta-lactamases, while methionine 69 is not. Informational suppression experiments show that replacement of M69 by a leucine, or that of R244 by most other amino acids lead to clavulanic acid-resistant phenotypes. The arginyl 244 side chain is tightly held in a network of interactions within the active site. Its replacement by a glutamine or a threonine perturbs the enzyme kinetics but to a smaller extent than would have been predicted if it were directly involved in substrate binding. Clavulanic acid and sulbactam still interact specifically with the mutant enzymes but are much less efficiently metabolized. Substitutions at position 244 also unveil interactions between the C6 substituent of substrates and the Asn132/Glu104 region of the active site. Methionine 69 is located in a region of strong structural constraints and presents an unusual conformation. Molecular dynamics simulation showed that its replacement by a leucine does not release the strain in this area and induces only minor structural changes. Accordingly, the kinetic behavior of the mutant is only marginally perturbed, except for suicide inhibitors. Both clavulanic acid and sulbactam are well degraded by the mutant enzyme, while irreversible inactivation is dramatically decreased. The contribution of both residues to catalysis is discussed in the light of the kinetic and structural data.

The crystal structure of beta-lactamase TEM1 from E. coli has been solved to 2.5 A resolution by X-ray diffraction methods and refined to a crystallographic R-factor of 22.7%. The structure was determined by multiple isomorphous replacement using four heavy atom derivatives. The solution from molecular replacement, using a polyalanine model constructed from the C alpha coordinates of S. Aureus PCl enzyme, provided a set of phases used for heavy atom derivatives analysis. The E. coli beta-lactamase TEM1 is made up of two domains whose topology is similar to that of the PCl enzyme. However, global superposition of the two proteins shows significant differences.

A gene encoding an N-terminally extended precursor of 107 residues of the human immunodeficiency virus type 1 protease (PR107) was chemically synthesized and cloned into a bacterial expression vector, under the control of the araB promoter. PR107 was expressed alone or fused in phase to the amino or carboxy terminus of the bacterial beta-galactosidase (beta-gal). The yield of protease and beta-gal was found to be significantly higher when the gene for PR107 was cloned upstream of the Escherichia coli lacZ gene (PR107-beta-gal). Comparisons of the level of cloned protein expression between protease precursor and mature form suggested that this enhanced expression was due to the additional 5' sequence of the PR107 gene, and occurred at the post-transcriptional level. Autoprocessing of protease precursor and its release from the beta-gal fusion protein were analysed using wild-type and mutated cleavage sites. Mutations were introduced at amino acids downstream of the F-P scissile bond, at positions P4' and P5' in the C-terminal site (TLNF*PISP), and at position P3' in a consensus N-terminal site (TLNF*PQITL) placed at the protease-beta-gal junction. The data obtained suggested that (i) autoprocessing at the carboxy-terminal F-P bond was not significantly influenced by the presence of the N-terminal precursor sequence, (ii) P4' and P5' substitutions in the C-terminal site had no effect on cleavage, and (iii) P3' in the N-terminal site tolerated a wide variety of substitutions.

Two crystal forms of Gram- bacteria TEM beta-lactamase have been obtained. The tetragonal form has a very large unit cell and diffracts to 3.0 A resolution. Orthorhombic crystals, grown using ammonium sulfate and a small amount of acetone as precipitating agents, belong to space group P2(1)2(1)2(1) with cell parameters a = 43.1 A, b = 64.4 A, c = 91.2 A and diffract to 1.7 A resolution. A seeding procedure has been designed that ensures reproducibility of the crystal properties. Molecular replacement, using a model reconstructed from the C alpha co-ordinates from Staphylococcus aureus PC1 beta-lactamase, gives a solution that satisfies crystal packing constraints.

Class A beta-lactamases are the major cause of bacterial resistance to beta-lactam antibiotics. In these active-site serine hydrolases, glutamic acid 166 has been hypothesized to act as a general acid-base catalyst. Replacing this residue by tyrosine in TEM-1 beta-lactamase yields an enzyme the activity of which is substantially lowered and strongly dependent on pH, thus confirming the alleged role of Glu166 in catalysis. This substitution also resulted in a spectacular change in substrate profile, the mutant enzyme being more active on cephalosporins than on penicillins. In fact, the E166Y enzyme behaves much like a class C enzyme, with high affinity and low hydrolytic activity towards second and third generation cephalosporins. Glu166 therefore seems to play a major part in defining the substrate profile of class A beta-lactamases.

A set of 11 expression vectors was constructed, each of them harbouring a cloning cassette under the control of the araB promoter. Some of these vectors enable expression of foreign proteins in the cytoplasm, while others include a synthetic sequence coding for a very efficient secretion signal sequence. Other features are an f1 origin of replication (in plus or minus orientation) and a promoter(up) mutation that enhances the already very high level of expression from these vectors. With such a versatile vector family, cloning, sequencing and site-directed mutagenesis can be performed on the same vector, and the level of expression can be defined according to the specific constraints of a given protein.

Lysine 234 is a residue highly conserved in all beta-lactamases, except in the carbenicillin-hydrolyzing enzymes, in which it is replaced by an arginine. Informational suppression has been used to create amino acid substitutions at this position in the broad spectrum Escherichia coli beta-lactamase TEM-1, in order to elucidate the role of this residue which lies on the wall at the closed end of the active site cavity. The mutants K234R and K234T were constructed and their kinetic constants measured. Replacement of lysine 234 by arginine yields an enzyme with similar activity toward cephalosporins and most penicillins, except toward the carboxypenicillins for which the presence of the guanidine group enhances the transition state binding. The removal of the basic group in the mutant K234T yields a protein variant which retains a low activity toward penicillins, but losts drastically its ability to hydrolyze cephalosporins. Moreover, these two mutations largely decreased the affinity of the enzyme for penicillins (10-fold for K234R and 50-fold for K234T). This can be correlated with the disruption of the predicted electrostatic binding between the C3 carboxylic group of penicillins and the amine function of the lysine. Therefore, lysine 234 in the E. coli beta-lactamase TEM-1 is involved both in the initial recognition of the substrate and in transition state stabilization.

A full length tat gene was constructed by a combination of polymerase chain reaction (PCR) for the first exon and chemical synthesis for the second exon. This gene was expressed in E. coli under the control of the strongly regulated araB promoter, either directly or fused to a secretion signal encoding sequence. We then defined a rapid, three-step procedure for the purification of the Tat protein.