RESUMEN

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.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/metabolismo , Alelos , Proteínas Bacterianas/metabolismo , Endonucleasas/metabolismo , Mycobacterium/enzimología , Mycobacterium/genética , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/aislamiento & purificación , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Secuencia de Bases , Tampones (Química) , Clonación Molecular , Secuencia Conservada , Endonucleasas/genética , Endonucleasas/aislamiento & purificación , Evolución Molecular , Transferencia de Gen Horizontal/genética , Genes Bacterianos/genética , Concentración de Iones de Hidrógeno , Modelos Genéticos , Datos de Secuencia Molecular , Recombinación Genética/genética , Alineación de Secuencia , Especificidad por Sustrato

RESUMEN

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.

Asunto(s)

Proteínas Bacterianas/genética , Girasa de ADN/genética , Genoma Bacteriano , Mycobacterium/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Girasa de ADN/química , Girasa de ADN/metabolismo , Datos de Secuencia Molecular , Mycobacterium/metabolismo , Empalme de Proteína , Rec A Recombinasas/genética , Alineación de Secuencia

RESUMEN

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.

Asunto(s)

Proteínas Bacterianas , Proteínas Portadoras , Citoplasma/enzimología , Proteínas de Escherichia coli , Escherichia coli/enzimología , Hexosiltransferasas/química , Hexosiltransferasas/metabolismo , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Muramoilpentapéptido Carboxipeptidasa , Peptidoglicano Glicosiltransferasa , Peptidil Transferasas/química , Peptidil Transferasas/metabolismo , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina , Secuencia de Aminoácidos , Antibacterianos/farmacología , Aztreonam/farmacología , Cefaloridina/farmacología , Cefalosporinas/farmacología , Dimerización , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Hexosiltransferasas/genética , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Complejos Multienzimáticos/genética , Mutación , Proteínas de Unión a las Penicilinas , Peptidil Transferasas/genética

RESUMEN

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.

Asunto(s)

Catálisis , Técnicas Químicas Combinatorias , Bases de Datos Genéticas , Evolución Molecular Dirigida , Bacterias/clasificación , Bacterias/enzimología , Bacterias/genética , Bacterias/metabolismo , Biotecnología/métodos , Candida/enzimología , Candida/genética , Industrias , Thermococcus/enzimología , Thermococcus/genética

Asunto(s)

Acetiltransferasas/química , Acetiltransferasas/metabolismo , Bleomicina/química , Zinc/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Bleomicina/metabolismo , Isótopos de Carbono , Escherichia coli , Hidrógeno , Isótopos de Nitrógeno , Resonancia Magnética Nuclear Biomolecular/métodos , Conformación Proteica , Zinc/metabolismo

RESUMEN

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.

Asunto(s)

ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Empalme de Proteína , Thermococcus/enzimología , Alelos , Secuencias de Aminoácidos , Secuencia de Bases , Secuencia de Consenso , Secuencia Conservada , ADN Polimerasa I/química , ADN Polimerasa I/genética , ADN Polimerasa I/metabolismo , ADN Polimerasa Dirigida por ADN/química , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes , Mutación Puntual , Estructura Terciaria de Proteína , Pyrococcus/enzimología , Pyrococcus/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Eliminación de Secuencia , Especificidad por Sustrato , Temperatura , Thermococcus/genética

RESUMEN

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.

Asunto(s)

Intrones , Mycobacterium/genética , Rec A Recombinasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Bacteriano , Genes Bacterianos , Datos de Secuencia Molecular , Mutagénesis

RESUMEN

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.

Asunto(s)

Receptores Opioides mu/fisiología , Saccharomyces cerevisiae/metabolismo , Esteroles/metabolismo , Colesterol/metabolismo , Ergosterol/metabolismo , Proteínas de Unión al GTP/fisiología , Humanos , Lípidos de la Membrana/análisis , Lípidos de la Membrana/fisiología , Conformación Proteica , Receptores Opioides mu/química , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética

RESUMEN

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.

Asunto(s)

Carboxiliasas/genética , Plásmidos/genética , Secuencias Reguladoras de Ácidos Nucleicos , Saccharomycetales/genética , Regiones no Traducidas 5' , Secuencia de Bases , Replicación del ADN , Regulación Fúngica de la Expresión Génica , Datos de Secuencia Molecular , Origen de Réplica , Saccharomyces cerevisiae/genética

RESUMEN

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.

Asunto(s)

ADN Polimerasa Dirigida por ADN/genética , Endonucleasas/genética , Thermococcus/enzimología , Secuencia de Bases , ADN Polimerasa Dirigida por ADN/metabolismo , Endonucleasas/antagonistas & inhibidores , Endonucleasas/metabolismo , Escherichia coli/genética , Inhibidores de la Síntesis del Ácido Nucleico , Empalme del ARN , Especificidad por Sustrato

RESUMEN

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.

Asunto(s)

Proteínas de Unión al GTP/metabolismo , Receptores Opioides mu/agonistas , Receptores Opioides mu/antagonistas & inhibidores , Sitios de Unión , Unión Competitiva , Buprenorfina/metabolismo , Diprenorfina/metabolismo , Encefalina Ala(2)-MeFe(4)-Gli(5) , Encefalinas/metabolismo , Etorfina/metabolismo , Expresión Génica/genética , Guanilil Imidodifosfato/metabolismo , Humanos , Unión Proteica , Isoformas de Proteínas/genética , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , Esferoplastos/metabolismo , Transformación Genética

RESUMEN

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.

Asunto(s)

Proteínas Bacterianas , Proteínas Portadoras , Cisteína , Disulfuros , Proteínas de Escherichia coli , Hexosiltransferasas/química , Hexosiltransferasas/metabolismo , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Muramoilpentapéptido Carboxipeptidasa , Peptidoglicano Glicosiltransferasa , Peptidil Transferasas/química , Peptidil Transferasas/metabolismo , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina , Cisteína/genética , Dimerización , Escherichia coli , Hexosiltransferasas/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Complejos Multienzimáticos/genética , Mutación , Proteínas de Unión a las Penicilinas , Peptidoglicano , Peptidil Transferasas/genética , Desnaturalización Proteica

RESUMEN

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.

Asunto(s)

Resistencia a Medicamentos , Xenobióticos/uso terapéutico , Animales , Antibacterianos/farmacología , Antimaláricos/farmacología , Antineoplásicos/farmacología , Reparación del ADN , Resistencia a Múltiples Medicamentos , Humanos , Inactivación Metabólica

RESUMEN

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.

Asunto(s)

Alanina/metabolismo , Proteínas Bacterianas , Proteínas Portadoras , Proteínas de Escherichia coli , Escherichia coli/enzimología , Hexosiltransferasas/metabolismo , Complejos Multienzimáticos/metabolismo , Muramoilpentapéptido Carboxipeptidasa , Peptidoglicano Glicosiltransferasa , Peptidil Transferasas/metabolismo , D-Ala-D-Ala Carboxipeptidasa de Tipo Serina , Sitios de Unión , Clonación Molecular , Hexosiltransferasas/biosíntesis , Hexosiltransferasas/genética , Complejos Multienzimáticos/biosíntesis , Complejos Multienzimáticos/genética , Mutagénesis Sitio-Dirigida , Proteínas de Unión a las Penicilinas , Peptidil Transferasas/biosíntesis , Peptidil Transferasas/genética

RESUMEN

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.

Asunto(s)

Selectina E/biosíntesis , Endotelio Vascular/metabolismo , Productos del Gen tat/farmacología , VIH-1 , Molécula 1 de Adhesión Intercelular/biosíntesis , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Cicloheximida/farmacología , Sinergismo Farmacológico , Endotelio Vascular/efectos de los fármacos , Citometría de Flujo , Humanos , Inhibidores de la Síntesis de la Proteína/farmacología , Espectrometría de Fluorescencia , Propiedades de Superficie , Factor de Necrosis Tumoral alfa/farmacología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana

RESUMEN

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.

Asunto(s)

Ensayo de Inmunoadsorción Enzimática/métodos , Inhibidores de la Proteasa del VIH/farmacología , Proteasa del VIH/análisis , VIH-1/enzimología , Aminoácidos/análisis , Biotina , Cromatografía Líquida de Alta Presión , Evaluación de Medicamentos , Péptidos/síntesis química , Péptidos/metabolismo

RESUMEN

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.

Asunto(s)

Acetiltransferasas , Dimerización , Prolina/fisiología , Conformación Proteica , Pliegue de Proteína , Secuencia de Aminoácidos , Animales , Aspartato Aminotransferasas/química , Proteínas Bacterianas/química , Bovinos , Pollos , Humanos , Mitocondrias Cardíacas/enzimología , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Virus de Plantas/química , Unión Proteica , Pirofosfatasas/química , Ribonucleasa Pancreática/química , Alineación de Secuencia , ATPasa Intercambiadora de Sodio-Potasio/química , Proteínas Estructurales Virales/química

RESUMEN

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.

Asunto(s)

Alanina/metabolismo , Farmacorresistencia Microbiana/genética , Mutagénesis/genética , Proteínas/química , Alanina/genética , Ampicilina/farmacología , Cloranfenicol/farmacología , Codón/genética , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Escherichia coli/genética , Oligodesoxirribonucleótidos/síntesis química , Oligodesoxirribonucleótidos/genética , Transformación Genética/genética

Asunto(s)

Clonación Molecular/métodos , Escherichia coli/genética , Secuencias Repetitivas de Ácidos Nucleicos , Secuencia de Bases , ADN Bacteriano , Datos de Secuencia Molecular

RESUMEN

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.

Asunto(s)

beta-Lactamasas/química , Secuencia de Aminoácidos , Secuencia de Bases , Cristalografía por Rayos X , Cinética , Espectrometría de Masas , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Conformación Proteica , beta-Lactamasas/genética , beta-Lactamasas/metabolismo