Biomedical subjects

B Joris

Publications and source records attributed to B Joris.

At least 19 recordsLinked to original sources

PubMed2001

Characterization of two genes encoding the mitochondrial alternative oxidase in Chlamydomonas reinhardtii.

Two cDNA clones (AOX1 and AOX2) and the corresponding genes encoding the alternative oxidases (AOXs) from Chlamydomonas reinhardtii were isolated and sequenced. The cDNAs, AOX1 and AOX2, contained open reading frames (ORFs) encoding putative proteins of 360 amino acids and 347 amino acids, respectively. For each of the ORFs, a potential mitochondrial-targeting sequence was found in the 5'-end regions. In comparison to AOX enzymes from plants and fungi, the predicted amino acid sequences of the ORFs showed their highest degree of identity with proteins from Aspergillus niger (38.1% and 37.2%) and Ajellomyces capsulatus (37% and 34.9%). Several residues supposed either to be Fe ligands or to be involved in the ubiquinol-binding site were fully conserved in both C. reinhardtii putative AOX proteins. In contrast, a cysteine residue conserved in the sequences of all higher plants and probably involved in the regulation of the enzyme activity was missing both from the AOX1 and AOX2 amino acid sequences and from protein sequences from various other microorganisms. The transcriptional expression of the AOX1 and AOX2 genes in wild-type cells and in mutant cells deficient in mitochondrial complex III activity was also investigated.

Amino Acid Sequence↗

PubMed2001

Use of an ALFexpress DNA sequencer to analyze protein-nucleic acid interactions by band shift assay.

Gel retardation analysis, or band shift assay, is technically the simplest method to investigate protein-nucleic acid interactions. In this report, we describe a nonradioactive band shift assay using a fluorescent DNA target and an ALFexpress automatic DNA sequencer in place of the current method that utilizes radioactively end-labeled DNA target and a standard electrophoresis unit. In our study, the dsDNA targets were obtained by annealing two synthetic oligonucleotides or by PCR. In both cases, a molecule of indodicarbocyanine (CY5) was attached at the 5' OH end of one of the two synthetic oligonucleotides, with a ratio of one molecule of fluorescent dye per molecule of dsDNA. To demonstrate the feasibility of this new band shift assay method, the DNA-binding proteins selected as models were the BlaI and AmpR repressors, which are involved in the induction of the Bacillus licheniformis 749/I and Citrobacter freundii beta-lactamases, respectively. The results show that the use of an automatic DNA sequencer allows easy gel retardation analysis and provides a fast, sensitive, and quantitative method. The ALFexpress DNA sequencer has the same limit of detection as a laser fluorescence scanner and can be used instead of a FluorImager or a Molecular Imager.

Autoanalysis↗

PubMed2000

Crystal structure of a D-aminopeptidase from Ochrobactrum anthropi, a new member of the 'penicillin-recognizing enzyme' family.

BACKGROUND: beta-Lactam compounds are the most widely used antibiotics. They inactivate bacterial DD-transpeptidases, also called penicillin-binding proteins (PBPs), involved in cell-wall biosynthesis. The most common bacterial resistance mechanism against beta-lactam compounds is the synthesis of beta-lactamases that hydrolyse beta-lactam rings. These enzymes are believed to have evolved from cell-wall DD-peptidases. Understanding the biochemical and mechanistic features of the beta-lactam targets is crucial because of the increasing number of resistant bacteria. DAP is a D-aminopeptidase produced by Ochrobactrum anthropi. It is inhibited by various beta-lactam compounds and shares approximately 25% sequence identity with the R61 DD-carboxypeptidase and the class C beta-lactamases. RESULTS: The crystal structure of DAP has been determined to 1.9 A resolution using the multiple isomorphous replacement (MIR) method. The enzyme folds into three domains, A, B and C. Domain A, which contains conserved catalytic residues, has the classical fold of serine beta-lactamases, whereas domains B and C are both antiparallel eight-stranded beta barrels. A loop of domain C protrudes into the substrate-binding site of the enzyme. CONCLUSIONS: Comparison of the biochemical properties and the structure of DAP with PBPs and serine beta-lactamases shows that although the catalytic site of the enzyme is very similar to that of beta-lactamases, its substrate and inhibitor specificity rests on residues of domain C. DAP is a new member of the family of penicillin-recognizing proteins (PRPs) and, at the present time, its enzymatic specificity is clearly unique.

Amino Acid Sequence↗

PubMed2000

A new variant of the Ntn hydrolase fold revealed by the crystal structure of L-aminopeptidase D-ala-esterase/amidase from Ochrobactrum anthropi.

BACKGROUND: The L-aminopeptidase D-Ala-esterase/amidase from Ochrobactrum anthropi (DmpA) releases the N-terminal L and/or D-Ala residues from peptide substrates. This is the only known enzyme to liberate N-terminal amino acids with both D and L stereospecificity. The DmpA active form is an alphabeta heterodimer, which results from a putative autocatalytic cleavage of an inactive precursor polypeptide. RESULTS: The crystal structure of the enzyme has been determined to 1.82 A resolution using the multiple isomorphous replacement method. The heterodimer folds into a single domain organised as an alphabetabetaalpha sandwich in which two mixed beta sheets are flanked on both sides by two alpha helices. CONCLUSIONS: DmpA shows no similarity to other known aminopeptidases in either fold or catalytic mechanism, and thus represents the first example of a novel family of aminopeptidases. The protein fold of DmpA does, however, show structural homology to members of the N-terminal nucleophile (Ntn) hydrolase superfamily. DmpA presents functionally equivalent residues in the catalytic centre when compared with other Ntn hydrolases, and is therefore likely to use the same catalytic mechanism. In spite of this homology, the direction and connectivity of the secondary structure elements differ significantly from the consensus Ntn hydrolase topology. The DmpA structure thus characterises a new subfamily, but supports the common catalytic mechanism for these enzymes suggesting an evolutionary relationship.

Aminopeptidases↗

PubMed2000

Technique for a rapid and efficient purification of the SHV-1 and PSE-2 beta-lactamases.

A simple procedure is described which results in an optimised resolution in molecular sieve chromatography. A sample exhibiting a large initial volume (about 20 ml) and conditioned in a buffer of low ionic strength (<20 mM) by filtration through a 53-ml G25 molecular sieve column, is adsorbed on a 1.7-ml ion-exchange (SOURCE) column. The proteins are released by a 10-ml pulse of 1 M NaCl and the eluate directly injected onto a 120-ml Sephacryl S100-HR column. The very low volume of the eluate ensures optimal conditions and resolution for the molecular sieving process. The method is applied as the polishing step in the purification of the SHV-1 and PSE-2 beta-lactamases. It could easily be scaled up for the treatment of larger samples.

Chromatography, Gel↗

PubMed2000

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.

Amino Acid Sequence↗

PubMed1999

The DmpA aminopeptidase from Ochrobactrum anthropi LMG7991 is the prototype of a new terminal nucleophile hydrolase family.

The DmpA (d-aminopeptidase A) protein produced by Ochrobactrum anthropi hydrolyses p-nitroanilide derivatives of glycine and d-alanine more efficiently than that of l-alanine. When regular peptides are utilized as substrates, the enzyme behaves as an aminopeptidase with a preference for N-terminal residues in an l configuration, thus exemplifying an interesting case of stereospecificity reversal. The best-hydrolysed substrate is l-Ala-Gly-Gly, but tetra- and penta-peptides are also efficiently hydrolysed. The gene encodes a 375-residue precursor, but the active enzyme contains two polypeptides corresponding to residues 2-249 (alpha-subunit) and 250-375 (beta-subunit) of the precursor. Residues 249 and 250 are a Gly and a Ser respectively, and various substitutions performed by site-directed mutagenesis result in the production of an uncleaved and inactive protein. The N-terminal Ser residue of the beta-subunit is followed by a hydrophobic peptide, which is predicted to form a beta-strand structure. All these properties strongly suggest that DmpA is an N-terminal amidohydrolase. An exploration of the databases highlights the presence of a number of open reading frames encoding related proteins in various bacterial genomes. Thus DmpA is very probably the prototype of an original family of N-terminal hydrolases.

Amidohydrolases↗

PubMed1999

Two new aminopeptidases from Ochrobactrum anthropi active on D-alanyl-p-nitroanilide.

Two new enzymes which hydrolyse D-alanyl-p-nitroanilide have been detected in Ochrobactrum anthropi LMG7991 extracts. The first enzyme, DmpB, was purified to homogeneity and found to be homologous to the Dap protein produced by O. anthropi SCRC C1-38 (ATCC49237). The second enzyme, DmpA, exhibits a similar substrate profile when tested on p-nitroanilide derivatives of glycine and L/D-alanine, but the amounts produced by the Ochrobactrum strain were not sufficient to allow complete purification. Interestingly, the DmpA preparation also exhibited an L-aminopeptidase activity on the tripeptide L-Ala-Gly-Gly but it was not possible to be certain that the same protein was responsible for both p-nitroanilide and peptide hydrolysing activities. The gene encoding the DmpA protein was cloned and sequenced. The deduced protein sequence exhibits varying degrees of similarity with those corresponding to several open reading frames found in the genomes of other prokaryotic organisms, including Mycobacteria. None of these gene products has been isolated or characterised, but a tentative relationship can be proposed with the NylC amidase from Flavobacterium sp. K172.

Amino Acid Sequence↗

PubMed1999

Crystallization and preliminary X-ray analysis of a new L-aminopeptidase-D-amidase/D-esterase activated by a Gly-Ser peptide bond hydrolysis.

Ochrobactrum anthropi possesses an L-aminopeptidase (DmpA) also able to act as a D-amidase/D-esterase. DmpA (40 kDa) is activated by auto-catalyzed protein splicing liberating an alpha-amino group presumably used as a general base in the catalytic mechanism. Two crystal forms were obtained at 294 K in 13-16% PEG 2000 mono-methylether at pH 9.0, adding either 0.2 M magnesium chloride or 1 M lithium chloride. Crystals of the first form belong to the space group C2221 and diffract to 3.0 A resolution, whereas crystals of the second form belong to the space group P21212 and diffract to 2.3 A resolution. Initial screening for heavy-atom derivatives on form II crystals, has led to a well substituted Hg derivative.

Aminopeptidases↗

PubMed1998

The diversity, structure and regulation of beta-lactamases.

beta-Lactamase production is responsible for the appearance of a large number of pathogenic bacterial strains exhibiting a high degree of resistance to beta-lactam antibiotics. A large number of enzymes have been described with very diverse primary structures and catalytic profiles. Nevertheless, all known three-dimensional structures of active-site serine beta-lactamases exhibit a high degree of similarity with apparently equivalent chemical functionalities in the same strategic positions. These groups might not, however, play identical roles in the various classes of enzymes. Structural data have also been recently obtained for the zinc metallo-beta-lactamases, but the detailed catalytic mechanisms might also differ widely, depending on the enzyme studied. Similarly, the induction of the synthesis of beta-lactamases is now better understood, but many questions remain to be answered.

Gene Expression Regulation, Bacterial↗

PubMed1997

The complete genome sequence of the gram-positive bacterium Bacillus subtilis.

Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.

Bacillus subtilis↗

PubMed1997

Use of an automatic DNA sequencer for S1 mapping: transcriptional analysis of the Streptomyces coelicolor A3(2) dnaK operon.

The transcription start point of the dnaK operon of Streptomyces coelicolor A3(2) has been determined by S1 mapping, using the EMBL automated fluorescent DNA sequencer. The -35 and -10 hexamers correspond to a sigma 70-type promoter. This promoter responds to heat shock and involves an inverted repeat different from the CIRCE sequence characteristic of the Gram-positive heat-shock promoters.

Base Sequence↗

PubMed1997

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

The catalytic pathway of class A beta-lactamases involves an acyl-enzyme intermediate where the substrate is ester-linked to the Ser-70 residue. Glu-166 and Lys-73 have been proposed as candidates for the role of general base in the activation of the serine OH group. The replacement of Glu-166 by an asparagine in the TEM-1 and by a histidine in the Streptomyces albus G beta-lactamases yielded enzymes forming stable acyl-enzymes with beta-lactam antibiotics. Although acylation of the modified proteins by benzylpenicillin remained relatively fast, it was significantly impaired when compared to that observed with the wild-type enzyme. Moreover, the E166N substitution resulted in a spectacular modification of the substrate profile much larger than that described for other mutations of Omega-loop residues. Molecular modeling studies indicate that the displacement of the catalytic water molecule can be related to this observation. These results confirm the crucial roles of Glu-166 and of the "catalytic" water molecule in both the acylation and the deacylation processes.

Acylation↗

PubMed1997

The two beta-lactamase genes of Streptomyces cacaoi, blaL and blaU, are under the control of the same regulatory system.

The production of beta-lactamase in Streptomyces cacaoi, which contains two beta-lactamase-encoding genes, blaL and blaU, is inducible by beta-lactam compounds. The two genes have been cloned independently in S. lividans TK24, a beta-lactamase-negative species. The blaU clone did not respond to the presence of beta-lactams, whereas the blaL clone appeared to be inducible in S. lividans. The latter clone contains two open reading frames, blaA and blaB, located just upstream of but transcribed divergently from blaL, which were shown to be required for the production as well as the induction of BlaL. The deduced BlaA protein belongs to the LysR family of transcription regulators. In order to examine the role of BlaA in regulation, we here report on over-expression of a GST-BlaA fusion protein in Escherichia coli and its use for antibody preparation. The GST-BlaA fusion protein was partially purified and bandshift assays showed that it bound the 197-bp blaL-blaA intergenic region. The BlaA DNA binding-site was further restricted to a 30-bp sequence containing a T-N11-A motif, a characteristic of LysR-type promoters. Another T-N11-A motif upstream of the blaU gene was also shown to bind BlaA. The affinities of these two T-N11-A motifs in BlaA binding were comparable. A plasmid bearing the blaU structural gene and the blaA-blaB regulatory region was constructed and shown to confer on an S. lividans host the capacity to produce inducible beta-lactamase. It can thus be concluded that the S. cacaoi blaL and blaU genes are controlled by the same regulatory system.

Bacterial Proteins↗

PubMed1997

The penicillin sensory transducer, BlaR, involved in the inducibility of beta-lactamase synthesis in Bacillus licheniformis is embedded in the plasma membrane via a four-alpha-helix bundle.

Prediction studies, conformational analyses and membrane-topology mapping lead to the conclusion that the penicillin sensory transducer, BlaR, involved in the inducibility of beta-lactamase synthesis in Bacillus licheniformis, is embedded in the plasma membrane bilayer via four transmembrane segments TM1-TM4 that form a four-alpha-helix bundle. The extracellular 262-amino-acid-residue polypeptide, S340-R601, that is fused at the carboxy end of TM4, possesses the amino acid sequence signature of a penicilloyl serine transferase. It probably functions as penicillin sensor. As an independent entity, this polypeptide behaves as a high-affinity penicillin-binding protein. As a component of the full-size BlaR, it adopts a different conformation presumably because of interactions with the extracellular 63-amino-acid-residue P53-S115 loop that connects TM2 and TM3. Reception of the penicillin-induced signal requires a precise conformation of the sensor but it does not involve penicilloylation of the serine residue S402 of motif STYK. Signal transmission through the plasma membrane by the four-alpha-helix bundle may proceed in a way comparable to that of the aspartate receptor, Tar. Signal emission in the cytosol by the intracellular 189-amino-acid-residue Y134-K322 loop that connects TM3 and TM4, may proceed via the activation of a putative metallopeptidase.

Amino Acid Sequence↗

PubMed1997

Dual multimodular class A penicillin-binding proteins in Mycobacterium leprae.

The ponA gene of cosmid L222 of the Mycobacterium leprae genome library encodes a multimodular class A penicillin-binding protein (PBP), PBP1. The PBP, labelled with a polyhistidine sequence, has been produced in Escherichia coli, extracted from the membranes with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS) and purified by Ni2(+)-nitrilotriacetic acid-agarose chromatography. In contrast to the pon1-encoded class A PBP1, PBP1 undergoes denaturation at temperatures higher than 25 degrees C, it catalyzes acyl transfer reactions on properly structured thiolesters, and it binds penicillin with high affinity.

Acylation↗

PubMed1996

A sequence-specific DNA-binding protein interacts with the xlnC upstream region of Streptomyces sp. strain EC3.

The alignment of the promoter region of several Streptomyces xylanases shows three conserved sequences which could be involved in gene regulation. By electromobility shift assays these specific sequences, present only in Streptomyces xylanolytic strains, were identified as protein-binding sites. The sequence required for efficient recognition by the retarding protein appeared to be a 4-bp inverted repeat: 5'-CTTT-Nx-AAAG-3'. The DNA-protein affinity was influenced by the culture conditions.

Bacterial Proteins↗

PubMed1996

Selection of hammerhead ribozymes for optimum cleavage of interleukin 6 mRNA.

Four GUC triplets in the coding region of the MRNA of interleukin 6 (IL-6) were examined for their suitabilty to serve as a target for hammerhead ribozome-mediated cleavage. This selection procedure was performed with the intention to downregulate IL-6 production as a potential treatment of those diseases in which IL-6 overexpression is involved. Hammerhead ribozymes and their respective short synthetic substrates (19-mers) were synthesized for these four GUC triplets. Notwithstanding the identical catalytic core sequences, the difference in base composition of the helices involved in substrate binding caused substantial variation in cleavage activity. The cleavage reactions on the 1035 nucleotide IL-6 mRNA transcript revealed that two ribozymes were able to cleave this substrate, showing a decrease in catalytic efficiency to 1/30 and 1/300 of the short substrate. This study indicates that the GUC triplet located at nucleotide 510 of the mRNA of IL-6 is the best site for hammerhead ribozyme-mediated cleavage. We suggest that in future targeting of chemically modified hammerhead ribosomes for cleavage of IL-6 RNA should be directed at this location.

Base Sequence↗