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H Schwab

Publications and source records attributed to H Schwab.

At least 37 records · Page 2Linked to original sources
PubMed1996

Cloning and characterization of the gene for the thermostable xylanase XynA from Thermomyces lanuginosus.

A thermostable xylanase from the filamentous fungus Thermomyces lanuginosus (DSM 5826) was purified. This enzyme has an apparent molecular weight of 24-26 kDa as determined by SDS polyacrylamide gel electrophoresis. cDNA and genomic DNA fragments coding for this enzyme were cloned and sequenced. The cDNA contains an open reading frame encoding a polypeptide of 225 amino acids and was functionally expressed in E. coli as a LacZ fusion protein. Comparison of the cDNA sequence with the genomic DNA sequence showed that the xylanase was encoded by two exons interrupted by an intron of 106 bp. Comparison of the deduced amino acid sequence to other published xylanases revealed high homology to xylanases of the family G glycanases.

Amino Acid Sequence↗
PubMed1996

Mechanism of cyanogenesis: the crystal structure of hydroxynitrile lyase from Hevea brasiliensis.

BACKGROUND: Over three thousand species of plants, including important food crops such as cassava, use cyanogenesis, the liberation of HCN upon tissue damage, as a defense against predation. Detoxification of cyanogenic food crops requires disruption of the cyanogenic pathway. Hydroxynitrile lyase is one of the key enzymes in cyanogenesis, catalyzing the decomposition of an alpha-cyanohydrin to form HCN plus the corresponding aldehyde or ketone. These enzymes are also of potential utility for industrial syntheses of optically pure chiral cyanohydrins, being used to catalyze the reverse reaction. We set out to gain insight into the catalytic mechanism of this important class of enzymes by determining the three-dimensional structure of hydroxynitrile lyase from the rubber tree, Hevea brasiliensis. RESULTS: The crystal structure of the enzyme has been determined to 1.9 A resolution. It belongs to the alpha/beta hydrolase superfamily, with an active site that is deeply buried within the protein and connected to the outside by a narrow tunnel. The catalytic triad is made up of Ser80, His235 and Asp207. By analogy with known mechanisms of other members of this superfamily, catalysis should involve an oxyanion hole formed by the main chain NH of Cys81 and the side chains of Cys81 and Thr11. Density attributed to a histidine molecule or ion is found in the active site. CONCLUSIONS: By analogy with other alpha/beta hydrolases, the reaction catalyzed by hydroxynitrile lyase involves a tetrahedral hemiketal or hemiacetal intermediate formed by nucleophilic attack of Ser80 on the substrate, stabilized by the oxyanion hole. The SH group of Cys81 is probably involved in proton transfer between the HCN and the hydroxynitrile OH. This mechanism is significantly different from the corresponding uncatalyzed solution reaction.

Aldehyde-Lyases↗
PubMed1996

Crystallization and preliminary X-ray diffraction studies of a hydroxynitrile lyase from Hevea brasiliensis.

Crystals of the hydroxynitrile lyase from Hevea brasiliensis overexpressed in Pichia pastoris have been obtained by the hanging-drop technique at 294 K with ammonium sulfate and PEG 400 as precipitants. The crystals belong to the orthorhombic space group C222(1) with cell dimensions of a = 47.6, b = 106.8 and c = 128.2 A. The crystals diffract to about 2.5 A resolution on a rotating-anode X-ray source.

Journal Article↗
PubMed1996

Molecular cloning of the full-length cDNA of (S)-hydroxynitrile lyase from Hevea brasiliensis. Functional expression in Escherichia coli and Saccharomyces cerevisiae and identification of an active site residue.

The full-length cDNA of (S)-hydroxynitrile lyase (Hnl) from leaves of Hevea brasiliensis (tropical rubber tree) was cloned by an immunoscreening and sequenced. Hnl from H. brasiliensis is involved in the biodegradation of cyanogenic glycosides and also catalyzes the stereospecific synthesis of aliphatic, aromatic, and heterocyclic cyanohydrins, which are important as precursors for pharmaceutical compounds. The open reading frame identified in a 1. 1-kilobase cDNA fragment codes for a protein of 257 amino acids with a predicted molecular mass of 29.2 kDa. The derived protein sequence is closely related to the (S)-hydroxynitrile lyase from Manihot esculenta (Cassava) and also shows significant homology to two proteins of Oryza sativa with as yet unknown enzymatic function. The H. brasiliensis protein was expressed in Escherichia coli and Saccharomyces cerevisiae and isolated in an active form from the respective soluble fractions. Replacement of cysteine 81 by serine drastically reduced activity of the heterologous enzyme, suggesting a role for this amino acid residue in the catalytic action of Hnl.

Aldehyde-Lyases↗
PubMed1996

Molecular cloning and homology modeling of protocatechuate 3,4-dioxygenase from Pseudomonas marginata.

The genes that encode the alpha and beta subunits of protocatechuate 3,4-dioxygenase (3,4-PCD [EC 1.13.11.3]) were cloned from a Pseudomonas marginata genomic library. These genes pcaG and pcaH, were found when screening the library for hydrolase genes. The two open reading frames of the PCD genes could be identified adjacent to an esterase gene by sequence homology. A 1.7-kb KpnI/ApaI fragment, carrying pcaG and pcaH, was subcloned and the genes were functionally expressed in Escherichia coli. The deduced amino acid sequence shows high homology to previously determined amino acid sequences of bacterial protocatechuate 3,4-dioxygenases. A homology model based on the available crystal structure of the protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa shows high similarity with the binding and catalytic sites.

Amino Acid Sequence↗
PubMed1995

Efficient secretion of Bacillus subtilis levanase by Saccharomyces cerevisiae.

The secretion of Bacillus subtilis (Bs) levanase (Lev) was studied in the yeast Saccharomyces cerevisiae. A set of different yeast expression plasmids, based on the constitutive PGK promoter and harbouring the Bs Lev-encoding gene (sacC), was constructed. In these plasmids, the original Bs signal sequence was either intact, partially deleted or entirely missing. With all constructs, Lev was produced from yeast transformants. However, only when the intact bacterial signal peptide was present was the synthesized enzyme secreted; around 20% was found in the periplasm and 30% in the culture medium. The secreted protein found in the periplasmic space was mainly core-glycosylated and unglycosylated, and had a size of 80-90 and 74 kDa, respectively. In contrast, Lev found in the culture medium was mainly hyper-glycosylated and had a size of 180-200 kDa. Yeast transformants harbouring sacC, but lacking parts of the bacterial signal sequence, only produced cytoplasmic protein which was not glycosylated and had a size of about 74 kDa. The deletion of the entire signal peptide and a further 22 amino acids at the N terminus of mature Lev resulted in a 71-kDa cytoplasmic protein which was not active.

Bacillus subtilis↗
PubMed1995

Comparison of ccd of F, parDE of RP4, and parD of R1 using a novel conditional replication control system of plasmid R1.

A number of plasmid-encoded gene systems are thought to stabilize plasmids by killing plasmid-free cells (also termed post-segregational killing or plasmid addiction). Here we analyse the mechanisms of plasmid stabilization by ccd of F, parDE of RP4 and parD of R1, and compare them to hok/sok of R1. To induce synchronous plasmid loss we constructed a novel plasmid replication-arrest system, which possesses the advantage that plasmid replication can be completely arrested by the addition of IPTG, a non-metabolizable inducer. Using isogenic plasmid constructions we have found, for the first time, consistent correlation between the effect on steady-state loss rates and the effect on cell proliferation in the plasmid replication-arrest assay for all three systems. The parDE system had the most pronounced effect both on plasmid stabilization and on plasmid retention after replication arrest. In contrast, ccd and parD both exhibited weaker effects than anticipated from previously published results. Thus, our results indicate that the function and efficiencies of some of the systems should be reconsidered. Our results are consistent with the previously postulated hypothesis that ccd and parDE act by killing plasmid-free segregants, whereas parD seems to act by inhibiting cell division of plasmid-free segregants.

Bacterial Proteins↗
PubMed1995

Purification and characterization of the Bacillus subtilis levanase produced in Escherichia coli.

The enzyme levanase encoded by the sacC gene from Bacillus subtilis was overexpressed in Escherichia coli with the strong, inducible tac promoter. The enzyme was purified from crude E. coli cell lysates by salting out with ammonium sulfate and chromatography on DEAE-Sepharose CL-6B, S-Sepharose, and MonoQ-Sepharose. The purified protein had an apparent molecular mass of 75,000 Da in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which is in agreement with that expected from the nucleotide sequence. Levanase was active on levan, inulin, and sucrose with Km values of 1.2 microM, 6.8 mM, and 65 mM, respectively. The pH optimum of the enzyme acting on inulin was 5.5, and the temperature optimum was 55 degrees C. Levanase was rapidly inactivated at 60 degrees C, but activity could be retained for longer times by adding fructose or glycerol. The enzyme activity was completely inactivated by Ag+ and Hg2+ ions, indicating that a sulfhydryl group is involved. A ratio of sucrase to inulinase activity of 1.2 was found for the purified enzyme with substrate concentrations of 50 mg/ml. The mechanism of enzyme action was investigated. No liberation of fructo-oligomers from inulin and levan could be observed by thin-layer chromatography and size exclusion chromatography-low-angle laser light scattering-interferometric differential refractive index techniques. This indicates that levanase is an exoenzyme acting by the single-chain mode.

Bacillus subtilis↗
PubMed1994

Complete nucleotide sequence of Birmingham IncP alpha plasmids. Compilation and comparative analysis.

The IncP alpha promiscuous plasmid (R18, R68, RK2, RP1 and RP4) comprises 60,099 bp of nucleotide sequence, encoding at least 74 genes. About 40 kb of the genome, designated the IncP core and including all essential replication and transfer functions, can be aligned with equivalent sequences in the IncP beta plasmid R751. The compiled IncP alpha sequence revealed several previously unidentified reading frames that are potential genes. IncP alpha plasmids carry genetic information very efficiently: the coding sequences of the genes are closely packed but rarely overlap, and occupy almost 86% of the genome's nucleotide sequence. All of the 74 genes should be expressed, although there is as yet experimental evidence for expression of only 60 of them. Six examples of tandem-in-frame initiation sites specifying two gene products each are known. Two overlapping gene arrangements occupy different reading frames of the same region. Intergenic regions include most of the 25 promoters; transcripts are usually polycistronic. Translation of most of the open reading frames seems to be initiated independently, each from its own ribosomal binding and initiation site, although, a few cases of coupled translation have been reported. The most frequently used initiation codon is AUG but translation for a few open reading frames begins at GUG or UUG. The most common stop-codon is UGA followed by UAA and then UAG. Regulatory circuits are complex and largely dependent on two components of the central control operon. KorA and KorB are transcriptional repressors controlling at least seven operons. KorA and KorB act synergistically in several cases by recognizing and binding to conserved nucleotide sequences. Twelve KorB binding sites were found around the IncP alpha sequence and these are conserved in R751 (IncP beta) with respect to both sequence and location. Replication of IncP alpha plasmids requires oriV and the plasmid-encoded initiator protein TrfA in combination with the host-encoded replication machinery. Conjugative plasmid transfer depends on two separate regions occupying about half of the genome. The primary segregational stability system designated Par/Mrs consists of a putative site-specific recombinase, a possible partitioning apparatus and a post-segregational lethality mechanism, all encoded in two divergent operons. Proteins related to the products of F sop and P1 par partitioning genes are separately encoded in the central control operon.

Amino Acid Sequence↗
PubMed1994

Analysis of the multimer resolution system encoded by the parCBA operon of broad-host-range plasmid RP4.

The broad-host-range plasmid RP4 encodes a highly efficient partitioning function, termed par, that is capable of stabilizing plasmids in a variety of Gram-negative bacteria independently of the nature of the replicon. The mechanism responsible for plasmid stabilization by this locus appears to be a complex system which includes a site-specific recombination system mediating resolution of plasmid multimers. In this report we present a detailed study on this multimer resolution system (mrs). The parA gene encodes two forms of a resolvase capable of catalysing site-specific recombination between specific sites situated in the promoter region of the parCBA operon. The two ParA proteins that are produced as a result of independent translation initiation at two different start codons within the same open reading frame were overexpressed in Escherichia coli and partially purified. Both forms of the enzyme are able to recombine a supercoiled cointegrate substrate containing two cis-acting elements with the same orientation in an in vitro resolution assay. ParA-mediated, site-specific recombination was found to be independent of any other gene product encoded by the RP4 par locus in vitro and in vivo. The DNA-binding sites for the ParA resolvase were determined using DNase I protection experiments. The results identified three binding sites within the mrs cis-acting region. Both the biochemical properties of the ParA protein and the organization of the cis-acting recombination site revealed a high degree of similarity to the site-specific recombination systems of Tn3-like transposable elements suggesting an evolutionary relationship.

Bacterial Proteins↗
PubMed1993

Stability of r-microbes: stabilization of plasmid vectors by the partitioning function of broad-host-range plasmid RP4.

The genes for biosynthesis of the biodegradable polymer poly-beta-hydroxybutyric acid (PHB) cloned from Alcaligenes eutrophus H16 were used for synthesis of PHB with recombinant Escherichia coli strains. It was recognized that the PHB-biosynthesis genes cause segregational instability to the plasmids used as vectors. Recombinant PHB-plasmids are rapidly lost from host cells and plasmid-free cells occur at high rates, even under conditions of selection for the plasmids. Cloning the partitioning region of plasmid RP4 onto such plasmids resulted in a high degree of stabilization. These par-stabilized recombinant PHB-plasmids could be maintained quite efficiently in batch cultivation experiments in the absence of any selection pressure.

Alcaligenes↗
PubMed1992

The divergent promoters mediating transcription of the par locus of plasmid RP4 are subject to autoregulation.

The partitioning region of broad-host-range plasmid RP4 contains four genes (parA, parB, parC, and parD) that encode products essential for partition activity. Two divergently arranged promoters located in the intercistronic region between parC and parD mediate transcription of these genes. The transcriptional initiation sites for both promoters were determined by primer extension. Transcriptional fusions were used to show that parA, parB, and parC are combined in an operon, while parD constitutes a separate transcription unit. Both parCBA (genes in order of transcription) and parD are negatively autoregulated at the level of transcription by the gene products of parA and parD, respectively. parD promoter mutants which have become insensitive to repression by parD were isolated. Comparison of wild type and the mutant parD promoter sequences indicated that three short repeats are likely involved in the negative regulation of this promoter. Potentially these sequence elements comprise target sites for the ParD protein.

Alleles↗