Cloning and over-expression of thermostable Bacillus sp. YM55-1 aspartase and site-directed mutagenesis for probing a catalytic residue.
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A thermostable aspartase gene (aspB) from Bacillus sp. YM55-1 was cloned and the gene sequenced. The aspB gene (1407 bp ORF) encodes a protein with a molecular mass of 51 627 Da, consisting of 468 amino-acid residues. An amino-acid sequence comparison revealed that Bacillus YM55-1 aspartase shared 71% homology with Bacillus subtilis aspartase and 49% with Escherichia coli and Pseudomonas fluorescens aspartases. The E. coli TK237/pUCASPB strain, which was obtained by transforming E. coli TK237 (aspartase-null strain) with a vector plasmid (pUCASPB) containing the cloned aspB gene, produced a large amount of the enzyme corresponding to > 10% of the total soluble protein. The over-expressed recombinant enzyme (native molecular mass: 200 kDa) was purified effectively and rapidly using heat treatment and affinity chromatography. In order to probe the catalytic residues of this enzyme, two conserved amino-acid residues, Lys183 and His134, were individually mutated to alanine. Although the tertiary structure of each mutant was estimated to be the same as that of wild-type aspartase in CD and fluorescence measurements, the Lys183Ala mutant lost its activity completely, whereas His134Ala retained full activity. This finding suggests that Lys183 may be involved in the catalytic activity of this thermostable Bacillus YM55-1 aspartase. (+info)
A monomeric L-aspartase obtained by in vitro selection.
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By mimicking the partial spatial structure of the dimer of the l-aspartase subunit, the central ten-helix bundle, and an "active site" between the cleft of domain 1 (D1) and domain 3 (D3) from different subunits, we designed l-aspartase variants, in which D1D2 and D2D3 were ligated with a random hexapeptide loop. As expected, we obtained the variant with the highest activity (relative activity is 21.3% of the native enzyme, named as drAsp017) by in vitro selection. The molecular weight of this variant, obtained from size-exclusion column chromatography, is about 81 kDa, which indicates that it is indeed a monomer, whereas native l-aspartase is a tetramer. The activity-reversibility of drAsp017 (10(-7) m) was 80% after incubation for 30 min at 50 degrees C, while native enzyme only retained about 17% under the same conditions. Reactivation of drAsp017 denatured in 4 m guanidine HCl was independent of protein concentration at up to 20 x 10(-8) m at 25 degrees C, whereas the protein concentration of native enzyme strongly affected its reactivation under the above conditions. The sensitivity of drAsp017 (10(-7) m) to effective factors in the fumarate-amination reaction compared with native enzyme was also determined. Half-saturating concentrations of the activator l-aspartate and Mg2+ for drAsp017 (0.8 and 0.5 mm, respectively) are much higher than that of the native enzyme (0.10 and 0.15 mm, respectively). The data show that a monomeric l-aspartase is obtained by in vitro selection. Thus, the conversion of oligomeric proteins into their functional monomers could have important applications. (+info)
Thermostable aspartase from a marine psychrophile, Cytophaga sp. KUC-1: molecular characterization and primary structure.
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We found that a psychrophilic bacterium isolated from Antarctic seawater, Cytophaga sp. KUC-1, abundantly produces aspartase [EC4.3.1.1], and the enzyme was purified to homogeneity. The molecular weight of the enzyme was estimated to be 192,000, and that of the subunit was determined to be 51,000: the enzyme is a homotetramer. L-Aspartate was the exclusive substrate. The optimum pH in the absence and presence of magnesium ions was determined to be pH 7.5 and 8.5, respectively. The enzyme was activated cooperatively by the presence of L-aspartate and by magnesium ions at neutral and alkaline pHs. In the deamination reaction, the K(m) value for L-aspartate was 1.09 mM at pH 7.0, and the S(1/2) value was 2.13 mM at pH 8.5. The V(max) value were 99.2 U/mg at pH 7.0 and 326 U/mg at pH 8.5. In the amination reaction, the K(m) values for fumarate and ammonium were 0.797 and 25.2 mM, respectively, and V(max) was 604 U/mg. The optimum temperature of the enzyme was 55 degrees C. The enzyme showed higher pH and thermal stabilities than that from mesophile: the enzyme was stable in the pH range of 4.5-10.5, and about 80% of its activity remained after incubation at 50 degrees C for 60 min. The gene encoding the enzyme was cloned into Escherichia coli, and its nucleotides were sequenced. The gene consisted of an open reading frame of 1,410-bp encoding a protein of 469 amino acid residues. The amino acid sequence of the enzyme showed a high degree of identity to those of other aspartases, although these enzymes show different thermostabilities. (+info)
Possible physiological roles of aspartase, NAD- and NADP-requiring glutamate dehydrogenases of Pseudomonas fluorescens.
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The levels of aspartase, NADP- and NAD-requiring glutamate dehydrogenases (GDHs) in Pseudomonas fluorescens grown under various nutritional conditions were determined. NADP-GDH showed the highest value on glucose-ammonium sulfate medium and markedly lower values on amino-acid and casamino-acids media, while the reverse was found for the NAD-GDH, as in the case of other microorganisms with two GDHs. Aspartase did not show a marked variation between the media examined. Glucose nutritionally induced NADP-GDH but suppressed NAD-GDH; and it had no effect on aspartase, which was slightly induced by casamino acids. Transfer of the cells grown on glucose-ammonium sulfate medium to casamino-acids medium clearly increased the levels of NAD-GDH and aspartase, while addition of chloramphenicol to the media abolished the increases, suggesting that the increases were due to de novo synthesis of the enzyme proteins. These results indicate that the aspartase of this microorganism has a different function from those in others, including Escherichia coli. (+info)
Enzymes involved in anaerobic respiration appear to play a role in Actinobacillus pleuropneumoniae virulence.
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Actinobacillus pleuropneumoniae, the etiological agent of porcine pleuropneumonia, is able to survive on respiratory epithelia, in tonsils, and in the anaerobic environment of encapsulated sequesters. It was previously demonstrated that a deletion of the anaerobic dimethyl sulfoxide reductase gene (dmsA) results in attenuation in acute disease (N. Baltes, S. Kyaw, I. Hennig-Pauka, and G. F. Gerlach, Infect. Immun. 71:6784-6792, 2003). In the present study, using two-dimensional polyacrylamide gel electrophoresis and quadrupole time-of-flight mass spectrometry, we identified an aspartate ammonia-lyase (AspA) which is upregulated upon induction with bronchoalveolar lavage fluid (BALF). This enzyme is involved in the production of fumarate, an alternative electron acceptor under anaerobic conditions. The coding gene (aspA) was cloned and shown to be present in all A. pleuropneumoniae serotype reference strains. The transcriptional start point was identified downstream of a putative FNR binding motif, and BALF-dependent activation of aspA was confirmed by construction of an isogenic A. pleuropneumoniae mutant carrying a chromosomal aspA::luxAB transcriptional fusion. Two aspA deletion mutants, A. pleuropneumoniae DeltaaspA and A. pleuropneumoniae DeltaaspADeltadmsA, were constructed, both showing reduced growth under anaerobic conditions in vitro. Pigs challenged with either of the two mutants in an aerosol infection model showed a lower lung lesion score than that of the A. pleuropneumoniae wild-type (wt) controls. Pigs challenged with A. pleuropneumoniae DeltaaspADeltadmsA had a significantly lower clinical score, and this mutant was rarely reisolated from unaltered lung tissue; in contrast, A. pleuropneumoniae DeltaaspA and the A. pleuropneumoniae wt were consistently reisolated in high numbers. These results suggest that enzymes involved in anaerobic respiration are necessary for the pathogen's ability to persist on respiratory tract epithelium and play an important role in A. pleuropneumoniae pathogenesis. (+info)
Deletion of the anaerobic regulator HlyX causes reduced colonization and persistence of Actinobacillus pleuropneumoniae in the porcine respiratory tract.
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Actinobacillus pleuropneumoniae, the etiological agent of porcine pleuropneumonia, is able to persist on respiratory epithelia, in tonsils, and in the anaerobic environment of encapsulated lung sequesters. We have demonstrated previously that putative HlyX-regulated genes, coding for dimethyl sulfoxide (DMSO) reductase and aspartate ammonia lyase, are upregulated during infection and that deletions in these genes result in attenuation of the organism. The study presented here investigates the role of HlyX, the fumarate nitrate reductase regulator (FNR) homologue of A. pleuropneumoniae. By constructing an isogenic A. pleuropneumoniae hlyX mutant, the HlyX protein is shown to be responsible for upregulated expression of both DMSO reductase and aspartate ammonia lyase (AspA) under anaerobic conditions. In a challenge experiment the A. pleuropneumoniae hlyX mutant is shown to be highly attenuated, unable to persist in healthy lung epithelium and tonsils, and impaired in survival inside sequestered lung tissue. Further, using an A. pleuropneumoniae strain carrying the luxAB genes as transcriptional fusion to aspA on the chromosome, the airway antioxidant glutathione was identified as one factor potentially responsible for inducing HlyX-dependent gene expression of A. pleuropneumoniae in epithelial lining fluid. (+info)
Cloning, nucleotide sequence, and expression of the Bacillus subtilis ans operon, which codes for L-asparaginase and L-aspartase.
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L-Aspartase was purified from Bacillus subtilis, its N-terminal amino acid sequence was determined to construct a probe for the aspartase gene, and the gene (termed ansB) was cloned and sequenced. A second gene (termed ansA) was found upstream of the ansB gene and coded for L-asparaginase. These two genes were in an operon designated the ans operon, which is 80% cotransformed with the previously mapped aspH1 mutation at 215 degrees. Primer extension analysis of in vivo ans mRNA revealed two transcription start sites, depending on the growth medium. In wild-type cells in log-phase growth in 2x YT medium (tryptone-yeast extract rich medium), the ans transcript began at -67 relative to the translation start site, while cells in log-phase growth or sporulating (t1 to t4) in 2x SG medium (glucose nutrient broth-based moderately rich medium) had an ans transcript which began at -73. The level of the -67 transcript was greatly increased in an aspH mutant grown in 2x YT medium; the -67 transcript also predominated when this mutant was grown in 2x SG medium, although the -73 transcript was also present. In vitro transcription of the ans operon by RNA polymerase from log-phase cells grown in 2x YT medium and log-phase or sporulating cells grown in 2x SG medium yielded only the -67 transcript. Depending on the growth medium, the levels of asparaginase and aspartase were from 2- to 40-fold higher in an aspH mutant than in wild-type cells, and evidence was obtained indicating that the gene defined by the aspH1 mutation codes for a trans-acting transcriptional regulatory factor. In wild-type cells grown in 2x SG medium, the levels of both aspartase and asparaginase decreased significantly by t0 of sporulation but then showed a small increase, which was mirrored by changes in the level of beta-galactosidase from an ansB-lacZ fusion. The increase in the activities of ans operon enzymes between t2 and t5 of sporulation was found primarily in the forespore, and the great majority of the increased was found in the mature spore. However, throughout sporulation the only ans transcript detected was the -73 form, and no sporulation-specific RNA polymerase tested yielded a -73 transcript in vitro. (+info)
A missense mutation causes aspartase deficiency in Yersinia pestis.
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