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PubMed2002

Identification of a disulfide isomerase protein of Leishmania major as a putative virulence factor.

Several approaches have been previously used to elucidate the genetic basis of Leishmania virulence. In general, they were based on laboratory Leishmania clones genetically modified or grown in the presence of selecting agents. In a previous study, we demonstrated that Leishmania major freshly isolated from human cutaneous lesions showed significant differences in the severity of the experimental disease induced in BALB/c mice. Here, using the mRNA differential display technique, we analyzed gene expression in L. major promastigotes showing different levels of virulence. We have identified a novel Leishmania gene encoding a 477-amino-acid protein exhibiting two distinct regions that are identical to the putative active-site sequence (CGHC) of the eukaryotic protein disulfide isomerase (PDI). The recombinant protein displayed a specific PDI enzymatic activity. This L. major disulfide isomerase protein (LmPDI) is predominantly expressed, at both the mRNA and protein levels, in highly virulent strains. Specific PDI inhibitors abolished the enzymatic activity of the recombinant protein and profoundly affected parasite growth. These findings suggest that LmPDI may play an important role in Leishmania natural pathogenicity and may constitute a new target for anti-Leishmania chemotherapy.

Amino Acid Sequence↗
PubMed1970

Purification of a D-mannose isomerase from Mycobacterium smegmatis.

An enzyme, d-mannose ketol isomerase, catalyzing the isomerization of d-mannose and d-fructose was purified approximately 60-fold from cells of Mycobacterium smegmatis grown on mannose as the sole carbon source. This enzyme was shown to catalyze the conversion of d-mannose and d-lyxose to ketoses. The ketose produced from mannose was identified as fructose by chemical and chromatographic methods. The reaction was shown to be reversible, the equilibrium ratio of fructose to mannose being approximately 65 to 35. The pH optimum was about 7.5, and the K(m) for mannose was estimated to be 7 x 10(-3)m. Mannose isomerase activity was greatest in cells grown on mannose, whereas cells grown on fructose had about 30% as much activity. Very low levels of activity were detected in cells grown on other substrates. There was an immediate increase in enzyme activity on transfer of cells from nutrient broth to a mannose mineral salts medium.

Calcium Phosphates↗
PubMed1971

Induction and repression of L-arabinose isomerase in Salmonella typhimurium.

As with other inducible enzymes, the induced synthesis of l-arabinose isomerase (l-arabinose ketol isomerase, EC 5.3.1.4) in Salmonella typhimurium is subject to catabolite repression. Of the three catabolite repressors tested, glucose produces maximum repression. Analogues of catabolite repressors like 2-deoxy-d-glucose and d-fucose also inhibit the synthesis of the enzyme. The catabolite repression is completely reversed in the presence of 1.5 x 10(-3)m cyclic 3',5'-adenosine monophosphate (AMP). The maximum repression is produced in glucose-grown cells in glucose-containing induction medium. Cyclic 3',5-AMP reverses this repression provided that the cells are treated with ethylenediaminetetraacetic acid (EDTA). In normal cells, cyclic 3',5'-AMP has no effect on the induction but in EDTA-treated cells the cyclic nucleotide enhances synthesis of the enzyme. The inhibition produced by d-fucose cannot be reversed by cyclic 3',5'-AMP. d-Fucose competes with the inducer l-arabinose in some step(s) involved in the process of induction.

Cell-Free System↗
PubMed1980

Mutants defective in isomerase and decarboxylase activities of the 4-hydroxyphenylacetic acid meta-cleavage pathway in Pseudomonas putida.

Degradation of 2-hydroxy-5-carboxymethylmuconic semialdehyde, the ring fission product of the 4-hydroxyphenylacetate meta-cleavage pathway, by mutant strains P23X19 and P23X16 of Pseudomonas putida NCI B 9865 was studied. Both mutants were unable to grow on either 4-hydroxyphenylacetate of 3,4-dihydroxyphenylacetate. Cell extracts of P23X19, grown in the presence of 3,4-dihydroxyphenylacetate, degraded the ring fission product to a compound that accumulated and had maximum UV absorption at 300 nm, pH 7.4, and 345 nm, pH 12. These are the spectral characteristics of 2-keto-5-carboxymethylhex-3-ene-1,6-dioate, the substrate for the decarboxylase in this pathway. This observation is consistent with P23X19's being decarboxylase defective. Cell extracts of P23X16, grown in the presence of 3,4-dihydroxyphenylacetate, degraded the ring fission product to a compound that accumulated and has maximum UV absorption at 295 nm, pH 7.4, and 345 nm, pH 12. This compound spontaneously degraded to a compound with the spectral properties of the decarboxylase substrate. The compound accumulated by P23X16 was also obtained when the decarboxylase substrate was treated with borate. It is suggested that the compound accumulated by P23X16 is the substrate of an isomerase. The results are consistent with P23X16's being unable to synthesize a functional isomerase while retaining decarboxylase activity and establish the physiological importance of an enzyme-catalyzed isomerization in the meta-cleavage degradation of 4-hydroxyphenylacetate.

Carboxy-Lyases↗
PubMed1981

Wild-type isopropylmalate isomerase in Salmonella typhimurium is composed of two different subunits.

The isopropylmalate isomerase in Salmonella typhimurium is the second enzyme specific for leucine biosynthesis. It is a complex enzyme composed of two subunits which are coded for by two genes of the leucine operon, leuC and leuD. The two polypeptides have been shown to copurify through successive ammonium sulfate fractionations and have been identified on sodium dodecyl sulfate-polyacrylamide gels as having molecular weights of 51,000 (leuC gene product) and 23,500 (leuD gene product). They have also been shown to be fairly stable, since in vitro complementation of cell-free extracts of leuC and leuD mutant strains was demonstrated, with only a 40% loss of activity 16 h after preparation of the extracts. The native isopropylmalate isomerase was shown to have a Km for its substrate alpha-isopropylmalate of 3 x 10(-4)M.

Genes, Bacterial↗
PubMed1989

Alternative route for biosynthesis of amino sugars in Escherichia coli K-12 mutants by means of a catabolic isomerase.

By inserting a lambda placMu bacteriophage into gene glmS encoding glucosamine 6-phosphate synthetase (GlmS), the key enzyme of amino sugar biosynthesis, a nonreverting mutant of Escherichia coli K-12 that was strictly dependent on exogenous N-acetyl-D-glucosamine or D-glucosamine was generated. Analysis of suppressor mutations rendering the mutant independent of amino sugar supply revealed that the catabolic enzyme D-glucosamine-6-phosphate isomerase (deaminase), encoded by gene nagB of the nag operon, was able to fulfill anabolic functions in amino sugar biosynthesis. The suppressor mutants invariably expressed the isomerase constitutively as a result of mutations in nagR, the locus for the repressor of the nag regulon. Suppression was also possible by transformation of glmS mutants with high-copy-number plasmids expressing the gene nagB. Efficient suppression of the glmS lesion, however, required mutations in a second locus, termed glmX, which has been localized to 26.8 min on the standard E. coli K-12 map. Its possible function in nitrogen or cell wall metabolism is discussed.

Aldose-Ketose Isomerases↗
PubMed1994

Cloning and nucleotide sequences of the genes encoding triose phosphate isomerase, phosphoglycerate mutase, and enolase from Bacillus subtilis.

The Bacillus subtilis genes tpi, pgm, and eno, encoding triose phosphate isomerase, phosphoglycerate mutase (PGM), and enolase, respectively, have been cloned and sequenced. These genes are the last three in a large putative operon coding for glycolytic enzymes; the operon includes pgk (coding for phosphoglycerate kinase) followed by tpi, pgm, and eno. The triose phosphate isomerase and enolase from B. subtilis are extremely similar to those from all other species, both eukaryotic and prokaryotic. However, B. subtilis PGM bears no resemblance to mammalian, fungal, or gram-negative bacterial PGMs, which are dependent on 2,3-diphosphoglycerate (DPG) for activity. Instead, B. subtilis PGM, which is DPG independent, is very similar to a DPG-independent PGM from a plant species but differs from the latter in the absolute requirement of B. subtilis PGM for Mn2+. The cloned pgm gene has been used to direct up to 25-fold overexpression of PGM in Escherichia coli; this should facilitate purification of large amounts of this novel Mn(2+)-dependent enzyme. Inactivation of pgm plus eno in B. subtilis resulted in extremely slow growth either on plates or in liquid, but growth of these mutants was enhanced by supplementation of media with malate. However, these mutants were asporogenous with or without malate supplementation.

Amino Acid Sequence↗
PubMed1994

Cloning, nucleotide sequence, and overexpression of the gene coding for delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B.

The structural gene coding for the delta 5-3-ketosteroid isomerase (KSI) of Pseudomonas putida biotype B has been cloned, and its entire nucleotide sequence has been determined by a dideoxynucleotide chain termination method. A 2.1-kb DNA fragment containing the ksi gene was cloned from a P. putida biotype B genomic library in lambda gt11. The open reading frame of ksi encodes 393 nucleotides, and the amino acid sequence deduced from the nucleotide sequence agrees with the directly determined amino acid sequence (K. Linden and W. F. Benisek, J. Biol. Chem. 261:6454-6460, 1986). A putative purine-rich ribosome binding site was found 8 bp upstream of the ATG start codon. Escherichia coli BL21(DE3) transformed with the pKK-KSI plasmid containing the ksi gene expressed a high level of isomerase activity when induced by isopropyl-beta-D-thiogalactopyranoside. KSI was purified to homogeneity by a simple and rapid procedure utilizing fractional precipitation and an affinity column of deoxycholate-ethylenediamine-agarose as a major chromatographic step. The molecular weight of KSI was 14,535 (calculated, 14,536) as determined by electrospray mass spectrometry. The purified KSI showed a specific activity (39,807 mumol min-1 mg-1) and a Km (60 microM) which are close to those of KSI originally obtained from P. putida biotype B.

Amino Acid Sequence↗
PubMed1996

Molecular cloning of the Haemophilus influenzae gmhA (lpcA) gene encoding a phosphoheptose isomerase required for lipooligosaccharide biosynthesis.

We have determined that gene HI#1181 of Haemophilus influenzae is a homolog of Escherichia coli gmhA (previously designated lpcA) (J. S. Brooke and M. A. Valvano, J. Biol. Chem. 271:3608-3614, 1996), which encodes a phosphoheptose isomerase catalyzing the first step of the biosynthesis of ADP-L-glycero-D-manno heptose. Mutations in this gene are associated with a heptoseless core lipopolysaccharide which determines an increased outer membrane permeability to hydrophobic compounds. The cloned H. influenzae gmhA restored the synthesis of a complete core in the gmhA-deleted E. coli strain chi711. Amino acid sequence comparisons of the GmhA proteins of E. coli and H. influenzae with other proteins in the databases revealed the existence of a novel family of phosphosugar a1do-keto isomerases.

Aldose-Ketose Isomerases↗
PubMed1997

Reduction of the periplasmic disulfide bond isomerase, DsbC, occurs by passage of electrons from cytoplasmic thioredoxin.

The Escherichia coli periplasmic protein DsbC is active both in vivo and in vitro as a protein disulfide isomerase. For DsbC to attack incorrectly formed disulfide bonds in substrate proteins, its two active-site cysteines should be in the reduced form. Here we present evidence that, in wild-type cells, these two cysteines are reduced. Further, we show that a pathway involving the cytoplasmic proteins thioredoxin reductase and thioredoxin and the cytoplasmic membrane protein DsbD is responsible for the reduction of these cysteines. Thus, reducing potential is passed from cytoplasmic electron donors through the cytoplasmic membrane to DsbC. This pathway does not appear to utilize the cytoplasmic glutathione-glutaredoxin pathway. The redox state of the active-site cysteines of DsbC correlates quite closely with its ability to assist in the folding of proteins with multiple disulfide bonds. Analysis of the activity of mutant forms of DsbC in which either or both of these cysteines have been altered further supports the role of DsbC as a disulfide bond isomerase.

Aprotinin↗
PubMed1997

Interruption of the phosphoglucose isomerase gene results in glucose auxotrophy in Mycobacterium smegmatis.

Two glycerol utilization mutants of Mycobacterium smegmatis that were unable to utilize most carbon sources except glucose were isolated. Supplementation of these media with small amounts of glucose restored growth in the mutants; these strains are therefore glucose auxotrophs. The mutant phenotype is complemented by the gene encoding phosphoglucose isomerase (pgi), and direct measurement of enzyme activities in the mutants suggests that this gene product is absent in the auxotrophic strains. Mapping of the mutant allele by Southern analysis demonstrates the presence of a 1-kb deletion extending into the coding sequence of pgi. The possible roles of phosphoglucose isomerase in mycobacterial cell wall synthesis and metabolic regulation are discussed.

Amino Acid Sequence↗
PubMed1999

Involvement of the cis/trans isomerase Cti in solvent resistance of Pseudomonas putida DOT-T1E.

Pseudomonas putida DOT-T1E is a solvent-resistant strain that is able to grow in the presence of high concentrations of toluene. We have cloned and sequenced the cti gene of this strain, which encodes the cis/trans isomerase, termed Cti, that catalyzes the cis-trans isomerization of esterified fatty acids in phospholipids, mainly cis-oleic acid (C(16:1,9)) and cis-vaccenic acid (C(18:1,11)), in response to solvents. To determine the importance of this cis/trans isomerase for solvent resistance a Cti-null mutant was generated and characterized. This mutant showed a longer lag phase when grown with toluene in the vapor phase; however, after the lag phase the growth rate of the mutant strain was similar to that of the wild type. The mutant also showed a significantly lower survival rate when shocked with 0.08% (vol/vol) toluene. In contrast to the wild-type strain, which grew in liquid culture medium at temperatures up to 38.5 degrees C, the Cti-null mutant strain grew significantly slower at temperatures above 37 degrees C. An in-frame fusion of the Cti protein with the periplasmic alkaline phosphatase suggests that this constitutively expressed enzyme is located in the periplasm. Primer extension studies confirmed the constitutive expression of Cti. Southern blot analysis of total DNA from various pseudomonads showed that the cti gene is present in all the tested P. putida strains, including non-solvent-resistant ones, and in some other Pseudomonas species.

Bacterial Proteins↗
PubMed2000

Mutations in multidrug efflux homologs, sugar isomerases, and antimicrobial biosynthesis genes differentially elevate activity of the sigma(X) and sigma(W) factors in Bacillus subtilis.

The sigma(X) and sigma(W) extracytoplasmic function sigma factors regulate more than 40 genes in Bacillus subtilis. sigma(W) activates genes which function in detoxification and the production of antimicrobial compounds, while sigma(X) activates functions that modify the cell envelope. Transposon mutagenesis was used to identify loci which negatively regulate sigma(W) or sigma(X) as judged by up-regulation from the autoregulatory promoter site P(W) or P(X). Fourteen insertions that activate P(W) were identified. The largest class of insertions are likely to affect transport. These include insertions in genes encoding two multidrug efflux protein homologs (yqgE and yulE), a component of the oligopeptide uptake system (oppA), and two transmembrane proteins with weak similarity to transporters (yhdP and yueF). Expression from P(W) is also elevated as a result of inactivation of at least one member of the sigma(W) regulon (ysdB), an ArsR homolog (yvbA), a predicted rhamnose isomerase (yulE), and a gene (pksR) implicated in synthesis of difficidin, a polyketide antibiotic. In a parallel screen, we identified seven insertions that up-regulate P(X). Remarkably, these insertions were in functionally similar genes, including a multidrug efflux homolog (yitG), a mannose-6-phosphate isomerase gene (yjdE), and loci involved in antibiotic synthesis (srfAB and possibly yogA and yngK). Significantly, most insertions that activate P(W) have little or no effect on P(X), and conversely, insertions that activate P(X) have no effect on P(W). This suggests that these two regulons respond to distinct sets of molecular signals which may include toxic molecules which are exported, cell density signals, and antimicrobial compounds.

Anti-Bacterial Agents↗
PubMed2000

Proteome analysis of the effect of mucoid conversion on global protein expression in Pseudomonas aeruginosa strain PAO1 shows induction of the disulfide bond isomerase, dsbA.

Pseudomonas aeruginosa strains that cause chronic pulmonary infections in cystic fibrosis patients typically undergo mucoid conversion. The mucoid phenotype indicates alginate overproduction and is often due to defects in MucA, an antisigma factor that controls the activity of sigma-22 (AlgT [also called AlgU]), which is required for the activation of genes for alginate biosynthesis. In this study we hypothesized that mucoid conversion may be part of a larger response that activates genes other than those for alginate synthesis. To address this, a two-dimensional (2-D) gel analysis was employed to compare total proteins in strain PAO1 to those of its mucA22 derivative, PDO300, in order to identify protein levels enhanced by mucoid conversion. Six proteins that were clearly more abundant in the mucoid strain were observed. The amino termini of such proteins were determined and used to identify the gene products in the genomic database. Proteins involved in alginate biosynthesis were expected among these, and two (AlgA and AlgD) were identified. This result verified that the 2-D gel approach could identify gene products under sigma-22 control and upregulated by mucA mutation. Two other protein spots were also clearly upregulated in the mucA22 background, and these were identified as porin F (an outer membrane protein) and a homologue of DsbA (a disulfide bond isomerase). Single-copy gene fusions were constructed to test whether these proteins were enhanced in the mucoid strain due to increased transcription. The oprF-lacZ fusion showed little difference in levels of expression in the two strains. However, the dsbA-lacZ fusion showed two- to threefold higher expression in PDO300 than in PAO1, suggesting that its promoter was upregulated by the deregulation of sigma-22 activity. A dsbA-null mutant was constructed in PAO1 and shown to have defects predicted for a cell with reduced disulfide bond isomerase activity, namely, reduction in periplasmic alkaline phosphatase activity, increased sensitivity to dithiothreitol, reduced type IV pilin-mediated twitching motility, and reduced accumulation of extracellular proteases, including elastase. Although efficient secretion of elastase in the dsbA mutant was still demonstrable, the elastase produced appeared to be unstable, possibly as a result of mispaired disulfide bonds. Disruption of dsbA in the mucoid PDO300 background did not affect alginate production. Thus, even though dsbA is coregulated with mucoid conversion, it was not required for alginate production. This suggests that mucA mutation, which deregulates sigma-22, results in a global response that includes other factors in addition to increasing the production of alginate.

Adenosine Triphosphatases↗
PubMed1970

Induction and repression of L-arabinose isomerase in bacteriophage-infected Salmonella typhimurium.

The induction of l-arabinose isomerase in Salmonella typhimurium (LT2) is repressed on infection with clear plaque forming mutants (C(1) and C(2)) of the temperate phage P22 (C(+)). However, after infection with C(+) leading to lysogeny, there is a temporary repression. During this period, messenger ribonucleic acid (RNA) for l-arabinose isomerase accumulates. DNA-RNA hybridization data suggest that there is transcription of host DNA during the period of repression. Interference at the level of translation might be responsible for the cessation of induced enzyme synthesis.

Arabinose↗
PubMed2005

The fusion-controlling disulfide bond isomerase in retrovirus Env is triggered by protein destabilization.

The membrane fusion function of murine leukemia virus (MLV) is carried by the Env protein. This protein is composed of three SU-TM subunit complexes. The fusion activity is loaded into the transmembrane TM subunit and controlled by the peripheral, receptor-binding SU subunit. It is assumed that TM adopts a metastable conformation in the native Env and that fusion activation involves the folding of TM into a stable form. Activation is suppressed by the associated SU and triggered by its dissociation, which follows receptor binding. Recently we showed that the two subunits are disulfide linked and that SU dissociation and triggering of the fusion function are caused by a switch of the intersubunit disulfide into an intrasubunit disulfide isomer using an isomerization-active CWLC motif in SU (M. Wallin, M. Ekstrom, and H. Garoff, EMBO J. 23:54-65, 2004). In the present work we address how the SU disulfide isomerase is activated. Using Moloney MLV, we show that isomerization of the SU-TM disulfide bond can be triggered by heat, urea, or guanidinium hydrochloride. Such protein perturbation treatments also significantly increase the kinetics and efficiency of viral fusion. The threshold conditions for the effects on isomerization and fusion are virtually the same. This finding indicates that destabilization of interactions in the SU oligomer induces the disulfide bond isomerase and the subsequent activation of the fusion function in TM.

Animals↗
PubMed1996

Molecular and industrial aspects of glucose isomerase.

Glucose isomerase (GI) (D-xylose ketol-isomerase; EC. 5.3.1.5) catalyzes the reversible isomerization of D-glucose and D-xylose to D-fructose and D-xylulose, respectively. The enzyme has the largest market in the food industry because of its application in the production of high-fructose corn syrup (HFCS). HFCS, an equilibrium mixture of glucose and fructose, is 1.3 times sweeter than sucrose and serves as a sweetener for use by diabetics. Interconversion of xylose to xylulose by GI serves a nutritional requirement in saprophytic bacteria and has a potential application in the bioconversion of hemicellulose to ethanol. The enzyme is widely distributed in prokaryotes. Intensive research efforts are directed toward improving its suitability for industrial application. Development of microbial strains capable of utilizing xylan-containing raw materials for growth or screening for constitutive mutants of GI is expected to lead to discontinuation of the use of xylose as an inducer for the production of the enzyme. Elimination of Co2+ from the fermentation medium is desirable for avoiding health problems arising from human consumption of HFCS. Immobilization of GI provides an efficient means for its easy recovery and reuse and lowers the cost of its use. X-ray crystallographic and genetic engineering studies support a hydride shift mechanism for the action of GI. Cloning of GI in homologous as well as heterologous hosts has been carried out, with the prime aim of overproducing the enzyme and deciphering the genetic organization of individual genes (xylA, xylB, and xylR) in the xyl operon of different microorganisms. The organization of xylA and xylB seems to be highly conserved in all bacteria. The two genes are transcribed from the same strand in Escherichia coli and Bacillus and Lactobacillus species, whereas they are transcribed divergently on different strands in Streptomyces species. A comparison of the xylA sequences from several bacterial sources revealed the presence of two signature sequences, VXW(GP)GREG(YSTAE)E and (LIVM)EPKPX(EQ)P. The use of an inexpensive inducer in the fermentation medium devoid of Co2+ and redesigning of a tailor-made GI with increased thermostability, higher affinity for glucose, and lower pH optimum will contribute significantly to the development of an economically feasible commercial process for enzymatic isomerization of glucose to fructose. Manipulation of the GI gene by site-directed mutagenesis holds promise that a GI suitable for biotechnological applications will be produced in the foreseeable future.

Aldose-Ketose Isomerases↗
PubMed1975

A comparative study on the heat stability of triosephosphate isomerase in psychrophilic, psychrotrophic, and mesophilic clostridia.

The temperature stability of triosephosphate isomerase (EC 5.3.1.1) in the cell-free extracts of psychrophilic, psychrotrophic, and mesophilic Clostridium species has been investigated. Even though this enzyme was found to be heat labile in the psychrophilic isolates, no detectable loss in activity was evident when cell-free extracts were heated for 1/2 h at the maximum temperature of growth for the organisms. Two organisms, each with a maximum growth temperature of 23 degrees C, showed different heat stability of triosephosphate isomerase. However, a trend is evident that as the maximum growth temperature increases, the thermostability also increases. It is suggested that the heat liability of this enzyme is not a controlling factor in psychrophilism, but rather an adaptation to the cold habitat of these organisms.

Adaptation, Physiological↗