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At least 73 records · Page 4Linked to original sources
PubMed1998

High-efficiency incorporation in vivo of tyrosine analogues with altered hydroxyl acidity in place of the catalytic tyrosine-14 of Delta 5-3-ketosteroid isomerase of Comamonas (Pseudomonas) testosteroni: effects of the modifications on isomerase kinetics.

Versions of the Y55F/Y88F modified form of Delta 5-3-ketosteroid isomerase in which the active-site tyrosine-14 is replaced by 2-fluorotyrosine, 3-fluorotyrosine, and 2,3-difluorotyrosine, amino acids having progressively greater acidity of their phenolic hydroxyls, have been expressed in an Escherichia coli host and purified to high homogeneity. The steady-state kinetic properties of Y55F/Y88F KSI and its fluorotyrosine modified forms have been determined. The mechanistic implications of the results are presented and discussed.

Amino Acid Substitution↗
PubMed2005

Phylogenetic analyses identify 10 classes of the protein disulfide isomerase family in plants, including single-domain protein disulfide isomerase-related proteins.

Protein disulfide isomerases (PDIs) are molecular chaperones that contain thioredoxin (TRX) domains and aid in the formation of proper disulfide bonds during protein folding. To identify plant PDI-like (PDIL) proteins, a genome-wide search of Arabidopsis (Arabidopsis thaliana) was carried out to produce a comprehensive list of 104 genes encoding proteins with TRX domains. Phylogenetic analysis was conducted for these sequences using Bayesian and maximum-likelihood methods. The resulting phylogenetic tree showed that evolutionary relationships of TRX domains alone were correlated with conserved enzymatic activities. From this tree, we identified a set of 22 PDIL proteins that constitute a well-supported clade containing orthologs of known PDIs. Using the Arabidopsis PDIL sequences in iterative BLAST searches of public and proprietary sequence databases, we further identified orthologous sets of 19 PDIL sequences in rice (Oryza sativa) and 22 PDIL sequences in maize (Zea mays), and resolved the PDIL phylogeny into 10 groups. Five groups (I-V) had two TRX domains and showed structural similarities to the PDIL proteins in other higher eukaryotes. The remaining five groups had a single TRX domain. Two of these (quiescin-sulfhydryl oxidase-like and adenosine 5'-phosphosulfate reductase-like) had putative nonisomerase enzymatic activities encoded by an additional domain. Two others (VI and VIII) resembled small single-domain PDIs from Giardia lamblia, a basal eukaryote, and from yeast. Mining of maize expressed sequence tag and RNA-profiling databases indicated that members of all of the single-domain PDIL groups were expressed throughout the plant. The group VI maize PDIL ZmPDIL5-1 accumulated during endoplasmic reticulum stress but was not found within the intracellular membrane fractions and may represent a new member of the molecular chaperone complement in the cell.

Base Sequence↗
PubMed2005

Characterization of the highly efficient sucrose isomerase from Pantoea dispersa UQ68J and cloning of the sucrose isomerase gene.

Sucrose isomerase (SI) genes from Pantoea dispersa UQ68J, Klebsiella planticola UQ14S, and Erwinia rhapontici WAC2928 were cloned and expressed in Escherichia coli. The predicted products of the UQ14S and WAC2928 genes were similar to known SIs. The UQ68J SI differed substantially, and it showed the highest isomaltulose-producing efficiency in E. coli cells. The purified recombinant WAC2928 SI was unstable, whereas purified UQ68J and UQ14S SIs were very stable. UQ68J SI activity was optimal at pH 5 and 30 to 35 degrees C, and it produced a high ratio of isomaltulose to trehalulose (>22:1) across its pH and temperature ranges for activity (pH 4 to 7 and 20 to 50 degrees C). In contrast, UQ14S SI showed optimal activity at pH 6 and 35 degrees C and produced a lower ratio of isomaltulose to trehalulose (<8:1) across its pH and temperature ranges for activity. UQ68J SI had much higher catalytic efficiency; the Km was 39.9 mM, the Vmax was 638 U mg(-1), and the Kcat/Km was 1.79 x 10(4) M(-1) s(-1), compared to a Km of 76.0 mM, a Vmax of 423 U mg(-1), and a Kcat/Km of 0.62 x 10(4) M(-1) s(-1) for UQ14S SI. UQ68J SI also showed no apparent reverse reaction producing glucose, fructose, or trehalulose from isomaltulose. These properties of the P. dispersa UQ68J enzyme are exceptional among purified SIs, and they indicate likely differences in the mechanism at the enzyme active site. They may favor the production of isomaltulose as an inhibitor of competing microbes in high-sucrose environments, and they are likely to be highly beneficial for industrial production of isomaltulose.

Amino Acid Sequence↗
PubMed1975

Two ribose-5-phosphate isomerases from Escherichia coli K12: partial characterisation of the enzymes and consideration of their possible physiological roles.

Two physically and genetically distinct forms of ribosephosphate isomerase have been identified in Escherichia coli K12. The constitutive ribosephosphate isomerase A has a Km for ribose 5-phosphate (4.4 +/- 0.5 mM) six times greater than that of the inducible ribosephosphate isomerase B (0.83 +/- 0.13 mM). Treatment of the enzymes with 1.25 mM iodoacetate resulted in 100% loss of activity for ribosephosphate isomerase B, whereas ribosephosphate isomerase A was unaffected. Various cellular metabolites were tested and found to be without significant effect on either enzyme. The two enzymes could be separated by filtration on Sephadex G75 superfine and their apparent molecular weights were 45000 for ribosephosphate isomerase A and 32000-34000 for ribosephosphate isomerase B. Under certain conditions the two enzymes showed different patterns of heat inactivation but the results with ribosephosphate isomerase A varied in an unusual way with the protein concentration. Ribosephosphate isomerase B was formed inducibly in a mutant lacking ribosephosphate isomerase A but there was no evidence for the production of ribosephosphate isomerase B in wild-type cells. The formation of ribosephosphate isomerase B was not a consequence of the ribosephosphate isomerase B mutation, since strains could be constructed which formed both enzymes constitutively in the anticipated amounts. The ribosephosphate isomerase formed by a secondary mutant obtained from a ribosephosphate-isomerase-A-negative strain was identified as ribosephosphate isomerase B on the basis of its Km, elution profile from Sephadex G75, inhibition of iodoacetate, and heat inactivation. The ribosephosphate isomerases of another Escherichia coli K12 strain, X289, were investigated, since their properties were reported to be different from many of these described here for ribosephosphate isomerases A and B. In our hands strain X289 contained two ribosephosphate isomerases apparently identical to ribosephosphate isomerases A and B. The evidence to date suggests that ribosephosphate isomerase A catalyses the formation of ribose 5-phosphate from ribulose 5-phosphate and also participates in the reverse reaction during ribose and adenosine catabolism. The normal physiological role of the inducible ribosephosphate isomerase B is still uncertain.

Aldose-Ketose Isomerases↗
PubMed1998

Both the immunosuppressant SR31747 and the antiestrogen tamoxifen bind to an emopamil-insensitive site of mammalian Delta8-Delta7 sterol isomerase.

SR31747 is a novel agent that elicits immunosuppressive and anti-inflammatory effects. This drug was shown to inhibit Delta8-Delta7 sterol isomerase in yeast. To test whether this enzyme could also be an SR31747 target in mammals, the binding, antiproliferative and sterol biosynthesis inhibitory properties of various drugs were studied in recombinant sterol isomerase-producing yeast cells. Our results clearly show that SR31747 is a high affinity ligand of recombinant mammalian sterol isomerase (Kd = 1 nM). Tridemorph, a sterol biosynthesis inhibitor that is widely used in agriculture as an antifungal agent, is also a powerful inhibitor of murine and human sterol isomerases (IC50 value in the nanomolar range). Some drugs, like cis-flupentixol, trifluoperazine, 7-ketocholestanol and tamoxifen, inhibit SR31747 binding only with the mammalian enzymes, whereas other drugs, like haloperidol and fenpropimorph, are much more effective with the yeast enzyme than with the mammalian ones. Emopamil, a high affinity ligand of human sterol isomerase, is inefficient in inhibiting SR31747 binding to its mammalian target, suggesting that the SR31747 and emopamil binding sites on mammalian sterol isomerase do not overlap. In contrast, SR31747 binding inhibition by tamoxifen is very efficient and competitive (IC50 value in the nanomolar range), indicating that mammalian sterol isomerase contains a so-called antiestrogen binding site. Tamoxifen is found to selectively inhibit sterol biosynthesis at the sterol isomerase step in the cells that are producing the mammalian enzyme in place of their own sterol isomerase. Finally, we also show that tridemorph, a sterol biosynthesis inhibitor widely used in agriculture as an antifungal agent, is not selective of yeast Delta8-Delta7 sterol isomerase but is also highly efficient against murine Delta8-Delta7 sterol isomerase or human Delta8-Delta7 sterol isomerase. This observation contrasts with our already published results showing that fenpropimorph, another sterol isomerase inhibitor used in agriculture, is only poorly efficient against the mammalian enzymes.

Animals↗
PubMed1996

Species-specific inhibition of homologous enzymes by modification of nonconserved amino acids residues. The cysteine residues of triosephosphate isomerase.

The possibility of using non-conserved amino acid residues to produce selective inhibition of homologous enzymes from different species has been further explored with triosephosphate isomerase. S-phenyl-p-toluenethiosulfonate (MePhSO2-SPh), which produces phenyl disulfides with accessible Cys residues, inhibits the activity of rabbit triosephosphate isomerase. The inhibition is due to derivatization of one of the five Cys residues of rabbit triosephosphate isomerase. The effect of MePhSO2-SPh on triosephosphate isomerase from Saccharomyces cerevisiae, Escherichia coli, chicken and Schizosaccharomyces pombe was also determined. MePhSO2-SPh did not affect the activity of triosephosphate isomerase from S. cerevisiae and E. coli but it inhibited triosephosphate isomerase from chicken and S. pombe. From an analysis of the Cys content of the various triosephosphate isomerases, it was evident that amongst the ones studied only those that have a Cys in position 217 (or in an equivalent position) were sensitive to MePhSO2-SPh. Methyl metanethiosulfonate (MeSO2-SMe), which produces methyl disulfides, had no effect on triosephosphate isomerases that lack Cys217 (S. cerevisiae and E. coli). In triosephosphate isomerases that have Cys217, MeSO2-SMe inhibited by 40-50% the activity of that from S. pombe, 20-25% that from rabbit but had no effect on the chicken enzyme. In the three latter triosephosphate isomerases, MeSO2-SMe protected against the strong inhibiting action of MePhSO2-SPh. The latter observations suggest that MeSO2-SMe and MePhSO2-SPh derivatize the same Cys and that significant inhibition of activity requires perturbation by the relatively large phenyl group. The intrinsic fluorescence of rabbit triosephosphate isomerase that had been derivatized to a phenyl disulfide was almost identical to that of the native enzyme. Thus, modification of Cys217 did not produce gross structural alterations, albeit it brought about important kinetic alterations, i.e. a nearly fivefold increase in the K(m) for glyceraldehyde 3-phosphate and a 65% decrease in Vmax. The effect of derivatizating Cys217 differs markedly from that produced by derivatization of Cys14 (another non-conserved cysteine). The differences may be explained from their position in the three-dimensional structure of the enzyme.

Animals↗