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At least 199 records · Page 11Linked to original sources
PubMed1994

Roles of peptidyl-prolyl cis-trans isomerase and calcineurin in the mechanisms of antimalarial action of cyclosporin A, FK506, and rapamycin.

The immunosuppressive peptide cyclosporin A inhibits the growth of malaria parasites in vitro and in vivo, but little is known about its mechanism of antimalarial action. The immunosuppressive action of cyclosporin A is believed to result from binding of the drug to cyclophilins (intracellular peptidyl-prolyl cis-trans isomerases), and inhibition of the protein phosphatase calcineurin by the cyclosporin A-cyclophilin complex. Two immunosuppressive macrolides, FK506 and rapamycin, bind to a distinct isomerase, FKBP12, and the FK506-FKBP complex also inhibits calcineurin. Calcineurin itself is apparently involved in signal transduction between the T-cell membrane and nucleus, and its inhibition blocks T-cell activation. Rapamycin inhibits a later step in T-cell proliferation. Peptidyl-propyl cis-trans isomerase activity was detected in extracts of Plasmodium falciparum. It was completely inhibited by concentrations of cyclosporin A above 0.1 microM, but not by FK506 or rapamycin, and probably represented one or more cyclophilins. Comparison of the antimalarial and anti-isomerase activities of a series of cyclosporin analogues failed to reveal a correlation between the two properties. Cyclosporin A and its more active 8'-oxymethyl-dihydro-derivative, in combination with the cyclophilin-containing P. falciparum extract, inhibited the protein phosphatase activity of bovine calcineurin. Therefore inhibition of a putative P. falciparum calcineurin by a complex of CsA and cyclophilin might be responsible for the antimalarial action of the drug. The most active cyclosporin, however, was a 3'-keto-derivative of cyclosporin D (SDZ PSC-833) which inhibited P. falciparum growth with a 50% inhibitory concentration (IC50) of 0.032 microM (compared with 0.30 microM for cyclosporin A), but was a poor inhibitor of the parasite isomerase. 3'-Keto-cyclosporin D has negligible immunosuppressive activity, but it strongly inhibits the P-glycoprotein of multi-drug resistant mammalian tumour cells. FK506 and rapamycin were also active antimalarials (IC50 of 1.9 and 2.6 microM, respectively) but in the absence of detectable FKBP in P. falciparum extracts, their mechanisms of antimalarial action remain unclear.

Amino Acid Isomerases↗
PubMedDate not supplied

Effect of membrane lipid environment on the activity of bovine adrenal 3-oxo-delta 5-steroid isomerase.

The 3-oxo-delta 5-steroid isomerase (EC 5.3.3.1) activity from bovine adrenal cortex microsomes can be extracted in soluble form by the use of appropriate detergents, although recovery of enzyme activity is low (ca. 2%). Activity is restored upon removal of detergent and reconstitution of the enzyme into phospholipid vesicles. Both Km and Vmax of 3-oxo-delta 5-steroid isomerase of intact microsomes increase as the pH is raised from 7.5 to 9.5, with a particularly sharp increase (6- to 8-fold) above pH 8.5. The kinetic parameters of a detergent-solubilized isomerase preparation show little increase from pH 7.5 to 9.0, but isomerase reconstituted into artificial phospholipid vesicles demonstrates a 6- to 10-fold increase in both Km and Vmax over this pH range. Addition of Ca++ (1 mM) enhances the pH dependence of both Km and Vmax of the membrane-bound isomerase, causing a slight rise in Vmax/Km.

Adrenal Cortex↗
PubMed2005

Intrinsic enoyl-CoA isomerase activity of rat acyl-CoA oxidase I.

Rat peroxisomal acyl-CoA oxidase I is a key enzyme for the beta-oxidation of fatty acids, and the deficiency of this enzyme in patient has been previously reported. It was found that rat acyl-CoA oxidase I has intrinsic enoyl-CoA isomerase activity, which was confirmed using incubation followed with HPLC analysis in this study. Various 3-enoyl-CoA substrates with cis or trans configuration were synthesized and used in the study of enzyme substrate specificity. The isomerase activity of the enzyme was characterized through studies of kinetics, pH dependence, and enzyme inhibition. Most k(cat)/K(M) values of rat peroxisomal acyl-CoA oxidase I for isomerization reaction are comparable with those of authentic rat liver peroxisomal Delta(3)-Delta(2)-enoyl-CoA isomerase and rat liver peroxisomal multifunctional enzyme 1 when hexenoyl-CoA and octenoyl-CoA with cis- or trans-configuration were used as substrate. Glu421 was found to be the catalytic residue for both oxidase and isomerase activities of the enzyme. The isomerase activity of rat peroxisomal acyl-CoA oxidase I is probably due to a spontaneous process driven by thermodynamic equilibrium with formation of a conjugated structure after deprotonation of substrate alpha-proton. The energy level of transition state may be lowered by a stable dienolate intermediate, which gain further stabilization via charge transfer with electron-deficient FAD cofactor of the enzyme.

Acyl-CoA Oxidase↗
PubMed1983

Photoaffinity modification of delta 5-3-ketosteroid isomerase by light-activatable steroid ketones covalently coupled to agarose beads.

In order to identify the minor site(s) of photoattachment of unsaturated steroid ketones to delta 5-3-ketosteroid isomerase from Pseudomonas testosteroni, we have developed a solid-state photoaffinity labeling technique. Two solid-state reagents, O-carboxymethylagarose-ethylenediamine-succinyl-17 beta-O-19-nortestosterone and O-carboxymethylagarose-ethylenediamine-succinyl-17 beta-O-4,6-androstadien-3-one, have been synthesized. Under anaerobic conditions, isomerase bound to these resins is photoinactivated by UV light (lambda greater than 290 nm) whereas isomerase bound to O-carboxymethylagarose-ethylenediamine-deoxycholate or isomerase in the presence of O-carboxymethylagarose-ethylenediamine-acetate is almost completely stable to irradiation under the same conditions. Photoinactivation under anaerobic condition promoted by the resin-bound steroid ketones results from a reaction at the active site since the competitive inhibitor, sodium cholate, which does not absorb light above 290 nm, provides protection toward photoinactivation. Preliminary analysis of isomerase that has been photolyzed in the presence of O-carboxymethylagarose-ethylenediamine-succinyl-17 beta-O-4,6-androstadiene-3-one has established that the enzyme is converted to at least two different forms. One form binds more tightly to the resin than does the native enzyme. This form can be eluted by a sodium dodecyl sulfate containing buffer. The second form is not eluted by this buffer but can be released from the resin by cleavage of the ester bond linking the steroid to the derivatized agarose. We presume that the latter form is covalently coupled to the resin-linked steroid. In the presence of oxygen, additional nonspecific inactivation reactions occur, but these can be suppressed by the singlet oxygen trap, L-histidine. The application of solid-state photoaffinity reagents to some areas of receptor isolation and characterization is discussed.

Enzymes, Immobilized↗
PubMed1986

Detection of a transient enzyme-steroid complex during active-site-directed irreversible inhibition of 3-oxo-delta 5-steroid isomerase.

The reaction of the active-site-directed irreversible inhibitor (17S)-spiro[estra-1,3,5(10),6,8-pentaene-17,2'-oxiran]-3-ol (5 beta) with 3-oxo-delta 5-steroid isomerase has been monitored by repetitive scanning ultraviolet spectroscopy of a solution of 5 beta plus isomerase against a blank containing only 5 beta. Upon initial mixing of 5 beta with the isomerase an absorbance maximum at ca. 250 nm appears. With time, this peak decreases and is replaced with a new peak near 280 nm. These results directly demonstrate the existence of a transient enzyme-steroid intermediate in the inactivation reaction. The ultraviolet spectrum suggests that the steroid in the transient complex resembles the ionized phenol, while the phenolic group in the irreversibly bound complex is un-ionized. These spectral studies support our previous proposal that there are two enzyme-steroid complexes that are related by a 180 degree rotation about an axis perpendicular to the plane of the steroid nucleus. This hypothesis offers an explanation for the reaction of 17 beta-oxiranes with the same residue (Asp-38) that is thought to be involved in the catalytic mechanism. Two new oxiranes, (17S)-spiro[estra-1,3,5(10)-triene-17,2'-oxiran]-3 beta-ol (6 beta) and (17S)-spiro[5 alpha-androstane-17,2'-oxiran]-3-one (8 beta), were also found to be potent active-site-directed irreversible inhibitors of the isomerase (k3/KI = 31 M-1 s-1 and 340 M-1 s-1, respectively). The relationship of these results to the nature of the active site of the isomerase is discussed.

Binding Sites↗
PubMed1985

Irreversible inhibition of delta 5-3-oxosteroid isomerase by 2-substituted progesterones.

2 alpha-Cyanoprogesterone (I) and 2-hydroxymethyleneprogesterone (II) were synthesized and screened as irreversible active-site-directed inhibitors of the delta 5-3-oxosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni. Both compounds were found to inhibit the purified bacterial enzyme in a time-dependent manner. In either case the inactivated enzyme could be dialysed without return of activity, indicating that a stable covalent bond had formed between the inhibitor and the enzyme. Inactivation mediated by compounds (I) and (II) followed pseudo-first-order kinetics, and at higher inhibitor concentrations saturation was observed. The competitive inhibitor 17 beta-oestradiol offered protection against the inactivation mediated by both compounds, and initial-rate studies indicated that compounds (I) and (II) can also act as competitive inhibitors yielding Ki values identical with those generated during inactivation experiments. 2 alpha-Cyanoprogesterone (I) and 2-hydroxymethyleneprogesterone (II) thus appear to be active-site-directed. To compare the reactivity of these 2-substituted progesterones with other irreversible inhibitors of the isomerase, 3 beta-spiro-oxiranyl-5 alpha-pregnan-20 beta-ol (III) was synthesized as the C21 analogue of 3 beta-spiro-oxiranyl-5 alpha-androstan-17 beta-ol, which is a potent inactivator of the isomerase [Pollack, Kayser & Bevins (1979) Biochem. Biophys. Res. Commun. 91, 783-790]. Comparison of the bimolecular rate constants for inactivation (k+3/Ki) mediated by compounds (I)-(III) indicated the following order of reactivity: (III) greater than (II) greater than (I). 2-Mercaptoethanol offers complete protection against the inactivation of the isomerase mediated by 2 alpha-cyanoprogesterone (I). Under the conditions of inactivation compound (I) appears to be completely stable, and no evidence could be obtained for enolate ion formation in the presence or absence of enzyme. It is suggested that cyanoprogesterone inactivates the isomerase after direct nucleophilic attack at the electropositive 2-position, and that tautomerization plays no role in the inactivation event. By contrast, 2-mercaptoethanol offers no protection against the inactivation mediated by 2-hydroxymethyleneprogesterone, and under the conditions of inactivation this compound appears to exist in the semi-enolized form.

Binding Sites↗
PubMed1989

Preparation and characterization of dog pancreas microsomal membranes specifically depleted of protein disulphide-isomerase.

1. The selective release of protein disulphide-isomerase from dog pancreas and rat liver microsomal membranes was studied to throw light on the mechanisms of retention of this enzyme within the endoplasmic reticulum, and in order to prepare microsomal membranes specifically depleted of the enzyme. 2. Protein disulphide-isomerase was quantitatively released from dog pancreas microsomal membranes by washing at pH 9 and above, as demonstrated both by enzyme assay and by immunoblotting analysis. 3. Integral membrane proteins implicated in the process of translocation and segregation of secretory proteins were retained in pH 9-washed dog pancreas microsomal membranes. 4. After pH 9 washing, dog pancreas microsomal membranes were fully active in the translocation, segregation and processing of nascent secretory proteins; these membranes therefore provide a useful experimental system for testing the action of protein disulphide-isomerase on nascent secretory proteins. 5. Protein disulphide-isomerase was not released from rat liver microsomal membranes by pH 9 washing, and was much less readily released from these membranes by sonication, washing etc. than from dog pancreas microsomal membranes. 6. The mechanism of retention of protein disulphide-isomerase, and of other resident proteins of the lumen of the endoplasmic reticulum, is discussed in the light of these findings.

Animals↗
PubMed1995

Isolation of Schizosaccharomyces pombe isopentenyl diphosphate isomerase cDNA clones by complementation and synthesis of the enzyme in Escherichia coli.

Isopentenyl diphosphate (IPP) isomerase catalyzes an essential activation step in the isoprene biosynthetic pathway. The Saccharomyces cerevisiae gene for IPP isomerase, IDI1, was recently isolated and characterized (Anderson, M. S., Muehlbacher, M., Street, I. P., Proffitt, J., and Poulter, C. D. (1989) J. Biol. Chem. 264, 19169-19175), and the wild-type gene, IDI1, was disrupted with a LEU2 marker to create a diploid yeast strain heterozygous for the idi1::leu2 disruption, which revealed that IDI1 was an essential single-copy gene (Mayer, M.P., Hahn, F. M., Stillman, D. J., and Poulter, C. D. (1992) Yeast 8, 743-748). We now report the isolation of a cDNA clone from Schizosaccharomyces pombe by a plasmid shuffle-mediated complementation of the LEU2 disrupted yeast gene. The S. pombe clone encoded a 26,864-dalton polypeptide of 227 amino acids with a high degree of similarity to the S. cerevisiae IDI1 enzyme. S. pombe IPP isomerase contained the essential Cys and Glu catalytic residues identified in yeast isomerase (Street, I. P., Coffman, H. R., Baker, J., and Poulter, C. (1994) Biochemistry 33, 4212-4217) but was significantly smaller than the S. cerevisiae enzyme. The plasmid shuffle technique is an excellent procedure for screening expression libraries for IPP isomerase activity by complementation of the idi1 mutation.

Amino Acid Sequence↗
PubMed1996

A novel abetalipoproteinemia genotype. Identification of a missense mutation in the 97-kDa subunit of the microsomal triglyceride transfer protein that prevents complex formation with protein disulfide isomerase.

The microsomal triglyceride transfer protein (MTP) is a heterodimer composed of the ubiquitous multifunctional protein, protein disulfide isomerase, and a unique 97-kDa subunit. Mutations that lead to the absence of a functional 97-kDa subunit cause abetalipoproteinemia, an autosomal recessive disease characterized by a defect in the assembly and secretion of apolipoprotein B (apoB) containing lipoproteins. Previous studies of abetalipoproteinemic patient, C.L., showed that the 97-kDa subunit was undetectable. In this report, [35S]methionine labeling showed that this tissue was capable of synthesizing the 97-kDa MTP subunit. Electrophoretic analysis showed two bands, one with a molecular mass of the wild type 97-kDa subunit and the other with a slightly lower molecular weight. Sequence analysis of cDNAs from additional intestinal biopsies showed this patient to be a compound heterozygote. One allele contained a perfect in-frame deletion of exon 10, explaining the lower molecular weight band. cDNAs of the second allele were found to contain 3 missense mutations: His297 --> Gln, Asp384 --> Ala, and Arg540 --> His. Transient expression of each mutant showed that only the Arg540 --> His mutant was non-functional based upon its inability to reconstitute apoB secretion in a cell culture system. The other amino acid changes are silent polymorphisms. High level coexpression in a baculovirus system of the wild type 97-kDa subunit or the Arg540 --> His mutant along with human protein disulfide isomerase showed that the wild type was capable of forming an active MTP complex while the mutant was not. Biochemical analysis of lysates from these cells showed that the Arg to His conversion interrupted the interaction between the 97-kDa subunit and protein disulfide isomerase. Replacement of Arg540 with a lysine residue maintained the ability of the 97-kDa subunit to complex with protein disulfide isomerase and form the active MTP holoprotein. These results indicate that a positively charged amino acid at position 540 in the 97-kDa subunit is critical for the productive association with protein disulfide isomerase. Of the 13 mutant MTP 97-kDa subunit alleles described to date, this is the first encoding a missense mutation.

Apolipoproteins B↗
PubMed1997

Cloning and subcellular localization of hamster and rat isopentenyl diphosphate dimethylallyl diphosphate isomerase. A PTS1 motif targets the enzyme to peroxisomes.

To date, isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IPP isomerase; EC 5.3.3.2) is presumed to have a cytosolic localization. However, we have recently shown that in permeabilized cells lacking cytosolic components, mevalonate can be converted to cholesterol, implying that all of the enzymes required for the conversion of mevalonate to farnesyl diphosphate are found in the peroxisome. To provide unequivocal evidence for the subcellular localization of IPP isomerase, in this study, we have cloned the rat and hamster homologues of IPP isomerase and identified the signal that targets this enzyme to peroxisomes. In addition, we also demonstrate that IPP isomerase is regulated at the mRNA level.

Amino Acid Sequence↗
PubMed1976

Lipid requirement of membrane-bound 3-oxosteroid delta4-delta5-isomerase. Studies on beef adrenocortical microsomes.

The role of phospholipid in the beef adrenal microsomal 3-oxosteroid delta4-delta5-isomerase (EC 5.3.1.1) has been investigated with the use of phospholipase A to alter the microsomal phospholipids. The byproducts of phospholipase A digestion have been removed with a wash solution containing bovine serum albumin. Removal of 80-85% of the phospholipid leads to loss of 80-90% of the 3-oxosteroid delta4-delta5-isomerase activity. Reconstitution experiments have been performed by introduction of lipid aqueous dispersions in the enzymatic assay. Asolectin, a commercially available preparation of soy phosphatides, is able to stimulate the enzymatic activity but does not restore the 3-oxosteroid delta4-delta5-isomerase activity in phospholipase-A-treated membranes. In contrast, the introduction of aqueous dispersions of microsomal total lipid mixtures in the enzymatic assay brings about a complete restoration of the 3-oxosteroid delta4-delta5-isomerase activity in the lipid-depleted membranes. It is concluded that the bovine adrenal microsomal 3-oxosteroid delta4-delta5-isomerase requires phospholipid(s) to exhibit its full catalytic activity.

Adrenal Cortex↗
PubMed1976

Human placental 3beta-hydroxysteroid dehydrogenase: delta5-isomerase. Demonstration of an intermediate in the conversion of 3beta-hydroxypregn-5-en-20-one to pregn-4-ene-3,20-dione.

3beta-Hydroxypregn-5-en-20-one (pregnenolone) and NAD+ were incubated with a solubilized preparation of the coupled enzyme 3beta-hydroxysteroid:NAD(P) oxidoreductase-3-ketosteroid delta4,delta5-isomerase (3beta-hydroxysteroid dehydrogenase: delta5-isomerase) from the mitochondrial fraction of human placenta. Unconverted pregnenolone, pregn-4-ene-3,20-dione (rogesterone), and a small but detectable amount of pregn-5-ene-3,20-dione were isolated from the medium by Sephadex LH-20 chromomatography. The identification of pregn-5-ene-3,20-dione, confirmed by mass fragmentography, has provided the first direct evidence for the formation of the hypothetical delta5,3-ketone intermediate in the conversion of pregnenolone to progesterone. When tritium-labeled pregnenolone and [4-14C]pregnenolone were incubated simultaneously the 3H:14C ratio in isolated pregn-5-ene-3,20-dione was 4.6 times greater than in isolated progesterone and pregnenolone, indicating a kinetic isotope effect in the enzymatic isomerization of tritium-labeled pregn-5-ene-3,20-dione. Exposure of the enzyme to two steroids which inhibit the overall enzyme reaction, 2alpha-cyano-17beta-hydroxy-4,4,17alpha-trimethylandrost-5-en-3-one (cyanoketone) and 3-hydroxyestra-1,3,5(10),6,8-pentaen-17-one (equilenin), increased the relative yield of labeled pregn-5-ene-3,20-dione as well as the recovery of radioactivity remaining as unconverted pregnenolone, suggesting that both the dehydrogenase and isomerase activities were inhibited. Exposure of the enzyme to equilenin increased the ratio of isolated pregn-5-ene-3,20-dione radioactivity to progesterone radioactivity as progesterone synthesis was inhibited. Equilenin also diminished the tritium isotope effect on the isomerase reaction. Both findings suggest that it is possible to inhibit the isomerase to a greater extent than the dehydrogenase. In order to measure the rate of progesterone produced by the coupled enzymes, we have modified a radiochemical method which involves precipitation of pregnenolone by digitonin. Digitonin precipitation proved to be effective in separating unconverted pregnenolone from the steroid products of both enzyme reactions, progesterone and pregn-5-ene-3,20-dione. Neither the steroidal inhibitors nor the kinetic isotope effect altered the accuracy of the method for routine measurement of the overall rate of conversion of delta5,3beta-hydroxysteroid to delta4,3-ketosteroid.

Female↗
PubMed1987

A single polypeptide acts both as the beta subunit of prolyl 4-hydroxylase and as a protein disulfide-isomerase.

A single polypeptide is shown to act both as the beta subunit of the proline hydroxylase (EC 1.14.11.2) and as a protein disulfide-isomerase (EC 5.3.4.1). When isolated from chick embryos or rat liver, the beta subunit of prolyl 4-hydroxylase and the enzyme protein disulfide-isomerase have identical molecular weights and peptide maps as produced by digestion with Staphylococcus aureus V8 protease. The apparent molecular weights of both proteins isolated from human placental tissue are slightly higher, and the human beta subunit and one of its peptides have molecular weights about Mr 500 higher than the protein disulfide-isomerase and its corresponding peptide. Experiments with polyclonal and monoclonal antibodies also suggest a structural identity between the two proteins. The beta subunit isolated from the prolyl 4-hydroxylase tetramer has protein disulfide-isomerase activity similar to protein disulfide-isomerase itself, and even the beta subunit when present in the prolyl 4-hydroxylase tetramer has one-half of this activity.

Animals↗
PubMed1994

Delta 3,5, delta 2,4-dienoyl-CoA isomerase from rat liver mitochondria. Purification and characterization of a new enzyme involved in the beta-oxidation of unsaturated fatty acids.

Mitochondrial delta 3,5, delta 2,4-dienoyl-CoA isomerase, which catalyzes the conversion of 3,5-octadienoyl-CoA to 2,4-octadienoyl-CoA, was purified from rat liver 370-fold at almost 30% yield by a six-step purification procedure. The final preparation appeared to be homogeneous as judged by gel electrophoresis. The molecular weights of the native enzyme and its subunit(s) were estimated to be 126,000 and 32,000, respectively. The purification of delta 3,5, delta 2,4-dienoyl-CoA isomerase completes the characterization of the enzymes functioning in the NADPH-dependent pathway for the beta-oxidation of unsaturated fatty acids with double bonds extending from odd-numbered carbon atoms. This novel pathway may not be operative in peroxisomes because delta 3,5, delta 2,4-dienoyl-CoA isomerase was only detected in mitochondria. Substrates of this pathway are 2,5-dienoyl-CoAs formed from 5-enoyl-CoAs by acyl-CoA dehydrogenase. Two sequential isomerization reactions catalyzed by delta 3, delta 2-enoyl-CoAs isomerase and delta 3,5, delta 2,4-dienoyl-CoA isomerase, respectively, convert 2,5-dienoyl-CoAs to 2,4-dienoyl-CoAs, which are reduced by NADPH-dependent 2,4-dienoyl-CoA reductase (EC 1.3.1.34) before reentering the beta-oxidation spiral.

Animals↗
PubMed1991

The adaptability of the active site of trypanosomal triosephosphate isomerase as observed in the crystal structures of three different complexes.

Crystals of triosephosphate isomerase from Trypanosoma brucei brucei have been used in binding studies with three competitive inhibitors of the enzyme's activity. Highly refined structures have been deduced for the complexes between trypanosomal triosephosphate isomerase and a substrate analogue (glycerol-3-phosphate to 2.2 A), a transition state analogue (3-phosphonopropionic acid to 2.6 A), and a compound structurally related to both (3-phosphoglycerate to 2.2 A). The active site structures of these complexes were compared with each other, and with two previously determined structures of triosephosphate isomerase either free from inhibitor or complexed with sulfate. The comparison reveals three conformations available to the "flexible loop" near the active site of triosephosphate isomerase: open (no ligand), almost closed (sulfate), and fully closed (phosphate/phosphonate complexes). Also seen to be sensitive to the nature of the active site ligand is the catalytic residue Glu-167. The side chain of this residue occupies one of two discrete conformations in each of the structures so far observed. A "swung out" conformation unsuitable for catalysis is observed when sulfate, 3-phosphoglycerate, or no ligand is bound, while a "swung in" conformation ideal for catalysis is observed in the complexes with glycerol-3-phosphate or 3-phosphonopropionate. The water structure of the active site is different in all five structures. The results are discussed with respect to the triosephosphate isomerase structure function relationship, and with respect to an on-going drug design project aimed at the selective inhibition of glycolytic enzymes of T. brucei.

Animals↗
PubMed1997

Structure and mechanism of L-fucose isomerase from Escherichia coli.

The three-dimensional structure of L-fucose isomerase from Escherichia coli has been determined by X-ray crystallography at 2.5 A resolution. This ketol isomerase converts the aldose L-fucose into the corresponding ketose L-fuculose using Mn2+ as a cofactor. Being a hexamer with 64,976 Da per subunit, L-fucose isomerase is the largest structurally known ketol isomerase. The enzyme shows neither sequence nor structural similarity with other ketol isomerases. The hexamer obeys D3 symmetry and forms the crystallographic asymmetric unit. The strict and favorably oriented local symmetry allowed for a computational phase extension from 7.3 A to 2.5 A resolution. The structure was solved with an L-fucitol molecule bound to the catalytic center such that the hydroxyl groups at positions 1 and 2 are ligands of the manganese ion. Most likely, L-fucitol mimics a bound L-fucose molecule in its open chain form. The protein environment suggests strongly that the reaction belongs to the ene-diol type.

Aldose-Ketose Isomerases↗
PubMed2001

High enzymatic activity and chaperone function are mechanistically related features of the dimeric E. coli peptidyl-prolyl-isomerase FkpA.

We have recently described the existence of a chaperone activity for the dimeric peptidyl-prolyl cis/trans isomerase FkpA from the periplasm of Escherichia coli that is independent of its isomerase activity. We have now investigated the molecular mechanism of these two activities in vitro in greater detail. The isomerase activity with a protein substrate (RNaseT1) is characterized by a 100-fold higher k(cat)/K(M) value than with a short tetrapeptide substrate. This enhanced activity with a protein is due to an increased affinity towards the protein substrate mediated by a polypeptide-binding site that is distinct from the active site. The chaperone activity is also mediated by interaction of folding and unfolding intermediates with a binding site that is most likely identical to the polypeptide-binding site which enhances catalysis. Both activities are thus mechanistically related, being based on the transient interaction with this high-affinity polypeptide-binding site. Only the isomerase activity, but not the chaperone activity, with the substrate citrate synthase can be inhibited by FK520. Experiments with the isolated domains of FkpA imply that both the isomerase and the chaperone site are located on the highly conserved FKBP domain. The additional amino-terminal domain mediates the dimerization and thus places the two active sites of the FKBP domains in juxtaposition, such that they can simultaneously interact with a protein, and this is required for full catalytic activity.

Binding Sites↗
PubMed2006

A quasi-Laue neutron crystallographic study of D-xylose isomerase.

The location of hydrogen atoms in enzyme structures can bring critical understanding of catalytic mechanism. However, whilst it is often difficult to determine the position of hydrogen atoms using X-ray crystallography even with subatomic (<1.0 A) resolution data available, neutron crystallography provides an experimental tool to directly localize hydrogen/deuterium atoms in biological macromolecules at resolution of 1.5-2.0 A. D-Xylose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5) is a 43 kDa enzyme that catalyses the first reaction in the catabolism of D-xylose. Linearization and isomerization of D-xylose at the active site of D-xylose isomerase rely upon a complex hydrogen transfer. Neutron quasi-Laue data at 2.2 A resolution were collected at room temperature on a partially deuterated Streptomyces rubiginosus D-xylose isomerase crystal using the LADI instrument at ILL with the objective to provide insight into the enzymatic mechanism. The neutron structure shows unambiguously that residue His 53 is doubly protonated at the active site of the enzyme. This suggests that the reaction proceeds through an acid catalyzed opening of the sugar ring, which is in accord with the mechanism suggested by Fenn et al. (Biochemistry 43(21): 6464-6474, 2004). This is the first report of direct observation of double protonation of His 53 and the first validation of the ring opening mechanism at the active site of D-xylose isomerase.

Aldose-Ketose Isomerases↗