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At least 829 records · Page 46Linked to original sources
PubMed2005

Chicken retinas contain a retinoid isomerase activity that catalyzes the direct conversion of all-trans-retinol to 11-cis-retinol.

Vertebrate retinas contain two types of light-detecting cells. Rods subserve vision in dim light, while cones provide color vision in bright light. Both contain light-sensitive proteins called opsins. The light-absorbing chromophore in most opsins is 11-cis-retinaldehyde, which is isomerized to all-trans-retinaldehyde by absorption of a photon. Restoration of light sensitivity requires chemical re-isomerization of retinaldehyde by an enzymatic pathway called the visual cycle in the retinal pigment epithelium. The isomerase in this pathway uses all-trans-retinyl esters synthesized by lecithin retinol acyl transferase (LRAT) as the substrate. Several lines of evidence suggest that cone opsins regenerate by a different mechanism. Here we demonstrate the existence of two catalytic activities in chicken retinas. The first is an isomerase activity that effects interconversion of all-trans-retinol and 11-cis-retinol. The second is an ester synthase that effects palmitoyl coenzyme A-dependent synthesis of all-trans- and 11-cis-retinyl esters. Kinetic analysis of these two activities suggests that they act in concert to drive the formation of 11-cis-retinoids in chicken retinas. These activities may be part of a new visual cycle for the regeneration of chromophores in cones.

Animals↗
PubMed2006

Uronate isomerase: a nonhydrolytic member of the amidohydrolase superfamily with an ambivalent requirement for a divalent metal ion.

Uronate isomerase, a member of the amidohydrolase superfamily, catalyzes the isomerization of D-glucuronate and D-fructuronate. During the interconversion of substrate and product the hydrogen at C2 of D-glucuronate is transferred to the pro-R position at C1 of the product, D-fructuronate. The exchange of the transferred hydrogen with solvent deuterium occurs at a rate that is 4 orders of magnitude slower than the interconversion of substrate and product. The enzyme catalyzes the elimination of fluoride from 3-deoxy-3-fluoro-D-glucuronate. These results have been interpreted to suggest a chemical reaction mechanism in which an active site base abstracts the proton from C2 of D-glucuronate to form a cis-enediol intermediate. The conjugate acid then transfers this proton to C1 of the cis-enediol intermediate to form D-fructuronate. The loss of fluoride from 3-deoxy-3-fluoro-D-glucuronate is consistent with a stabilized carbanion at C2 of the substrate during substrate turnover. The slow exchange of the transferred hydrogen with solvent water is consistent with a shielded conjugate acid after abstraction of the proton from either D-glucuronate or D-fructuronate during the isomerization reaction. This conclusion is supported by the competitive inhibition of the enzymatic reaction by D-arabinaric acid and the monohydroxamate derivative with Ki values of 13 and 670 nM, respectively. There is no evidence to support a hydride transfer mechanism for uronate isomerase. The wild type enzyme was found to contain 1 equiv of zinc per subunit. The divalent cation could be removed by dialysis against the metal chelator, dipicolinate. However, the apoenzyme has the same catalytic activity as the Zn-substituted enzyme and thus the divalent metal ion is not required for enzymatic activity. This is the only documented example of a member in the amidohydrolase superfamily that does not require one or two divalent cations for enzymatic activity.

Aldose-Ketose Isomerases↗
PubMed1996

13C NMR relaxation studies of backbone and side chain motion of the catalytic tyrosine residue in free and steroid-bound delta 5-3-ketosteroid isomerase.

Side chain and backbone dynamics of the catalytic residue, Tyr-14, in free and steroid-bound delta 5-3-ketosteroid isomerase (EC 5.3.3.1, homodimer, M(r) = 26.8 kDa) have been examined by measurements of longitudinal and transverse 13C relaxation rates and nuclear Overhauser effects at both 500 and 600 MHz (proton frequencies). The data, analyzed using the model-free formalism, yielded an optimized correlation time for overall molecular rotation (tau m) of 17.9 ns, in agreement with the result (18 ns) of fluorescence anisotropy decay measurements [Wu, P., Li, Y.-K., Talalay, P., & Brand, L. (1994) Biochemistry 33, 7415-7422] and Stokes' law calculation (20 ns). The order parameter of the side chain C epsilon of Tyr-14 (S2 = 0.74), which is a measure of the restriction of its high-frequency (nanosecond to picosecond) motion, was significantly lower than that of the backbone C alpha (S2 = 0.82), indicating greater restriction of backbone motion. Upon binding of the steroid ligand, 19-nortestosterone hemisuccinate, a product analog and substrate of the reverse isomerase reaction, S2 of the side chain C epsilon increased from 0.74 to 0.86, while that of the backbone C alpha did not change significantly. Thus, in the steroid complex, the amplitude of high-frequency side chain motion of Tyr-14 became more restricted than that of its backbone which could lower the entropy barrier to catalysis. Lower-frequency (millisecond to microsecond) motion of Tyr-14 at rates comparable to kcat were detected by exchange contributions to transverse relaxation of both C epsilon and C alpha. Steroid binding produced no change in this low-frequency motion of the side chain of Tyr-14, which could contribute to substrate binding and product release, but decreased the exchange contribution to transverse relaxation of the backbone.

Androstenedione↗
PubMed1997

Effect of prolyl isomerase on the folding reactions of staphylococcal nuclease.

The low-temperature fluorescence-detected refolding of staphylococcal nuclease (SNase) can be described by three slow kinetic phases. The slowest phase is absent in the P117G mutant of SNase. Peptidyl prolyl cis-trans isomerase (cyclophilin), which has been shown to catalyze the slow folding reactions of some proteins, was employed to determine which of the refolding reactions of SNase and P117G SNase involve proline isomerization. We report here that all three folding phases of the wild type and the slower phase of P117G SNase are catalyzed by prolyl isomerase, indicating that proline isomerization is involved in these fluorescence-detected phases in the refolding of SNase. Since the rates of these phases are denaturant-dependent, we conclude that the slow folding steps involve isomerization of non-native cis proline peptide bonds and are tightly coupled to denaturant-sensitive structural changes.

Catalysis↗
PubMed1998

Electrophilic assistance by Asp-99 of 3-oxo-Delta 5-steroid isomerase.

3-Oxo-Delta 5-steroid isomerase (Delta 5-3-ketosteroid isomerase, KSI; EC 5.3.3.1) catalyzes the conversion of a variety of beta, gamma-unsaturated 3-oxosteroids to their corresponding alpha, beta-unsaturated isomers at rates that approach the diffusion limit for specific substrates. The reaction proceeds through a dienolate intermediate, with two amino acid residues (Asp-38 and Tyr-14) known to be involved in catalysis. When the complete three-dimensional structure of KSI was determined recently by NMR methods, an additional polar residue (Asp-99) was found in the active site and this group was shown to be important for catalytic activity. In this work, we examine the properties of several mutant KSIs to determine the nature of catalysis by Asp-99 of KSI. The electrophoretic mobilities of wild-type (WT) KSI and several mutants (D99A, D99N, D38N, and D38N/D99A) on native gels were determined at pH values ranging from 6.0 to 8.5. The results demonstrate that the pKa of Asp-99 is >8.5 in wild-type KSI. The pH-rate profiles for the D99A, D99N, and D38H/D99A mutants of KSI were also determined. For all three mutants, kcat and kcat/KM do not decrease at high pH, in contrast to those for WT and D38H, which lose activity above pH 9 and 8, respectively. Mutation of Asp-99 to Asn decreases kcat for the substrate 5-androstene-3,17-dione by 27-fold and kcat/Km by 23-fold, substantially less than the loss of activity (3000-fold in kcat and 2200-fold in kcat/Km) observed when Asp-99 is mutated to Ala, consistent with a hydrogen bonding role for Asp-99. Taken together, these results provide evidence that Asp-99 participates in catalysis in its protonated form, with a pKa of >9 in WT and approximately 8.5 in the D38H mutant. Asp-99 likely donates a hydrogen bond to O-3 of the steroid, helping to stabilize the transition state(s) of the KSI-catalyzed reaction.

Alanine↗
PubMed1999

Unfolding of Plasmodium falciparum triosephosphate isomerase in urea and guanidinium chloride: evidence for a novel disulfide exchange reaction in a covalently cross-linked mutant.

The conformational stability of Plasmodium falciparum triosephosphate isomerase (TIMWT) enzyme has been investigated in urea and guanidinium chloride (GdmCl) solutions using circular dichroism, fluorescence, and size-exclusion chromatography. The dimeric enzyme is remarkably stable in urea solutions. It retains considerable secondary, tertiary, and quaternary structure even in 8 M urea. In contrast, the unfolding transition is complete by 2.4 M GdmCl. Although the secondary as well as the tertiary interactions melt before the perturbation of the quaternary structure, these studies imply that the dissociation of the dimer into monomers ultimately leads to the collapse of the structure, suggesting that the interfacial interactions play a major role in determining multimeric protein stability. The Cm(urea)/Cm(GdmCl) ratio (where Cm is the concentration of the denaturant required at the transition midpoint) is unusually high for triosephosphate isomerase as compared to other monomeric and dimeric proteins. A disulfide cross-linked mutant protein (Y74C) engineered to form two disulfide cross-links across the interface (13-74') and (13'-74) is dramatically destablized in urea. The unfolding transition is complete by 6 M urea and involves a novel mechanism of dimer dissociation through intramolecular thiol-disulfide exchange.

Animals↗
PubMed1998

Solution structure of Delta 5-3-ketosteroid isomerase complexed with the steroid 19-nortestosterone hemisuccinate.

The solution structure of the ketosteroid isomerase homodimer complexed with the product analogue 19-nortestosterone hemisuccinate (19-NTHS) was solved by heteronuclear multidimensional NMR methods using 1647 distance restraints, 77 dihedral angle (phi) restraints, and 67 hydrogen bond restraints per monomer. The refined secondary structure of each subunit consists of three alpha-helices, eight beta-strands, four turns, and two beta-bulges. The beta-strands form a mixed beta-sheet. One of the five proline residues, Pro-39, is cis and begins a nonclassical turn. A self-consistent ensemble of 15 tertiary/quaternary structures of the enzyme dimer-steroid complex, with no distance violations greater than 0.35 A, was generated by simulated annealing and energy minimization with the program X-PLOR. The mean pairwise RMSD of the secondary structural elements was 0.63 A for the average subunit and 1.25 A for the dimer. Within each subunit, the three alpha-helices are packed onto the concave surface of the beta-sheet with a groove between them into which the steroid binds at a site defined by 14 intermolecular distances. In the productive complex, Tyr-14, from alpha-helix 1, approaches both Asp-99 and the 3-keto group of 19-NTHS while, from beta-strand 1, the carboxylate of Asp-38 approaches the beta-face of the steroid near C4 and C6, between which it transfers a proton during catalysis. Thus the solution structure of the isomerase-steroid complex can accommodate the catalytic diad mechanism in which Asp-99 donates a hydrogen bond to Tyr-14 which in turn is hydrogen bonded to the 3-oxygen of the steroid. While direct hydrogen bonding of Asp-99 to the steroid oxygen is less likely, it cannot be excluded. All other interactions of the steroid with the enzyme are hydrophobic. The dimer interface, which is between the convex surfaces of the beta-sheets, is defined by 28 intersubunit NOEs between hydrophobic residues in the 13C-filtered NOESY-HSQC spectrum of a 13C/12C-heterolabeled dimer. Both hydrophobic and polar interactions occur at the dimer interface which contains no space that would permit additional steroid binding. Comparison of the complexed enzyme with the solution structure of the free enzyme [Wu et al. (1997) Science 276, 415-418] reveals that the three helices change position in the steroid complex, becoming more closely packed onto the concave surface of the beta-sheet, thus bringing Tyr-14 closer to Asp-99 and the substrate. Comparison of the enzyme-steroid complex in solution with the free enzyme in the crystalline state reveals similar differences between the positions of the helices.

Binding Sites↗
PubMed2000

Crystal structure of rabbit phosphoglucose isomerase, a glycolytic enzyme that moonlights as neuroleukin, autocrine motility factor, and differentiation mediator.

The multifunctional protein phosphoglucose isomerase, also known as neuroleukin, autocrine motility factor, and differentiation and maturation mediator, has different roles inside and outside the cell. In the cytoplasm, it catalyzes the second step in glycolysis. Outside the cell, it serves as a nerve growth factor and cytokine. We have determined the three-dimensional structure of rabbit muscle phosphoglucose isomerase complexed with the competitive inhibitor D-gluconate 6-phosphate by X-ray crystallography at 2.5 A resolution. The structure shows that the enzyme is a dimer with two alpha/beta-sandwich domains in each subunit. The location of the bound D-gluconate 6-phosphate inhibitor leads to the identification of residues involved in substrate specificity (Ser209, Ser159, Thr214, Thr217, and Thr211). The results of previously published kinetic studies suggest that a lysine and a histidine are involved in the catalytic mechanism. The crystal structure suggests active site residues Lys518 and His388 might be these residues. In addition, the positions of amino acid residues that are substituted in the genetic disease nonspherocytic hemolytic anemia suggest how these substitutions can result in altered catalysis or protein stability.

Amino Acid Sequence↗
PubMed2004

Temperature effects on the catalytic activity of the D38E mutant of 3-oxo-Delta5-steroid isomerase: favorable enthalpies and entropies of activation relative to the nonenzymatic reaction catalyzed by acetate ion.

3-oxo-Delta5-steroid isomerase (ketosteroid isomerase, KSI) catalyzes the isomerization of 5-androstene-3,17-dione (1) to 4-androstene-3,17-dione (3) via a dienolate intermediate (2-). KSI catalyzes this conversion about 13 orders of magnitude faster than the corresponding reaction catalyzed by acetate ion, a difference in activation energy (DeltaG) of approximately 18 kcal/mol. To evaluate whether the decrease in DeltaG by KSI is due to enthalpic or entropic effects, the activation parameters for the isomerization of 1 catalyzed by the D38E mutant of KSI were determined. A linear Arrhenius plot of kcat/KM versus 1/T gives the activation enthalpy (DeltaH = 5.9 kcal/mol) and activation entropy (TDeltaS = -2.6 kcal/mol). Relative to catalysis by acetate, D38E reduces DeltaH by approximately 10 kcal/mol and increases TDeltaS by approximately 5 kcal/mol. The activation parameters for the microscopic rate constants for D38E catalysis were also determined and compared to those for the acetate ion-catalyzed reaction. Enthalpic stabilization of 2- and favorable entropic effects in both chemical transition states by D38E result in an overall energetically more favorable enzymatic reaction relative to that catalyzed by acetate ion.

Catalysis↗
PubMed1991

Crystallographic and molecular modeling studies on trypanosomal triosephosphate isomerase: a critical assessment of the predicted and observed structures of the complex with 2-phosphoglycerate.

In the continuation of a project aimed at the rational design of drugs against diseases caused by trypanosomes, the crystal structure of trypanosomal triosephosphate isomerase in complex with the active site inhibitor 2-phosphoglycerate has been determined. Two alternative modeling protocols have been attempted to predict the mode of binding of this ligand. In the first protocol, certain key interactions were restrained in the modeling procedure. In the second protocol, a full search of ligand conformational space was performed. In both cases the protein scaffold was kept static. Both protocols produced models which were reasonably close to the observed structure (rms difference less than 2.0 A). Nevertheless, some essential features were missed by each of the protocols. The crystallographic structure of the 2-PGA TIM complex shows that the ligand binds fully within the active site of TIM, with partners for all but one of the ligand's strongly hydrogen bonding groups. Several of the interactions between the ligand and the active site of TIM are seen to be common to all of the complexes so far structurally characterized between trypanosomal triosephosphate isomerase and competitive inhibitors. Such key interactions appear to be the best guide in the prediction of the binding mode of a new inhibitor.

Animals↗
PubMed2003

Production of tagatose by a recombinant thermostable L-arabinose isomerase from Thermus sp. IM6501.

A gene (thaI) corresponding to L-arabinose isomerase from Thermus strain IM6501 was cloned by PCR. It comprised 1488 nucleotides and encoded a polypeptide of 496 residues with a predicted molecular weight of 56019 Da. The deduced amino acid sequence had 96.8% identity with the L-arabinose isomerase of Geobacillus stearothermophilus. Recombinant ThaI with N-terminal hexa-tistidine tags was over-expressed in Escherichia coli and purified by affinity chromatography using Ni-NTA resin. The purified ThaI was thermostable with maximal activity at 60 degrees C at pH 8 for 30 min of reaction. Zn2+ and Ni2+ inactivated the catalytic activity of ThaI, 5 mM Mn2+ enhanced the bioconversion yield by 90%. The bioconversion yield of 54% from D-galactose to D-tagatose was obtained by recombinant ThaI at 60 degrees C over 3 d.

Aldose-Ketose Isomerases↗
PubMed1988

Mouse glucose-6-phosphate isomerase and neuroleukin have identical 3' sequences.

Neuroleukin is a neurotrophic factor of relative molecular mass (Mr) 56,000 (56K) found in skeletal muscle, brain, heart and kidneys which supports the survival of embryonic spinal neurones, skeletal motor neurones and sensory neurones. Neuroleukin is also a lymphokine product of lectin-stimulated T cells and induces immunoglobulin secretion by cultured human peripheral blood mononuclear cells. Mouse neuroleukin has been cloned, the complete nucleotide sequence has been determined and its complementary DNA has been transiently expressed in monkey COS-1 cells. The serum-free supernatant of the transfected, but not of control mock-transfected, cells was shown to mimic the properties of neuroleukin isolated from mouse salivary glands. In our work on the molecular genetics of carbohydrate metabolism we have recently isolated a mouse glucose-6-phosphate isomerase (or phosphoglucose isomerase, PGI) cDNA clone using the yeast PGI gene (PGI 1) as a probe. We report here that there is complete sequence identity between the 759 nucleotides at the 3' end of this clone (coding and non-coding) and the sequence of mouse neuroleukin.

Amino Acid Sequence↗
PubMed2000

Crystal structure of the protein disulfide bond isomerase, DsbC, from Escherichia coli.

DsbC is one of five Escherichia coli proteins required for disulfide bond formation and is thought to function as a disulfide bond isomerase during oxidative protein folding in the periplasm. DsbC is a 2 x 23 kDa homodimer and has both protein disulfide isomerase and chaperone activity. We report the 1.9 A resolution crystal structure of oxidized DsbC where both Cys-X-X-Cys active sites form disulfide bonds. The molecule consists of separate thioredoxin-like domains joined via hinged linker helices to an N-terminal dimerization domain. The hinges allow relative movement of the active sites, and a broad uncharged cleft between them may be involved in peptide binding and DsbC foldase activities.

Amino Acid Sequence↗
PubMed2002

The hsp70 chaperone DnaK is a secondary amide peptide bond cis-trans isomerase.

Peptidyl prolyl cis-trans isomerases can enzymatically assist protein folding, but these enzymes exclusively target the peptide bond preceding proline residues. Here we report the identification of the Hsp70 chaperone DnaK as the first member of a novel enzyme class of secondary amide peptide bond cis-trans isomerases (APIases). APIases selectively accelerate the cis-trans isomerization of nonprolyl peptide bonds. Results from independent experiments support the APIase activity of DnaK: (i) exchange crosspeaks between the cis-trans conformers appear in 2D (1)H NMR exchange spectra of oligopeptides (ii) the rate constants for the cis-trans isomerization of various dipeptides increase and (iii) refolding of the RNase T1 P39A variant is catalyzed. The APIase activity shows both regio and stereo selectivity and is stimulated two-fold in the presence of the complete DnaK/GrpE/DnaJ/ATP refolding system. Moreover, known DnaK-binding oligopeptides simultaneously affect the APIase activity of DnaK and the refolding yield of denatured firefly luciferase in the presence of DnaK/GrpE/DnaJ/ATP. These results suggest a new role for the chaperone as a regioselective catalyst for bond rotation in polypeptides.

Adenosine Triphosphate↗
PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗
PubMed2001

Mutation of yeast Eug1p CXXS active sites to CXXC results in a dramatic increase in protein disulphide isomerase activity.

Protein disulphide isomerase (PDI) is an essential protein which is localized to the endoplasmic reticulum of eukaryotic cells. It catalyses the formation and isomerization of disulphide bonds during the folding of secretory proteins. PDI is composed of domains with structural homology to thioredoxin and with CXXC catalytic motifs. EUG1 encodes a yeast protein, Eug1p, that is highly homologous to PDI. However, Eug1p contains CXXS motifs instead of CXXC. In the current model for PDI function both cysteines in this motif are required for PDI-catalysed oxidase activity. To gain more insight into the biochemical properties of this unusual variant of PDI we have purified and characterized the protein. We have furthermore generated a number of mutant forms of Eug1p in which either or both of the active sites have been mutated to a CXXC sequence. To determine the catalytic capacity of the wild-type and mutant forms we assayed activity in oxidative refolding of reduced and denatured procarboxypeptidase Y as well as refolding of bovine pancreatic trypsin inhibitor. The wild-type protein showed very little activity, not only in oxidative refolding but also in assays where only isomerase activity was required. This was surprising, in particular since mutant forms of Eug1p containing a CXXC motif displayed activity close to that of genuine PDI. These results lead us to propose that general disulphide isomerization is not the main function of Eug1p in vivo.

Amino Acid Motifs↗
PubMed2000

Bioconversion of D-galactose into D-tagatose by expression of L-arabinose isomerase.

D-Tagatose is a potential bulking agent in food as a non-calorific sweetener. To produce D-tagatose from cheaper resources, plasmids harbouring the L-arabinose isomerase gene (araA) from Escherichia coli, Bacillus subtilis and Salmonella typhimurium were constructed because L-arabinose isomerase was suggested previously as an enzyme that mediates the bioconversion of galactose into tagatose as well as that of arabinose to ribulose. The constructed plasmids were named pTC101, pTC105 and pTC106, containing araA from E. coli, B. subtilis and S. typhimurium respectively. In the cultures of recombinant E. coli with pTC101, pTC105 and pTC106, tagatose was produced from galactose in 9.9, 7.1 and 6.9% yields respectively. The enzyme extract of E. coli with the plasmid pTC101 also converted galactose into tagatose with a 96.4% yield.

Aldose-Ketose Isomerases↗
PubMed2001

Improvement of tagatose conversion rate by genetic evolution of thermostable galactose isomerase.

To enhance the isomerization rate of galactose into tagatose, a thermostable galactose isomerase, which was isolated from bacteria growing in a hot spring, was genetically improved using the error-prone PCR method. From 500 mutant clones, a clone showing improved conversion activity was selected. The sequence of the selected clone had five amino acid changes: His(228)-->Asp, Gly(384)-->Asp, Ser(393)-->Thr, Lys(428)-->Asn and Asp(475)-->Lys. The improved galactose isomerase had an 11-fold higher reaction rate than the original.

Aldose-Ketose Isomerases↗