HUMANGGP:026099 - FACTA Search

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Query: HUMANGGP:026099 (oxidoreductase)
7,952 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Xylose reductase is a homodimeric oxidoreductase dependent on NADPH or NADH and belongs to the largely monomeric aldo-keto reductase superfamily of proteins. It catalyzes the first step in the assimilation of xylose, an aldose found to be a major constituent monosaccharide of renewable plant hemicellulosic material, into yeast metabolic pathways. It does this by reducing open chain xylose to xylitol, which is reoxidized to xylulose by xylitol dehydrogenase and metabolically integrated via the pentose phosphate pathway. No structure has yet been determined for a xylose reductase, a dimeric aldo-keto reductase or a family 2 aldo-keto reductase. The structures of the Candida tenuis xylose reductase apo- and holoenzyme, which crystallize in spacegroup C2 with different unit cells, have been determined to 2.2 A resolution and an R-factor of 17.9 and 20.8%, respectively. Residues responsible for mediating the novel dimeric interface include Asp-178, Arg-181, Lys-202, Phe-206, Trp-313, and Pro-319. Alignments with other superfamily members indicate that these interactions are conserved in other dimeric xylose reductases but not throughout the remainder of the oligomeric aldo-keto reductases, predicting alternate modes of oligomerization for other families. An arrangement of side chains in a catalytic triad shows that Tyr-52 has a conserved function as a general acid. The loop that folds over the NAD(P)H cosubstrate is disordered in the apo form but becomes ordered upon cosubstrate binding. A slow conformational isomerization of this loop probably accounts for the observed rate-limiting step involving release of cosubstrate. Xylose binding (K(m) = 87 mM) is mediated by interactions with a binding pocket that is more polar than a typical aldo-keto reductase. Modeling of xylose into the active site of the holoenzyme using ordered waters as a guide for sugar hydroxyls suggests a convincing mode of substrate binding.
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PMID:The structure of apo and holo forms of xylose reductase, a dimeric aldo-keto reductase from Candida tenuis. 1210 21

Cinnamoyl-coenzyme A:NADP oxidoreductase (CCR, EC 1.2.1.44), the entry-point enzyme into the monolignol biosynthetic pathway, was purified to apparent electrophoretic homogeneity from differentiating xylem of Eucalyptus gunnii Hook. The purified protein is a monomer of 38 kD and has an isoelectric point of 7. Although Eucalyptus gunnii CCR has approximately equal affinities for all possible substrates (p-coumaroyl-coenzyme A, feruloyl-coenzyme A, and sinapoyl-coenzyme A), it is approximately three times more effective at converting feruloyl-coenzyme A than the other substrates. To gain a better understanding of the catalytic regulation of Eucalyptus CCR, a variety of compounds were tested to determine their effect on CCR activity. CCR activity is inhibited by NADP and coenzyme A. Effectors that bind lysine and cysteine residues also inhibit CCR activity. As a prerequisite to the study of the regulation of CCR at the molecular level, polyclonal antibodies were obtained.
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PMID:Purification and Characterization of Cinnamoyl-Coenzyme A:NADP Oxidoreductase in Eucalyptus gunnii. 1223 55

Multiple sequence alignment and analysis of evolutionary relationships have been used to characterize a family of polyol-specific long-chain dehydrogenases/reductases (PSLDRs). At the present time, 66 known and putative NAD(P)H-dependent oxidoreductases of mainly prokaryotic origin and between 357 and 544 amino acids in length constitute this family. The family is shown to include D-mannitol 2-dehydrogenase, D-mannonate 5-oxidoreductase, D-altronate 5-oxidoreductase, D-arabinitol 4-dehydrogenase, and D-mannitol-1-phosphate 5-dehydrogenase which form individual sub-families (defined by internal sequence identity of >/=30%) having distant origin and divergent substrate specificity but clearly displaying entire-chain relationship. When all forms are aligned, only three residues, Gly-33, Asp-230, and Lys-295 (in the numbering of Pseudomonas fluorescens D-mannitol 2-dehydrogenase (PsM2DH)) are strictly conserved. By combining sequence alignment with the known structure of PsM2DH and results from site-directed mutagenesis, we have developed a structure/function analysis for the family. Gly-33 is in the N-terminal coenzyme-binding domain and part of a nucleotide fingerprint region for the family, and Asp-230 and Lys-295 are at an interdomain segment contributing to the active site in which the lysine likely functions as the catalytic general acid/base. PSLDRs do not require a metal cofactor for activity and are specific for transferring the 4-pro-S hydrogen from NAD(P)H. Comparisons reveal that the core part of the two-domain fold has been conserved throughout all family members, perhaps reflecting the recruitment of a stable oxidoreductase structure and extensive trimming thereof to acquire functional properties specific to each sub-family. They also identify interactions that define the chemical mechanism of oxidoreduction and likely contribute to substrate and co-substrate specificities and are thus relevant for protein engineering.
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PMID:Pseudomonas fluorescens mannitol 2-dehydrogenase and the family of polyol-specific long-chain dehydrogenases/reductases: sequence-based classification and analysis of structure-function relationships. 1260 42

The nicotinamide adenine dinucleotide-specific glutamate dehydrogenase (l-glutamate:NAD(+) oxidoreductase, EC 1.4.1.2) of Chlorella sorokiniana was purified 1,000-fold to electrophoretic homogeneity. The native enzyme was shown to have a molecular weight of 180,000 and to be composed of four identical subunits with a molecular weight of 45,000. The N-terminal amino acid was determined to be lysine. The pH optima for the aminating and deaminating reactions were approximately 8 and 9, respectively. The K(m) values for alpha-ketoglutarate, NADH, NH(4) (+), NAD(+), and l-glutamate were 2 mm, 0.15 mm, 40 mm, 0.15 mm, and 60 mm, respectively. Whereas the K(m) for alpha-ketoglutarate and l-glutamate increased 10-fold, 1 pH unit above or below the pH optima for the aminating or deaminating reactions, respectively, the K(m) values for NADH and NAD(+) were independent of change in pH from 7 to 9.6. By initial velocity, product inhibition, and equilibrium substrate exchange studies, the kinetic mechanism of enzyme was shown to be consistent with a bi uni uni uni ping-pong addition sequence. Although this kinetic mechanism differs from that reported for any other glutamate dehydrogenase, the chemical mechanism still appears to involve the formation of a Schiff base between alpha-ketoglutarate and an epsilon-amino group of a lysine residue in the enzyme. The physical, chemical, and kinetic properties of this enzyme differ greatly from those reported for the NH(4) (+)-inducible glutamate dehydrogenase in this organism.
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PMID:Physical and Kinetic Properties of the Nicotinamide Adenine Dinucleotide-specific Glutamate Dehydrogenase Purified from Chlorella sorokiniana. 1666 Apr 36

Saccharopine dehydrogenase [N6-(glutaryl-2)-L-lysine:NAD oxidoreductase (L-lysine forming)] catalyzes the final step in the alpha-aminoadipate pathway for lysine biosynthesis. It catalyzes the reversible pyridine nucleotide-dependent oxidative deamination of saccharopine to generate alpha-Kg and lysine using NAD+ as an oxidizing agent. The proton shuttle chemical mechanism is proposed on the basis of the pH dependence of kinetic parameters, dissociation constants for competitive inhibitors, and isotope effects. In the direction of lysine formation, once NAD+ and saccharopine bind, a group with a pKa of 6.2 accepts a proton from the secondary amine of saccharopine as it is oxidized. This protonated general base then does not participate in the reaction again until lysine is formed at the completion of the reaction. A general base with a pKa of 7.2 accepts a proton from H2O as it attacks the Schiff base carbon of saccharopine to form the carbinolamine intermediate. The same residue then serves as a general acid and donates a proton to the carbinolamine nitrogen to give the protonated carbinolamine. Collapse of the carbinolamine is then facilitated by the same group accepting a proton from the carbinolamine hydroxyl to generate alpha-Kg and lysine. The amine nitrogen is then protonated by the group that originally accepted a proton from the secondary amine of saccharopine, and products are released. In the reverse reaction direction, finite primary deuterium kinetic isotope effects were observed for all parameters with the exception of V2/K(NADH), consistent with a steady-state random mechanism and indicative of a contribution from hydride transfer to rate limitation. The pH dependence, as determined from the primary isotope effect on DV2 and D(V2/K(Lys)), suggests that a step other than hydride transfer becomes rate-limiting as the pH is increased. This step is likely protonation/deprotonation of the carbinolamine nitrogen formed as an intermediate in imine hydrolysis. The observed solvent isotope effect indicates that proton transfer also contributes to rate limitation. A concerted proton and hydride transfer is suggested by multiple substrate/solvent isotope effects, as well as a proton transfer in another step, likely hydrolysis of the carbinolamine. In agreement, dome-shaped proton inventories are observed for V2 and V2/K(Lys), suggesting that proton transfer exists in at least two sequential transition states.
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PMID:A proposed proton shuttle mechanism for saccharopine dehydrogenase from Saccharomyces cerevisiae. 1722 9

Ignicoccus hospitalis is an autotrophic hyperthermophilic archaeon that serves as a host for another parasitic/symbiotic archaeon, Nanoarchaeum equitans. In this study, the biosynthetic pathways of I. hospitalis were investigated by in vitro enzymatic analyses, in vivo (13)C-labeling experiments, and genomic analyses. Our results suggest the operation of a so far unknown pathway of autotrophic CO(2) fixation that starts from acetyl-coenzyme A (CoA). The cyclic regeneration of acetyl-CoA, the primary CO(2) acceptor molecule, has not been clarified yet. In essence, acetyl-CoA is converted into pyruvate via reductive carboxylation by pyruvate-ferredoxin oxidoreductase. Pyruvate-water dikinase converts pyruvate into phosphoenolpyruvate (PEP), which is carboxylated to oxaloacetate by PEP carboxylase. An incomplete citric acid cycle is operating: citrate is synthesized from oxaloacetate and acetyl-CoA by a (re)-specific citrate synthase, whereas a 2-oxoglutarate-oxidizing enzyme is lacking. Further investigations revealed that several special biosynthetic pathways that have recently been described for various archaea are operating. Isoleucine is synthesized via the uncommon citramalate pathway and lysine via the alpha-aminoadipate pathway. Gluconeogenesis is achieved via a reverse Embden-Meyerhof pathway using a novel type of fructose 1,6-bisphosphate aldolase. Pentosephosphates are formed from hexosephosphates via the suggested ribulose-monophosphate pathway, whereby formaldehyde is released from C-1 of hexose. The organism may not contain any sugar-metabolizing pathway. This comprehensive analysis of the central carbon metabolism of I. hospitalis revealed further evidence for the unexpected and unexplored diversity of metabolic pathways within the (hyperthermophilic) archaea.
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PMID:Insights into the autotrophic CO2 fixation pathway of the archaeon Ignicoccus hospitalis: comprehensive analysis of the central carbon metabolism. 1740 Jul 48

Methionine synthase (MS) is a cobalamin-dependent enzyme. It transfers a methyl group from methyltetrahydrofolate to homocysteine forming methionine and tetrahydrofolate. On the basis of sequence similarity with Escherichia coli cobalamin-dependent MS (MetH), human MS comprises four discrete functional modules that bind from the N- to C-terminus, respectively, homocysteine, methyltetrahydrofolate, cobalamin, and S-adenosylmethionine (AdoMet). The C-terminal activation domain also interacts with methionine synthase reductase (MSR), a NADPH-dependent diflavin oxidoreductase required for the reductive regeneration of catalytically inert cob(II)alamin (which is formed every 200-1000 catalytic cycles of MS) to cob(I)alamin. We have investigated complex formation between the (i) MS activation domain and MSR and (ii) MS activation domain and the isolated FMN-binding domain of MSR. We show that the MS activation domain interacts directly with the FMN-binding domain of MSR. Binding is weakened at high ionic strength, emphasizing the importance of electrostatic interactions at the protein-protein interface. Mutagenesis of conserved lysine residues (Lys1071 and Lys987) in the human activation domain weakens this protein interaction. Chemical cross-linking demonstrates complex formation mediated by acidic residues (FMN-binding domain) and basic residues (activation domain). The activation domain and isolated FMN-domain form a 1:1 complex, but a 1:2 complex is formed with activation domain and MSR. The midpoint reduction potentials of the FAD and FMN cofactors of MSR are not perturbed significantly on forming this complex, implying that electron transfer to cob(II)alamin is endergonic. The kinetics of electron transfer in MSR and the MSR-activation domain complex are similar. Our studies indicate (i) conserved binding determinants, but differences in protein stoichiometry, between human MS and bacterial MetH in complex formation with redox partners; (ii) a substantial endergonic barrier to electron transfer in the reactivation complex; and (iii) a lack of control on the thermodynamics and kinetics of electron transfer in MSR exerted by complex formation with activation domain. The structural and functional consequences of complex formation are discussed in light of the known crystal structure of human activation domain and the inferred conformational heterogeneity of the multidomain MSR-MS complex.
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PMID:Protein interactions in the human methionine synthase-methionine synthase reductase complex and implications for the mechanism of enzyme reactivation. 1747 49

Binding of Zn2+ has been shown previously to inhibit the ubiquinol cytochrome c oxidoreductase (cyt bc1 complex). X-ray diffraction data in Zn-treated crystals of the avian cyt bc1 complex identified two binding sites located close to the catalytic Qo site of the enzyme. One of them (Zn01) might interfere with the egress of protons from the Qo site to the aqueous phase. Using Zn K-edge x-ray absorption fine-structure spectroscopy, we report here on the local structure of Zn2+ bound stoichiometrically to noncrystallized cyt bc1 complexes. We performed a comparative x-ray absorption fine-structure spectroscopy study by examining avian, bovine, and bacterial enzymes. A large number of putative clusters, built by combining information from first-shell analysis and metalloprotein databases, were fitted to the experimental spectra by using ab initio simulations. This procedure led us to identify the binding clusters with high levels of confidence. In both the avian and bovine enzyme, a tetrahedral ligand cluster formed by two His, one Lys, and one carboxylic residue was found, and this ligand attribution fit the crystallographic Zn01 location of the avian enzyme. In the chicken enzyme, the ligands were the His121, His268, Lys270, and Asp253 residues, and in the homologous bovine enzyme they were the His121, His267, Lys269, and Asp254 residues. Zn2+ bound to the bacterial cyt bc1 complex exhibited quite different spectral features, consistent with a coordination number of 6. The best-fit octahedral cluster was formed by one His, two carboxylic acids, one Gln or Asn residue, and two water molecules. It was interesting that by aligning the crystallographic structures of the bacterial and avian enzymes, this group of residues was found located in the region homologous to that of the Zn01 site. This cluster included the His276, Asp278, Glu295, and Asn279 residues of the cyt b subunit. The conserved location of the Zn2+ binding sites at the entrance of the putative proton release pathways, and the presence of His residues point to a common mechanism of inhibition. As previously shown for the photosynthetic bacterial reaction center, zinc would compete with protons for binding to the His residues, thus impairing their function as proton donors/acceptors.
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PMID:X-Ray absorption studies of Zn2+ binding sites in bacterial, avian, and bovine cytochrome bc1 complexes. 1757 35

Humans lack the ability to synthesize vitamin C (ascorbate) due to the absence of gulonolactone oxidase, the last enzyme in the biosynthetic pathway in most other mammals. The corresponding oxidoreductase in trypanosomes therefore represents a target that may be therapeutically exploitable. This is reinforced by our observation that Trypanosoma cruzi, the causative agent of Chagas' disease, lacks the capacity to scavenge ascorbate from its environment and is therefore dependent on biosynthesis to maintain intracellular levels of this vitamin. Here, we show that T. cruzi galactonolactone oxidase (TcGAL) can utilize both L-galactono-gamma-lactone and D-arabinono-gamma-lactone as substrates for synthesis of vitamin C, in reactions that obey Michaelis-Menten kinetics. It is >20-fold more active than the analogous enzyme from the African trypanosome Trypanosoma brucei. FMN is an essential cofactor for enzyme activity and binds to TcGAL non-covalently. In other flavoproteins, a histidine residue located within the N-terminal flavin-binding motif has been shown to be crucial for cofactor binding. Using site-directed mutagenesis, we show that the corresponding residue in TcGAL (Lys-55) is not essential for this interaction. In contrast, we find that histidine and tryptophan residues (His-447 and Trp-448), localized within a C-terminal motif (HWXK) that is a feature of ascorbate-synthesizing enzymes, are necessary for the FMN association. The conserved lysine residue within this motif (Lys-450) is not required for cofactor binding, but its replacement by glycine renders the protein completely inactive.
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PMID:The terminal step in vitamin C biosynthesis in Trypanosoma cruzi is mediated by a FMN-dependent galactonolactone oxidase. 1762 8

Adiponectin is an insulin-sensitizing adipokine with anti-diabetic, anti-atherogenic, anti-inflammatory and cardioprotective properties. This adipokine is secreted from adipocytes into the circulation as three oligomeric isoforms, including trimeric, hexameric and the HMW (high-molecular-mass) oligomeric complex consisting of at least 18 protomers. Each oligomeric isoform of adiponectin exerts distinct biological properties in its various target tissues. The HMW oligomer is the major active form mediating the insulin-sensitizing effects of adiponectin, whereas the central actions of this adipokine are attributed primarily to the hexameric and trimeric oligomers. In patients with Type 2 diabetes and coronary heart disease, circulating levels of HMW adiponectin are selectively decreased due to an impaired secretion of this oligomer from adipocytes. The biosynthesis of the adiponectin oligomers is a complex process involving extensive post-translational modifications. Hydroxylation and glycosylation of several conserved lysine residues in the collagenous domain of adiponectin are necessary for the intracellular assembly and stabilization of its high-order oligomeric structures. Secretion of the adiponectin oligomers is tightly controlled by a pair of molecular chaperones in the ER (endoplasmic reticulum), including ERp44 (ER protein of 44 kDa) and Ero1-Lalpha (ER oxidoreductase 1-Lalpha). ERp44 inhibits the secretion of adiponectin oligomers through a thiol-mediated retention. In contrast, Ero1-Lalpha releases HMW adiponectin trapped by ERp44. The PPARgamma (peroxisome-proliferator-activated receptor gamma) agonists thiazolidinediones selectively enhance the secretion of HMW adiponectin through up-regulation of Ero1-Lalpha. In the present review, we discuss the recent advances in our understanding of the structural and biological properties of the adiponectin oligomeric isoforms and highlight the role of post-translational modifications in regulating the biosynthesis of HMW adiponectin.
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PMID:Post-translational modifications of adiponectin: mechanisms and functional implications. 1817 70

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