Publications

• Biochemical characterization of the 2-ketoacid reductases encoded by ycdW and yiaE genes in Escherichia coli.

  Nunez M.F., Pellicer M.T., Badia J., Aguilar J., Baldoma L.

  Glyoxylate is an important intermediate of the central microbial metabolism formed from acetate, allantoin or glycolate. Depending on the physiological conditions, glyoxylate is incorporated into the central metabolism by the combined actions of the activity of malate synthase and the D-glycerate ... >> More

  Glyoxylate is an important intermediate of the central microbial metabolism formed from acetate, allantoin or glycolate. Depending on the physiological conditions, glyoxylate is incorporated into the central metabolism by the combined actions of the activity of malate synthase and the D-glycerate pathway, or alternatively it can be reduced to glycolate by constitutive glyoxylate reductase activity. At present no information is available on this latter enzyme in Escherichia coli, although similar enzymes, classified as 2-hydroxyacid dehydrogenases, have been characterized in other organisms. A BLAST search using as the query sequence the hydroxypyruvate/glyoxylate reductase from Cucumis sativus identified as an orthologue the yiaE gene of E. coli encoding a ketoaldonate reductase. Use of this sequence in a subsequent BLAST search yielded the ycdW gene as a good candidate to encode glyoxylate reductase in this bacterium. Cloning and overexpression of the ycdW gene showed that its product displayed a high NADPH-linked glyoxylate reductase activity, and also catalysed the reduction of hydroxypyruvate with a lower efficiency. Disruption of the ycdW gene by a chloramphenicol acetyltransferase ('CAT') cassette did not totally abolish the glyoxylate reductase activity, indicating that another enzyme accomplished this function. The similarity with YiaE led us to test whether this protein was responsible for the remaining glyoxylate reductase activity. Purification of YcdW and YiaE proteins permitted their kinetic characterization and comparison. Analysis of the catalytic power (k(cat)/K(m)) disclosed a higher ratio of YcdW for glyoxylate and of YiaE for hydroxypyruvate. << Less

  Biochem. J. 354:707-715(2001) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• An aldo-keto reductase with 2-keto-l-gulonate reductase activity functions in l-tartaric acid biosynthesis from vitamin C in Vitis vinifera.

  Jia Y., Burbidge C.A., Sweetman C., Schutz E., Soole K., Jenkins C., Hancock R.D., Bruning J.B., Ford C.M.

  Tartaric acid has high economic value as an antioxidant and flavorant in food and wine industries. l-Tartaric acid biosynthesis in wine grape (<i>Vitis vinifera</i>) uses ascorbic acid (vitamin C) as precursor, representing an unusual metabolic fate for ascorbic acid degradation. Reduction of the ... >> More

  Tartaric acid has high economic value as an antioxidant and flavorant in food and wine industries. l-Tartaric acid biosynthesis in wine grape (<i>Vitis vinifera</i>) uses ascorbic acid (vitamin C) as precursor, representing an unusual metabolic fate for ascorbic acid degradation. Reduction of the ascorbate breakdown product 2-keto-l-gulonic acid to l-idonic acid constitutes a critical step in this l-tartaric acid biosynthetic pathway. However, the underlying enzymatic mechanisms remain obscure. Here, we identified a <i>V. vinifera</i> aldo-keto reductase, Vv2KGR, with 2-keto-l-gulonic acid reductase activity. Vv2KGR belongs to the d-isomer-specific 2-hydroxyacid dehydrogenase superfamily and displayed the highest similarity to the hydroxyl pyruvate reductase isoform 2 in <i>Arabidopsis thaliana</i> Enzymatic analyses revealed that Vv2KGR efficiently reduces 2-keto-l-gulonic acid to l-idonic acid and uses NADPH as preferred coenzyme. Moreover, Vv2KGR exhibited broad substrate specificity toward glyoxylate, pyruvate, and hydroxypyruvate, having the highest catalytic efficiency for glyoxylate. We further determined the X-ray crystal structure of Vv2KGR at 1.58 Å resolution. Comparison of the Vv2KGR structure with those of d-isomer-specific 2-hydroxyacid dehydrogenases from animals and microorganisms revealed several unique structural features of this plant hydroxyl pyruvate reductase. Substrate structural analysis indicated that Vv2KGR uses two modes (A and B) to bind different substrates. 2-Keto-l-gulonic acid displayed the lowest predicted free-energy binding to Vv2KGR among all docked substrates. Hence, we propose that Vv2KGR functions in l-tartaric acid biosynthesis. To the best of our knowledge, this is the first report of a d-isomer-specific 2-hydroxyacid dehydrogenase that reduces 2-keto-l-gulonic acid to l-idonic acid in plants. << Less

  J. Biol. Chem. 294:15932-15946(2019) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.

• Purification and characterization of a novel NADPH(NADH)-dependent hydroxypyruvate reductase from spinach leaves. Comparison of immunological properties of leaf hydroxypyruvate reductases.

  Kleczkowski L.A., Randall D.D.

  A novel hydroxypyruvate reductase preferring NADPH to NADH as a cofactor was purified over 1500-fold from spinach leaf extracts. The enzyme was an oligomer of about 70 kDa, composed of two subunits of 38 kDa each. The Km for hydroxypyruvate (with NADPH) was about 0.8 mM in the pH range 5.5-6.5, an ... >> More

  A novel hydroxypyruvate reductase preferring NADPH to NADH as a cofactor was purified over 1500-fold from spinach leaf extracts. The enzyme was an oligomer of about 70 kDa, composed of two subunits of 38 kDa each. The Km for hydroxypyruvate (with NADPH) was about 0.8 mM in the pH range 5.5-6.5, and 0.3 mM at pH 8.2. The Vmax. was highest in the pH range 5.5-6.5 and decreased by about 65% at pH 8.2. Above pH 6.0, the enzyme was prone to a strong substrate inhibition by hydroxypyruvate. The reductase could use glyoxylate as an alternative substrate, with rates up to one-quarter of those with hydroxypyruvate. This glyoxylate-dependent activity preferred NADPH to NADH as a cofactor. Rabbit antibodies prepared against NADPH(NADH)-hydroxypyruvate reductase were highly specific for this enzyme and did not cross-react with peroxisomal NADH(NADPH)-dependent hydroxypyruvate reductase, as found by Western immunoblots of proteins from leaf extracts of spinach, pea and wheat. Antibodies raised against purified NADH(NADPH)-hydroxypyruvate reductase were also highly specific, recognizing only their own antigen. To our knowledge, this is the first report in the literature of the occurrence of NADPH(NADH)-hydroxypyruvate reductase in leaves, and the first to provide immunological comparison of leaf hydroxypyruvate reductases. Because of the relatively high rates of the novel reductase in leaf extracts (at least 20 mumol/h per mg of chlorophyll), this enzyme might be an important side-component of the glycollate pathway (photorespiration), possibly utilizing hydroxypyruvate 'leaked' from peroxisomes, and thus contributing to the glycerate pool derived from glycollate. Because of the glyoxylate-dependent activity, the enzyme may also contribute to glycollate formation in leaves. << Less

  Biochem J 250:145-152(1988) [PubMed] [EuropePMC]

• Tartaric acid metabolism. VII. Crystalline hydroxypyruvate reductase (D-glycerate dehydrogenase).

  Kohn L.D., Jakoby W.B.

  J Biol Chem 243:2494-2499(1968) [PubMed] [EuropePMC]

• Identification of hydroxypyruvate and glyoxylate reductases in maize leaves.

  Kleczkowski L.A., Edwards G.E.

  At least two hydroxypyruvate reductases (HPRs), differing in specificity for NAD(P)H and (presumably) utilizing glyoxylate as a secondary substrate, were identified by fractionation of crude maize leaf extracts with ammonium sulfate. The NADH-preferring enzyme, which most probably represented pero ... >> More

  At least two hydroxypyruvate reductases (HPRs), differing in specificity for NAD(P)H and (presumably) utilizing glyoxylate as a secondary substrate, were identified by fractionation of crude maize leaf extracts with ammonium sulfate. The NADH-preferring enzyme, which most probably represented peroxisomal HPR, was precipitated by 30 to 45% saturated ammonium sulfate, while most of the NADPH-dependent activity was found in a 45 to 60% precipitate. The HPRs had similar low K(m)s for hydroxypyruvate (about 0.1 millimolar), regardless of cofactor, while affinities of glyoxylate reductase (GR) reactions for glyoxylate varied widely (K(m)s of 0.4-12 millimolar) depending on cofactor. At high hydroxypyruvate concentrations, the NADPH-HPR from the 30 to 45% precipitate showed negative cooperativity with respect to this reactant, having a second K(m) of 6 millimolar. In contrast, NADPH-HPR from the 45 to 60% precipitate was inhibited at high hydroxypyruvate concentrations (K(1) of 3 millimolar) and, together with NADPH-GR, had only few, if any, common antigenic determinants with NADH-HPR from the 30 to 45% fraction. Both NADPH-HPR and NADPH-GR activities from the 45 to 60% precipitate were probably carried out by the same enzyme(s), as found by kinetic studies. Following preincubation with NADPH, there was a marked increase (up to sixfold) in activity of NADPH-HPR from either crude or fractionated extracts. Most of this increase could be attributed to an artefact resulting from an interference by endogeneous NADPH-phosphatase, which hydrolyzed NADPH to NADH, the latter being utilized by the NADH-dependent HPR. However, in the presence of 15 millimolar fluoride (phosphatase inhibitor), preincubation with NADPH still resulted in over 60% activation of NADPH-HPR. The NADPH treatment stimulated the V(max) of the reductase but had no effect on its K(m) for hydroxypyruvate. Enzyme distribution studies revealed that both NADH and NADPH-dependent HPR and GR activities were predominantly localized in the bundle sheath compartment. Rates of NADPH-HPR and NADPH-GR in this tissue (over 100 micromoles per hour per milligram of chlorophyll each) are in the upper range of values reported for leaves of C(3) species. << Less

  Plant Physiol 91:278-286(1989) [PubMed] [EuropePMC]