Uronate Oxidation by Phytopathogenic Pseudomonads

Kilgore, Wendell W.; Starr, Mortimer P.

doi:10.1038/1831412a0

Cited by 34 publications

(19 citation statements)

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“…Bacterial decomposition of D-glucuronic acid occurs on standing (Keutel, Schweisfurth & Litos, 1961), and Kilgore & Starr (1959) found that cellfree extracts of some phytopathic pseudomonads contained an enzyme capable of oxidizing Dglucuronate to a product believed to be D-glucaric acid. However, there was no change in the acidpotentiated P-glucuronidase inhibition by normal human urine after it had stood for 7 weeks at 00, or for 2 weeks at 200 (A. J. Hay, personal communication); moreover, after ingestion of D-glucuronolactone the excretion of D-glucarate in man increased 100-fold and that of uronic acid only 10-fold.…”

Section: Discussionmentioning

confidence: 99%

Metabolism of d-glucuronolactone in mammalian systems. Identification of d-glucaric acid as a normal constituent of urine

Marsh¹

1963

Biochemical Journal

174

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The inhibition of P-glucuronidase by mam ng animals maintained on normal diets in metabolism cages. steroids, from the fl-D-glucosiduronic acid con-The rat and guinea-pig exereta were separated by filtration, jugates. P-Glucuronidase assay is normally con-and the faeces washed with water, before the combined ducted with a chromogenic substrate, the enzymic urine and washings were tested. Urines were stored at 0' if hydrolysis of which may be diminished in the examined within 24 hr. of collection, or otherwise at -20°. glucuronidase, however, mammnalian urine con-for 40 min. after adjustment with 3N-HCl to pH 2-0-2-2, tains trueenzymeinhibitrs Abul-Fadl(1957) followed by readjustment with NaOH to pH 4-0-45, or tamns true enzyme inhibitors. Abul-Fadl (1957) (b) at 100°for 15 min. after adjustment with 0-1 N-NaOH to found both dialysable and non-dialysable material pH 7-5-8-0, follQwed by readjustment with HCI to pH 60-in human urine which was inhibitory to /-gluc-6-5. The inhibitory power was maximal after treatment

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Section: Discussionmentioning

confidence: 99%

Metabolism of d-glucuronolactone in mammalian systems. Identification of d-glucaric acid as a normal constituent of urine

Marsh¹

1963

Biochemical Journal

174

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“…The isomerization pathway has been previously reported to occur in bacteria, including Escherichia coli (7), Erwinia carotovora (18), Erwinia chrysanthemi (15), Klebsiella pneumoniae (9,23), and Serratia marcescens (28). In the oxidation pathway, aldohexuronate is oxidized to aldohexarate by uronate dehydrogenase (Udh) and further catabolized to pyruvate (2,5,7,9,18,19,24). Uronate dehydrogenase, the key enzyme of this pathway, has been investigated in two plant pathogen bacteria, Pseudomonas syringae and Agrobacterium tumefaciens.…”

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confidence: 99%

Cloning and Characterization of Uronate Dehydrogenases from Two Pseudomonads andAgrobacterium tumefaciensStrain C58

et al. 2009

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Uronate dehydrogenase has been cloned from Pseudomonas syringae pv. tomato strain DC3000, Pseudomonas putida KT2440, and Agrobacterium tumefaciens strain C58. The genes were identified by using a novel complementation assay employing an Escherichia coli mutant incapable of consuming glucuronate as the sole carbon source but capable of growth on glucarate. A shotgun library of P. syringae was screened in the mutant E. coli by growing transformed cells on minimal medium containing glucuronic acid. Colonies that survived were evaluated for uronate dehydrogenase, which is capable of converting glucuronic acid to glucaric acid. In this manner, a 0.8-kb open reading frame was identified and subsequently verified to be udh. Homologous enzymes in P. putida and A. tumefaciens were identified based on a similarity search of the sequenced genomes. Recombinant proteins from each of the three organisms expressed in E. coli were purified and characterized. For all three enzymes, the turnover number (k cat ) with glucuronate as a substrate was higher than that with galacturonate; however, the Michaelis constant (K m ) for galacturonate was lower than that for glucuronate. The A. tumefaciens enzyme was found to have the highest rate constant (k cat ‫؍‬ 1.9 ؋ 10 2 s ؊1 on glucuronate), which was more than twofold higher than those of both of the pseudomonad enzymes.

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“…The isomerase pathway (in Escherichia coli) converts D-galacturonic acid into pyruvate and D-glyceraldehyde 3-phosphate. The oxidative pathway has been described for Agrobacterium tumefaciens and Pseudomonas syringae (2,3). In this pathway, D-galacturonic acid is first oxidized into meso-galactaric acid and then converted in the following step to ␣-ketoglutarate.…”

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Crystal Structure of Uronate Dehydrogenase from Agrobacterium tumefaciens

Parkkinen

Boer

Jänis

et al. 2011

Journal of Biological Chemistry

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D-Galacturonic acid is the main component of pectin, a natural polymer that exists in primary cell walls of terrestrial plants. Citrus peel and sugar beet pulp are cheap raw materials, and both contain a large amount of pectin, which is currently exploited mainly as cattle feed. Pectin has the potential to be an important raw material for biotechnical conversions to fuels and chemicals. The microbial pathways of D-galacturonic acid catabolism have recently been described (1). Two different catabolic pathways, the isomerase pathway and the oxidative pathway, have been found in bacteria. The isomerase pathway (in Escherichia coli) converts D-galacturonic acid into pyruvate and D-glyceraldehyde 3-phosphate. The oxidative pathway has been described for Agrobacterium tumefaciens and Pseudomonas syringae (2, 3). In this pathway, D-galacturonic acid is first oxidized into meso-galactaric acid and then converted in the following step to ␣-ketoglutarate.Uronate dehydrogenase (EC 1.1.1.203) is the key enzyme in the oxidative pathway of D-galacturonic acid catabolism in bacteria. The enzyme catalyzes the oxidation of D-galacturonic acid into D-galactaric acid. Uronate dehydrogenases from A. tumefaciens (4) and P. syringae (5) have been purified and characterized, and the corresponding genes have also been identified (6, 7). A. tumefaciens (Rhizobium radiobacter) uronate dehydrogenase (AtUdh) 2 is specific for NAD ϩ as a cofactor but accepts both D-galacturonic acid and D-glucuronic acid as substrates with similar affinities. AtUdh belongs to the short-chain dehydrogenase/reductase (SDR) superfamily. SDR proteins are NAD(P)(H)-dependent enzymes with a wide spectrum of substrate specificities and also different enzyme classes (21). The sequence identities between the members of the SDR family are low, but they share a similar three-dimensional ␣/␤-structure.To date, no structural information on uronate dehydrogenase is available. Here, we present the first three-dimensional structure of uronate dehydrogenase, namely AtUdh. We determined the crystal structures of AtUdh in the apo-form and the ternary complex with NADH and product at 1.9 and 2.1 Å resolutions, respectively. In addition, we performed a site-directed mutagenesis study of the catalytic residue Tyr-136 and determined the NAD ϩ -bound crystal structure of the inactive mutant Y136A. This crystallographic information has enabled us to identify the active site of the enzyme and the molecular basis for cofactor and substrate recognition. We also propose a structure-based mechanism for the oxidation of D-galacturonic acid. This information can be used to improve the properties of the enzyme, especially the substrate specificity and enzymatic activity.

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Uronate Oxidation by Phytopathogenic Pseudomonads

Cited by 34 publications

References 5 publications

Metabolism of d-glucuronolactone in mammalian systems. Identification of d-glucaric acid as a normal constituent of urine

Metabolism of d-glucuronolactone in mammalian systems. Identification of d-glucaric acid as a normal constituent of urine

Cloning and Characterization of Uronate Dehydrogenases from Two Pseudomonads andAgrobacterium tumefaciensStrain C58

Crystal Structure of Uronate Dehydrogenase from Agrobacterium tumefaciens

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