title: Identification in Agrobacterium tumefaciens of the D-galacturonic acid dehydrogenase gene.
abstract: There are at least three different pathways for the catabolism of D-galacturonate in microorganisms. In the oxidative pathway, which was described in some prokaryotic species, D-galacturonate is first oxidised to meso-galactarate (mucate) by a nicotinamide adenine dinucleotide (NAD)-dependent dehydrogenase (EC 1.1.1.203). In the following steps of the pathway mucate is converted to 2-keto-glutarate. The enzyme activities of this catabolic pathway have been described while the corresponding gene sequences are still unidentified. The D-galacturonate dehydrogenase was purified from Agrobacterium tumefaciens, and the mass of its tryptic peptides was determined using MALDI-TOF mass spectrometry. This enabled the identification of the corresponding gene udh. It codes for a protein with 267 amino acids having homology to the protein family of NAD(P)-binding Rossmann-fold proteins. The open reading frame was functionally expressed in Saccharomyces cerevisiae. The N-terminally tagged protein was not compromised in its activity and was used after purification for a kinetic characterization. The enzyme was specific for NAD and accepted D-galacturonic acid and D-glucuronic acid as substrates with similar affinities. NMR analysis showed that in water solution the substrate D-galacturonic acid is predominantly in pyranosic form which is converted by the enzyme to 1,4 lactone of galactaric acid. This lactone seems stable under intracellular conditions and does not spontaneously open to the linear meso-galactaric acid.
authors.affiliation: VTT Technical Research Centre of Finland, Espoo, Finland.
authors.forename: Harry
authors.initials: H
authors.lastname: Boer
authors.forename: Hannu
authors.initials: H
authors.lastname: Maaheimo
authors.forename: Anu
authors.initials: A
authors.lastname: Koivula
authors.forename: Merja
authors.initials: M
authors.lastname: Penttilä
authors.forename: Peter
authors.initials: P
authors.lastname: Richard
mesh_terms: D016960:Agrobacterium tumefaciens / Q000201:enzymology*; D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D015870:Gene Expression; D020723:Glucuronic Acid / Q000378:metabolism; D006603:Hexuronic Acids / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D053858:Metabolic Networks and Pathways; D008954:Models, Biological; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013400:Sugar Acids / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D006603:Hexuronic Acids; D011993:Recombinant Fusion Proteins; D013400:Sugar Acids; D009243:NAD; C007819:galacturonic acid; D020723:Glucuronic Acid; C000090:galactaric acid; C023280:uronate dehydrogenase; D000445:Aldehyde Oxidoreductases