Identification in Agrobacterium tumefaciens of the D-galacturonic acid dehydrogenase gene.
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.
Boer
Harry
H
VTT Technical Research Centre of Finland, Espoo, Finland.
Maaheimo
Hannu
H
Koivula
Anu
A
Penttilä
Merja
M
Richard
Peter
P
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
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