Identification in Agrobacterium tumefaciens of the D-galacturonic acid dehydrogenase gene

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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 mesogalactarate (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.

Original languageEnglish
Pages (from-to)901-909
JournalApplied Microbiology and Biotechnology
Volume86
Issue number3
DOIs
Publication statusPublished - 1 Apr 2010
MoE publication typeA1 Journal article-refereed

Fingerprint

Agrobacterium tumefaciens
Oxidoreductases
NAD
uronate dehydrogenase
Lactones
Genes
Proteins
Enzymes
Glutarates
Glucuronic Acid
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Open Reading Frames
Saccharomyces cerevisiae
Mass Spectrometry
Amino Acids
Peptides
galacturonic acid
Water
galactaric acid

Keywords

  • EC 1.1.1.203
  • Lactone
  • Meso-galactaric acid
  • Mucic acid
  • Oxidative pathway

Cite this

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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 mesogalactarate (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.",
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T1 - Identification in Agrobacterium tumefaciens of the D-galacturonic acid dehydrogenase gene

AU - Boer, Harry

AU - Maaheimo, Hannu

AU - Koivula, Anu

AU - Penttilä, Merja

AU - Richard, Peter

N1 - CA2: TK404 CA2: TK402 CA2: TK400 ISI: BIOTECHNOLOGY & APPLIED MICROBIOLOGY

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N2 - 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 mesogalactarate (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.

AB - 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 mesogalactarate (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.

KW - EC 1.1.1.203

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