Applied Microbiology and Biotechnology

, Volume 86, Issue 3, pp 901–909 | Cite as

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

  • Harry Boer
  • Hannu Maaheimo
  • Anu Koivula
  • Merja Penttilä
  • Peter Richard
Biotechnologically Relevant Enzymes and Proteins


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 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.


meso-galactaric acid Mucic acid Lactone Oxidative pathway EC 



We thank Arja Kiema for technical assistance. This work was supported by the Academy of Finland through the following programmes: Finnish Centre of Excellence in White Biotechnology–Green Chemistry (decision number 118573) and an Academy Research Fellowship for P.R.


  1. Ashwell G, Wahba AJ, Hickman J (1960) Uronic acid metabolism in bacteria. I. Purification and properties of uronic acid isomerase in Escherichia coli. J Biol Chem 235:1559–1565Google Scholar
  2. Bateman DF, Kosuge T, Kilgore WW (1970) Purification and properties of uronate dehydrogenase from Pseudomonas syringae. Arch Biochem Biophys 136:97–105CrossRefGoogle Scholar
  3. Chang YF, Feingold DS (1969) Hexuronic acid dehydrogenase of Agrobacterium tumefaciens. J Bacteriol 99:667–673Google Scholar
  4. Chang YF, Feingold DS (1970) d-glucaric acid and galactaric acid catabolism by Agrobacterium tumefaciens. J Bacteriol 102:85–96Google Scholar
  5. Cynkin MA, Ashwell G (1960) Uronic acid metabolism in bacteria. IV. Purification and properties of 2-keto-3-deoxy-d-gluconokinase in Escherichia coli. J Biol Chem 235:1576–1579Google Scholar
  6. Doran-Peterson J, Cook DM, Brandon SK (2008) Microbial conversion of sugars from plant biomass to lactic acid or ethanol. Plant J 54:582–592CrossRefGoogle Scholar
  7. Goodner B et al (2001) Genome sequence of the plant pathogen and biotechnology agent Agrobacterium tumefaciens C58. Science 294:2323–2328CrossRefGoogle Scholar
  8. Hickman J, Ashwell G (1960) Uronic acid metabolism in bacteria. II. Purification and properties of d-altronic acid and d-mannonic acid dehydrogenases in Escherichia coli. J Biol Chem 235:1566–1570Google Scholar
  9. Hilditch S, Berghäll S, Kalkkinen N, Penttilä M, Richard P (2007) The missing link in the fungal D-galacturonate pathway: identification of the l-threo-3-deoxy-hexulosonate aldolase. J Biol Chem 282:26195–26201CrossRefGoogle Scholar
  10. Hubbard BK, Koch M, Palmer DR, Babbitt PC, Gerlt JA (1998) Evolution of enzymatic activities in the enolase superfamily: characterization of the (d)-glucarate/galactarate catabolic pathway in Escherichia coli. Biochemistry 37:14369–14375CrossRefGoogle Scholar
  11. Jeffcoat R (1975) Studies on the subunit structure of 4-deoxy-5-oxoglucarate hydro-lyase (decarboxylating) from Pseudomonas acidovorans. Biochem J 145:305–309Google Scholar
  12. Kuorelahti S, Kalkkinen N, Penttilä M, Londesborough J, Richard P (2005) Identification in the mold Hypocrea jecorina of the first fungal d-galacturonic acid reductase. Biochemistry 44:11234–11240CrossRefGoogle Scholar
  13. Kuorelahti S, Jouhten P, Maaheimo H, Penttilä M, Richard P (2006) l-galactonate dehydratase is part of the fungal path for d-galacturonic acid catabolism. Mol Microbiol 61:1060–1068CrossRefGoogle Scholar
  14. Liepins J, Kuorelahti S, Penttilä M, Richard P (2006) Enzymes for the NADPH-dependent reduction of dihydroxyacetone and d-glyceraldehyde and l-glyceraldehyde in the mould Hypocrea jecorina. FEBS J 273:4229–4235CrossRefGoogle Scholar
  15. Martens-Uzunova E (2008) Assessment of the pectinolytic network of Aspergillus niger by functional genomics. Insight from the transcriptome. Ph.D. thesis, University of Wageningen, WageningenGoogle Scholar
  16. Mata-Gilsinger M, Ritzenthaler P (1983) Physical mapping of the exuT and uxaC operators by use of exu plasmids and generation of deletion mutants in vitro. J Bacteriol 155:973–982Google Scholar
  17. Meloche HP, Wood WA (1964) Crystallization and characteristics of 2-keto-3-deoxy-6-phosphogluconic aldolase. J Biol Chem 239:3515–3518Google Scholar
  18. Moon TS, Yoon SH, Lanza AM, Roy-Mayhew JD, Prather KL (2009) Production of glucaric acid from a synthetic pathway in recombinant Escherichia coli. Appl Environ Microbiol 75:589–595CrossRefGoogle Scholar
  19. Richard P, Hilditch S (2009) d-Galacturonic acid catabolism in microorganisms and its biotechnological relevance. Appl Microbiol Biotechnol 82:597–604CrossRefGoogle Scholar
  20. Richard P, Londesborough J, Putkonen M, Kalkkinen N, Penttilä M (2001) Cloning and expression of a fungal l-arabinitol 4-dehydrogenase gene. J Biol Chem 276:40631–40637CrossRefGoogle Scholar
  21. Rosenfeld J, Capdevielle J, Guillemot JC, Ferrara P (1992) In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal Biochem 203:173–179CrossRefGoogle Scholar
  22. Sharma BS, Blumenthal HJ (1973) Catabolism of d-gluaric acid to alpha-ketoglutarate in Bacillus megaterium. J Bacteriol 116:1346–1354Google Scholar
  23. Smiley JD, Ashwell G (1960) Uronic acid metabolism in bacteria. III. Purification and properties of d-altronic acid and d-mannonic acid dehydrases in Escherichia coli. J Biol Chem 235:1571–1575Google Scholar
  24. Ueberschär KH, Blachnitzky EO, Kurz G (1974) Reaction mechanism of d-galactose dehydrogenases from Pseudomonas saccharophila and Pseudomonas fluorescens. Formation and rearrangement of aldono-1, 5-lactones. Eur J Biochem 48:389–405CrossRefGoogle Scholar
  25. Wagner G, Hollmann S (1976a) A new enzymatic method for the determination of free and conjugated glucuronic acid. J Clin Chem Clin Biochem 14:225–226Google Scholar
  26. Wagner G, Hollmann S (1976b) Uronic acid dehydrogenase from Pseudomonas syringae. Purification and properties. Eur J Biochem 61:589–596CrossRefGoogle Scholar
  27. Watanabe S, Yamada M, Ohtsu I, Makino K (2007) alpha-ketoglutaric semialdehyde dehydrogenase isozymes involved in metabolic pathways of d-glucarate, d-galactarate, and hydroxy-l-proline. Molecular and metabolic convergent evolution. J Biol Chem 282:6685–6695CrossRefGoogle Scholar
  28. Wood DW et al (2001) The genome of the natural genetic engineer Agrobacterium tumefaciens C58. Science 294:2317–2323CrossRefGoogle Scholar
  29. Yoon SH, Moon TS, Iranpour P, Lanza AM, Prather KJ (2008) Cloning and characterization of uronate dehydrogenases from two Pseudomonads and Agrobacterium tumefaciens str. C58. J Bacteriol 191:1565–1573CrossRefGoogle Scholar
  30. Zajic JE (1959) Hexuronic dehydrogenase of Agrobacterium tumefaciens. J Bacteriol. 78:734–735Google Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Harry Boer
    • 1
  • Hannu Maaheimo
    • 1
  • Anu Koivula
    • 1
  • Merja Penttilä
    • 1
  • Peter Richard
    • 1
  1. 1.VTT Technical Research Centre of FinlandEspooFinland

Personalised recommendations