, 20:541 | Cite as

Cloning, heterologous expression, and functional characterization of the nicotinate dehydrogenase gene from Pseudomonas putida KT2440

  • Yao Yang
  • Sheng Yuan
  • Ting Chen
  • Pengjuan Ma
  • Guangdong Shang
  • Yijun Dai
Original Paper


6-Hydroxynicotinate can be used for the production of drugs, pesticides and intermediate chemicals. Some Pseudomonas species were reported to be able to convert nicotinic acid to 6-hydroxynicotinate by nicotinate dehydrogenase. So far, previous reports on NaDH in Pseudomonas genus were confused and contradictory each other. Recently, Ashraf et al. reported an NaDH gene cloned from Eubacterium barkeri and suggested some deducted NaDH genes from other nine bacteria. But they did not demonstrate the activity of recombinant NaDH and did not mention NaDH gene in Pseudomonas. In this study we cloned the gene of NaDH, ndhSL, from Pseudomonas putida KT2440. NdhSL in P. putida KT2440 is composed of two subunits. The small subunit contains [2Fe2S] iron sulfur domain, while the large subunit contains domains of molybdenum cofactor and cytochrome c. Expression of recombinant ndhSL in P. entomophila L48, which lacks the ability to produce 6-hydroxynicotinate, enabled the resting cell and cell extract of engineering P. entomophila L48 to hydroxylate nicotinate. Gene knockout and recovery studies further confirmed the ndhSL function.


Nicotinate 6-Hydroxynicotinate Nicotinate dehydrogenase (NaDH) ndhSL Pseudomonas putida KT2440 



We thank Isabelle Vallet-Gely, Bruno Lemaitre laboratory, FRANCE, and He Jian, Nanjing Agriculture University, Department of Life Science, for kindly providing Pseudomonas putida L48 and Pseudomonas putida KT2440, respectively. This work was supported by the Key Fundamental Research Program of Jiangsu Higher Education Institution of China (06KJA21016), the Natural Science Foundation of Jiangsu Higher Education Institution of China (04KJB180071).


  1. Alhapel A, Darley DJ, Wagener N, Eckel E, Elsner N, Pierik AJ (2006) Molecular and functional analysis of nicotinate catabolism in Eubacterium barkeri. Proc Natl Acad Sci USA 103:12341–12346. doi: 10.1073/pnas.0601635103 PubMedCrossRefGoogle Scholar
  2. Amano T, Ochi N, Sato H, Sakaki S (2007) Oxidation reaction by xanthine oxidase: theoretical study of reaction mechanism. J Am Chem Soc 129:8131–8138. doi: 10.1021/ja068584d PubMedCrossRefGoogle Scholar
  3. Andreesen JR, Fetzner S (2002) The molybdenum-containing hydroxylases of nicotinate, isonicotinate, and nicotine. Met Ions Biol Syst 39:405–430PubMedGoogle Scholar
  4. Berry DF, Francis AJ, Bollag JM (1987) Microbial metabolism of homocyclic and heterocyclic aromatic compounds under anaerobic conditions. Microbiol Rev 51:43–59PubMedGoogle Scholar
  5. Blatny JM, Brautaset T, Winther-Larsen HC, Haugan K, Valla S (1997a) Construction and use of a versatile set of broad-host-range cloning and expression vectors based on the RK2 replicon. Appl Environ Microbiol 63:370–379PubMedGoogle Scholar
  6. Blatny JM, Brautaset T, Winther-Larsen HC, Karunakaran P, Valla S (1997b) Improved broad-host-range RK2 vectors useful for high and low regulated gene expression levels in gram-negative bacteria. Plasmid 38:35–51. doi: 10.1006/plas.1997.1294 PubMedCrossRefGoogle Scholar
  7. Boyer HW, Roulland-Dussoix D (1969) A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41:459–472. doi: 10.1016/0022-2836(69)90288-5 PubMedCrossRefGoogle Scholar
  8. Caponi L, Migliorini P (1999) Immunoblotting. In: Caponi L, Migliorini P (eds) Antibody usage in the laboratory. Springer, BerlinGoogle Scholar
  9. Davis RW, Botstein D, Roth JR (1980) A manual for genetic engineering: advanced bacterial genetics. Cold Spring Harbor, NYGoogle Scholar
  10. Dower WJ, Miller JF, Ragsdale CW (1988) High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res 16:6127–6145. doi: 10.1093/nar/16.13.6127 PubMedCrossRefGoogle Scholar
  11. Ensign JC, Rittenberg SC (1964) The pathway of nicotinic acid oxidation by a Bacillus species. J Biol Chem 239:2285–2291PubMedGoogle Scholar
  12. Harary I (1957a) Bacterial fermentation of nicotinic acid. I. End products. J Biol Chem 227:815–822PubMedGoogle Scholar
  13. Harary I (1957b) Bacterial fermantation of nicotinic acid. II. Anaerobic reversible hydroxylation of nicotinic acid to 6-hydroxynicotinic acid. J Biol Chem 227:823–831PubMedGoogle Scholar
  14. Hirschberg R, Ensign JC (1971a) Oxidation of nicotinic acid by a Bacillus species: source of oxygen atoms for the hydroxylation of nicotinic acid and 6-hydroxynicotinic acid. J Bacteriol 108:757–759PubMedGoogle Scholar
  15. Hirschberg R, Ensign JC (1971b) Oxidation of nicotinic acid by a Bacillus species: purification and properties of nicotinic acid and 6-hydroxynicotinic acid hydroxylases. J Bacteriol 108:751–756PubMedGoogle Scholar
  16. Hirschberg R, Ensign JC (1972) Oxidation of nicotinic acid by a Bacillus species: regulation of nicotinic acid and 6-hydroxynicotinic acid hydroxylases. J Bacteriol 112:392–397PubMedGoogle Scholar
  17. Holcenberg JS, Tsai L (1969) Nicotinic acid metabolism. IV. Ferredoxin-dependent reduction of 6-hydroxynicotinic acid to 6-oxo-1, 4, 5, 6-tetrahydronicotinic acid. J Biol Chem 244:1204–1211PubMedGoogle Scholar
  18. Hughes DE (1955) 6-Hydroxynicotinic acid as an intermediate in the oxidation of nicotinic acid by Pseudomonas fluorescens. Biochem J 60:303–310PubMedGoogle Scholar
  19. Hunt AL (1959) Purification of the nicotinic acid hydroxylase system of Pseudomonas fluorescens KB1. Biochem J 72:1–7PubMedGoogle Scholar
  20. Hunt AL, Hughes DE, Lowenstein JM (1958) The hydroxylation of nicotinic acid by Pseudomonas fluorescens. Biochem J 69:170–173PubMedGoogle Scholar
  21. Hurh BYT, Nagasawa T (1994) Purification and characterization of nicotinic acid dehydrogenase from Pseudomonas fluoescens TN5. Ferment Bioengin 78:19–26. doi: 10.1016/0922-338X(94)90172-4 CrossRefGoogle Scholar
  22. Hurh BOM, Yamane T et al (1994) Microbial production of 6-hydroxynicotinic acid, an impotrant building block for the synthesis of modern insecticides. J Ferment bioengin 77:382–385CrossRefGoogle Scholar
  23. Iwasaki K, Uchiyama H, Yagi O, Kurabayashi T, Ishizuka K, Takamura Y (1994) Transformation of Pseudomonas putida by electroporation. Biosci Biotechnol Biochem 58:851–854PubMedCrossRefGoogle Scholar
  24. Jiménez JI, Canales A, Jiménez-Barbero J, Ginalski K, Rychlewski L, García JL, Díaz E (2008) Deciphering the genetic determinants for aerobic nicotinic acid degradation: the nice cluster from Pseudomonas putida KT2440. Proc Natl Acad Sci USA 105(32):11329–11334. doi: 10.1073/pnas.0802273105 PubMedCrossRefGoogle Scholar
  25. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. doi: 10.1038/227680a0 PubMedCrossRefGoogle Scholar
  26. Lu WH, Wang X, Xu L, Dai YJ, Yuan S (2005) Induction of nicotinic acid hydroxylase activity of Pseudomonas putida NA-1 and optimization of transformation conditions. Acta Microbiol Sin 45:6–9Google Scholar
  27. Nagel M, Andreesen JR (1989) Molybdenum-dependent degradation of nicotinic acid by Bacillus sp. DSM 2923. FEMS Microbiol Lett 59:147–152. doi: 10.1111/j.1574-6968.1989.tb03099.x CrossRefGoogle Scholar
  28. Nagel M, Andreesen JR (1990) Purification and characterization of the molybdoenzymes nicotinate dehydrogenase and 6-hydroxynicotinate dehydrohenase from Bacillus niacini. Arch Microbiol 154:605–613. doi: 10.1007/BF00248844 CrossRefGoogle Scholar
  29. Nakano H, Wieser M, Hurh B, Kawai T, Yoshida T, Yamane T, Nagasawa T (1999) Purification, characterization and gene cloning of 6-hydroxynicotinate 3-monooxygenase from Pseudomonas fluorescens TN5. Eur J Biochem 260:120–126. doi: 10.1046/j.1432-1327.1999.00124.x PubMedCrossRefGoogle Scholar
  30. Nelson KE, Weinel C, Paulsen IT et al (2002) Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ Microbiol 4:799–808. doi: 10.1046/j.1462-2920.2002.00366.x PubMedCrossRefGoogle Scholar
  31. Pastan I, Tsai L, Stadtman ER (1964) Nicotinic acid metabolism. I. Distribution of isotope in fermentation products of labelled nicotinic acid. J Biol Chem 239:902–906PubMedGoogle Scholar
  32. Quenee L, Lamotte D, Polack B (2005) Combined sacB-based negative selection and cre-lox antibiotic marker recycling for efficient gene deletion in pseudomonas aeruginosa. Biotechniques 38:63–67. doi: 10.2144/05381ST01 PubMedCrossRefGoogle Scholar
  33. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, vol 2. Cold Spring Harbor Laboratory Press, NYGoogle Scholar
  34. Stemmer WP, Crameri A, Ha KD, Brennan TM, Heyneker HL (1995) Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene 164:49–53. doi: 10.1016/0378-1119(95)00511-4 PubMedCrossRefGoogle Scholar
  35. Tautz D, Renz M (1983) An optimized freeze-squeeze method for the recovery of DNA fragments from agarose gels. Anal Biochem 132:14–19. doi: 10.1016/0003-2697(83)90419-0 PubMedCrossRefGoogle Scholar
  36. Tsai L, Pastan I, Stadtman ER (1966) Nicotinic acid metabolism. II. The isolation and characterization of intermediates in the fermentation of nicotinic acid. J Biol Chem 241:1807–1813PubMedGoogle Scholar
  37. Vodovar N, Vallenet D, Cruveiller S et al (2006) Complete genome sequence of the entomopathogenic and metabolically versatile soil bacterium Pseudomonas entomophila. Nat Biotechnol 24:673–679. doi: 10.1038/nbt1212 PubMedCrossRefGoogle Scholar
  38. Wenzel SC, Gross F, Zhang Y, Fu J, Stewart AF, Muller R (2005) Heterologous expression of a myxobacterial natural products assembly line in pseudomonads via red/ET recombineering. Chem Biol 12:349–356. doi: 10.1016/j.chembiol.2004.12.012 PubMedCrossRefGoogle Scholar
  39. Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119. doi: 10.1016/0378-1119(85)90120-9 PubMedCrossRefGoogle Scholar
  40. Yoshida T, Nagasawa T (2000) Enzymatic functionalization of aromatic N-heterocycles: hydroxylation and carboxylation. J Biosci Bioeng 89:111–118. doi: 10.1016/S1389-1723(00)88723-X PubMedCrossRefGoogle Scholar
  41. Yuan S, Yang Y, Sun J et al (2005) A combined technology of growing culture hydroxylation of nicotinic acid and resting cells hydroxylation of 3-cyanopyridine by Comamonas testosterone JA1. Eng Life Sci 5:369–374. doi: 10.1002/elsc.200520063 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Yao Yang
    • 1
  • Sheng Yuan
    • 1
  • Ting Chen
    • 1
  • Pengjuan Ma
    • 1
  • Guangdong Shang
    • 1
  • Yijun Dai
    • 1
  1. 1.Nanjing Research Center for Microbial Engineering and Biotechnology, Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life SciencesNanjing Normal UniversityNanjingPeople’s Republic of China

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