Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Apr;175(7):2083–2086. doi: 10.1128/jb.175.7.2083-2086.1993

Alcaligenes eutrophus JMP134 "2,4-dichlorophenoxyacetate monooxygenase" is an alpha-ketoglutarate-dependent dioxygenase.

F Fukumori 1, R P Hausinger 1
PMCID: PMC204309  PMID: 8458850

Abstract

The Alcaligenes eutrophus JMP134 tfdA gene, encoding the enzyme responsible for the first step in 2,4-dichlorophenoxyacetic acid (2,4-D) biodegradation, was overexpressed in Escherichia coli, and several enzymatic properties of the partially purified gene product were examined. Although the tfdA-encoded enzyme is typically referred to as 2,4-D monooxygenase, we were unable to observe any reductant-dependent activity. Rather, we demonstrate that this enzyme is a ferrous ion-dependent dioxygenase that uses alpha-ketoglutarate as a cosubstrate. The alpha-ketoglutarate is converted to succinate concomitant with 2,4-D conversion to 2,4-dichlorophenol. By using [1-14C]alpha-ketoglutarate, we established that carbon dioxide is the second product derived from alpha-ketoglutarate. Finally, we verified the proposal that glyoxylate is the second product derived from 2,4-D.

Full text

PDF
2085

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Chamberlin B. A., Sweeley C. C. Metabolic profiles of urinary organic acids recovered from absorbent filter paper. Clin Chem. 1987 Apr;33(4):572–576. [PubMed] [Google Scholar]
  2. Don R. H., Pemberton J. M. Genetic and physical map of the 2,4-dichlorophenoxyacetic acid-degradative plasmid pJP4. J Bacteriol. 1985 Jan;161(1):466–468. doi: 10.1128/jb.161.1.466-468.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Don R. H., Pemberton J. M. Properties of six pesticide degradation plasmids isolated from Alcaligenes paradoxus and Alcaligenes eutrophus. J Bacteriol. 1981 Feb;145(2):681–686. doi: 10.1128/jb.145.2.681-686.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Don R. H., Weightman A. J., Knackmuss H. J., Timmis K. N. Transposon mutagenesis and cloning analysis of the pathways for degradation of 2,4-dichlorophenoxyacetic acid and 3-chlorobenzoate in Alcaligenes eutrophus JMP134(pJP4). J Bacteriol. 1985 Jan;161(1):85–90. doi: 10.1128/jb.161.1.85-90.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Frantz B., Chakrabarty A. M. Organization and nucleotide sequence determination of a gene cluster involved in 3-chlorocatechol degradation. Proc Natl Acad Sci U S A. 1987 Jul;84(13):4460–4464. doi: 10.1073/pnas.84.13.4460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Harker A. R., Olsen R. H., Seidler R. J. Phenoxyacetic acid degradation by the 2,4-dichlorophenoxyacetic acid (TFD) pathway of plasmid pJP4: mapping and characterization of the TFD regulatory gene, tfdR. J Bacteriol. 1989 Jan;171(1):314–320. doi: 10.1128/jb.171.1.314-320.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Haugland R. A., Sangodkar U. M., Sferra P. R., Chakrabarty A. M. Cloning and characterization of a chromosomal DNA region required for growth on 2,4,5-T by Pseudomonas cepacia AC1100. Gene. 1991 Apr;100:65–73. doi: 10.1016/0378-1119(91)90351-b. [DOI] [PubMed] [Google Scholar]
  8. Helaakoski T., Vuori K., Myllylä R., Kivirikko K. I., Pihlajaniemi T. Molecular cloning of the alpha-subunit of human prolyl 4-hydroxylase: the complete cDNA-derived amino acid sequence and evidence for alternative splicing of RNA transcripts. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4392–4396. doi: 10.1073/pnas.86.12.4392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Häggblom M. M. Microbial breakdown of halogenated aromatic pesticides and related compounds. FEMS Microbiol Rev. 1992 Sep;9(1):29–71. doi: 10.1111/j.1574-6968.1992.tb05823.x. [DOI] [PubMed] [Google Scholar]
  10. Jia S., VanDusen W. J., Diehl R. E., Kohl N. E., Dixon R. A., Elliston K. O., Stern A. M., Friedman P. A. cDNA cloning and expression of bovine aspartyl (asparaginyl) beta-hydroxylase. J Biol Chem. 1992 Jul 15;267(20):14322–14327. [PubMed] [Google Scholar]
  11. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Liu T., Chapman P. J. Purification and properties of a plasmid-encoded 2,4-dichlorophenol hydroxylase. FEBS Lett. 1984 Aug 6;173(2):314–318. doi: 10.1016/0014-5793(84)80797-8. [DOI] [PubMed] [Google Scholar]
  14. Loos M. A., Roberts R. N., Alexander M. Phenols as intermediates in the decomposition of phenoxyacetates by an Arthrobacter species. Can J Microbiol. 1967 Jun;13(6):679–690. doi: 10.1139/m67-090. [DOI] [PubMed] [Google Scholar]
  15. Matsuda J., Okabe S., Hashimoto T., Yamada Y. Molecular cloning of hyoscyamine 6 beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, from cultured roots of Hyoscyamus niger. J Biol Chem. 1991 May 25;266(15):9460–9464. [PubMed] [Google Scholar]
  16. Myllylä R., Pihlajaniemi T., Pajunen L., Turpeenniemi-Hujanen T., Kivirikko K. I. Molecular cloning of chick lysyl hydroxylase. Little homology in primary structure to the two types of subunit of prolyl 4-hydroxylase. J Biol Chem. 1991 Feb 15;266(5):2805–2810. [PubMed] [Google Scholar]
  17. Nurk A., Kasak L., Kivisaar M. Sequence of the gene (pheA) encoding phenol monooxygenase from Pseudomonas sp. EST1001: expression in Escherichia coli and Pseudomonas putida. Gene. 1991 Jun 15;102(1):13–18. doi: 10.1016/0378-1119(91)90531-f. [DOI] [PubMed] [Google Scholar]
  18. Perkins E. J., Gordon M. P., Caceres O., Lurquin P. F. Organization and sequence analysis of the 2,4-dichlorophenol hydroxylase and dichlorocatechol oxidative operons of plasmid pJP4. J Bacteriol. 1990 May;172(5):2351–2359. doi: 10.1128/jb.172.5.2351-2359.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Perkins E. J., Lurquin P. F. Duplication of a 2,4-dichlorophenoxyacetic acid monooxygenase gene in Alcaligenes eutrophus JMP134(pJP4). J Bacteriol. 1988 Dec;170(12):5669–5672. doi: 10.1128/jb.170.12.5669-5672.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Rüetschi U., Odelhög B., Lindstedt S., Barros-Söderling J., Persson B., Jörnvall H. Characterization of 4-hydroxyphenylpyruvate dioxygenase. Primary structure of the Pseudomonas enzyme. Eur J Biochem. 1992 Apr 15;205(2):459–466. doi: 10.1111/j.1432-1033.1992.tb16800.x. [DOI] [PubMed] [Google Scholar]
  21. Streber W. R., Timmis K. N., Zenk M. H. Analysis, cloning, and high-level expression of 2,4-dichlorophenoxyacetate monooxygenase gene tfdA of Alcaligenes eutrophus JMP134. J Bacteriol. 1987 Jul;169(7):2950–2955. doi: 10.1128/jb.169.7.2950-2955.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  23. van der Meer J. R., Eggen R. I., Zehnder A. J., de Vos W. M. Sequence analysis of the Pseudomonas sp. strain P51 tcb gene cluster, which encodes metabolism of chlorinated catechols: evidence for specialization of catechol 1,2-dioxygenases for chlorinated substrates. J Bacteriol. 1991 Apr;173(8):2425–2434. doi: 10.1128/jb.173.8.2425-2434.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES