Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Jun;178(11):3133–3139. doi: 10.1128/jb.178.11.3133-3139.1996

Site-directed mutagenesis of conserved amino acids in the alpha subunit of toluene dioxygenase: potential mononuclear non-heme iron coordination sites.

H Jiang 1, R E Parales 1, N A Lynch 1, D T Gibson 1
PMCID: PMC178063  PMID: 8655491

Abstract

The terminal oxygenase component of toluene dioxygenase from Pseudomonas putida F1 is an iron-sulfur protein (ISP(TOL)) that requires mononuclear iron for enzyme activity. Alignment of all available predicted amino acid sequences for the large (alpha) subunits of terminal oxygenases showed a conserved cluster of potential mononuclear iron-binding residues. These were between amino acids 210 and 230 in the alpha subunit (TodC1) of ISP(TOL). The conserved amino acids, Glu-214, Asp-219, Tyr-221, His-222, and His-228, were each independently replaced with an alanine residue by site-directed mutagenesis. Tyr-266 in TodC1, which has been suggested as an iron ligand, was treated in an identical manner. To assay toluene dioxygenase activity in the presence of TodC1 and its mutant forms, conditions for the reconstitution of wild-type ISP(TOL) activity from TodC1 and purified TodC2 (beta subunit) were developed and optimized. A mutation at Glu-214, Asp-219, His-222, or His-228 completely abolished toluene dioxygenase activity. TodC1 with an alanine substitution at either Tyr-221 or Tyr-266 retained partial enzyme activity (42 and 12%, respectively). In experiments with [14C]toluene, the two Tyr-->Ala mutations caused a reduction in the amount of Cis-[14C]-toluene dihydrodiol formed, whereas a mutation at Glu-214, Asp-219, His-222, or His-228 eliminated cis-toluene dihydrodiol formation. The expression level of all of the mutated TWO proteins was equivalent to that of wild-type TodC1 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analyses. These results, in conjunction with the predicted amino acid sequences of 22 oxygenase components, suggest that the conserved motif Glu-X3-4,-Asp-X2-His-X4-5-His is critical for catalytic function and the glutamate, aspartate, and histidine residues may act as mononuclear iron ligands at the site of oxygen activation.

Full Text

The Full Text of this article is available as a PDF (500.1 KB).

Selected References

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

  1. Asturias J. A., Díaz E., Timmis K. N. The evolutionary relationship of biphenyl dioxygenase from gram-positive Rhodococcus globerulus P6 to multicomponent dioxygenases from gram-negative bacteria. Gene. 1995 Apr 14;156(1):11–18. doi: 10.1016/0378-1119(94)00530-6. [DOI] [PubMed] [Google Scholar]
  2. Batie C. J., LaHaie E., Ballou D. P. Purification and characterization of phthalate oxygenase and phthalate oxygenase reductase from Pseudomonas cepacia. J Biol Chem. 1987 Feb 5;262(4):1510–1518. [PubMed] [Google Scholar]
  3. Bender C. J., Rosenzweig A. C., Lippard S. J., Peisach J. Nuclear hyperfine coupling of nitrogen in the coordination sphere of the diiron center of methane monooxygenase hydroxylase. J Biol Chem. 1994 Jun 10;269(23):15993–15998. [PubMed] [Google Scholar]
  4. Bill E., Bernhardt F. H., Trautwein A. X. Mössbauer studies on the active Fe ... [2Fe-2S] site of putidamonooxin, its electron transport and dioxygen activation mechanism. Eur J Biochem. 1981 Dec;121(1):39–46. doi: 10.1111/j.1432-1033.1981.tb06426.x. [DOI] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  6. Brunel F., Davison J. Cloning and sequencing of Pseudomonas genes encoding vanillate demethylase. J Bacteriol. 1988 Oct;170(10):4924–4930. doi: 10.1128/jb.170.10.4924-4930.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Crutcher S. E., Geary P. J. Properties of the iron--sulphur proteins of the benzene dioxygenase system from Pseudomonas putida. Biochem J. 1979 Feb 1;177(2):393–400. doi: 10.1042/bj1770393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Danganan C. E., Ye R. W., Daubaras D. L., Xun L., Chakrabarty A. M. Nucleotide sequence and functional analysis of the genes encoding 2,4,5-trichlorophenoxyacetic acid oxygenase in Pseudomonas cepacia AC1100. Appl Environ Microbiol. 1994 Nov;60(11):4100–4106. doi: 10.1128/aem.60.11.4100-4106.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Davidson E., Ohnishi T., Atta-Asafo-Adjei E., Daldal F. Potential ligands to the [2Fe-2S] Rieske cluster of the cytochrome bc1 complex of Rhodobacter capsulatus probed by site-directed mutagenesis. Biochemistry. 1992 Apr 7;31(13):3342–3351. doi: 10.1021/bi00128a006. [DOI] [PubMed] [Google Scholar]
  10. Dehmel U., Engesser K. H., Timmis K. N., Dwyer D. F. Cloning, nucleotide sequence, and expression of the gene encoding a novel dioxygenase involved in metabolism of carboxydiphenyl ethers in Pseudomonas pseudoalcaligenes POB310. Arch Microbiol. 1995 Jan;163(1):35–41. doi: 10.1007/BF00262201. [DOI] [PubMed] [Google Scholar]
  11. Denome S. A., Stanley D. C., Olson E. S., Young K. D. Metabolism of dibenzothiophene and naphthalene in Pseudomonas strains: complete DNA sequence of an upper naphthalene catabolic pathway. J Bacteriol. 1993 Nov;175(21):6890–6901. doi: 10.1128/jb.175.21.6890-6901.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ensley B. D., Gibson D. T. Naphthalene dioxygenase: purification and properties of a terminal oxygenase component. J Bacteriol. 1983 Aug;155(2):505–511. doi: 10.1128/jb.155.2.505-511.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Erickson B. D., Mondello F. J. Nucleotide sequencing and transcriptional mapping of the genes encoding biphenyl dioxygenase, a multicomponent polychlorinated-biphenyl-degrading enzyme in Pseudomonas strain LB400. J Bacteriol. 1992 May;174(9):2903–2912. doi: 10.1128/jb.174.9.2903-2912.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Finette B. A., Subramanian V., Gibson D. T. Isolation and characterization of Pseudomonas putida PpF1 mutants defective in the toluene dioxygenase enzyme system. J Bacteriol. 1984 Dec;160(3):1003–1009. doi: 10.1128/jb.160.3.1003-1009.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fukuda M., Yasukochi Y., Kikuchi Y., Nagata Y., Kimbara K., Horiuchi H., Takagi M., Yano K. Identification of the bphA and bphB genes of Pseudomonas sp. strains KKS102 involved in degradation of biphenyl and polychlorinated biphenyls. Biochem Biophys Res Commun. 1994 Jul 29;202(2):850–856. doi: 10.1006/bbrc.1994.2008. [DOI] [PubMed] [Google Scholar]
  16. Gassner G. T., Ballou D. P., Landrum G. A., Whittaker J. W. Magnetic circular dichroism studies on the mononuclear ferrous active site of phthalate dioxygenase from Pseudomonas cepacia show a change of ligation state on substrate binding. Biochemistry. 1993 May 11;32(18):4820–4825. doi: 10.1021/bi00069a017. [DOI] [PubMed] [Google Scholar]
  17. Geary P. J., Saboowalla F., Patil D., Cammack R. An investigation of the iron-sulphur proteins of benzene dioxygenase from Pseudomonas putida by electron-spin-resonance spectroscopy. Biochem J. 1984 Feb 1;217(3):667–673. doi: 10.1042/bj2170667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gibson D. T., Hensley M., Yoshioka H., Mabry T. J. Formation of (+)-cis-2,3-dihydroxy-1-methylcyclohexa-4,6-diene from toluene by Pseudomonas putida. Biochemistry. 1970 Mar 31;9(7):1626–1630. doi: 10.1021/bi00809a023. [DOI] [PubMed] [Google Scholar]
  19. Haak B., Fetzner S., Lingens F. Cloning, nucleotide sequence, and expression of the plasmid-encoded genes for the two-component 2-halobenzoate 1,2-dioxygenase from Pseudomonas cepacia 2CBS. J Bacteriol. 1995 Feb;177(3):667–675. doi: 10.1128/jb.177.3.667-675.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Haddock J. D., Gibson D. T. Purification and characterization of the oxygenase component of biphenyl 2,3-dioxygenase from Pseudomonas sp. strain LB400. J Bacteriol. 1995 Oct;177(20):5834–5839. doi: 10.1128/jb.177.20.5834-5839.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Han S., Eltis L. D., Timmis K. N., Muchmore S. W., Bolin J. T. Crystal structure of the biphenyl-cleaving extradiol dioxygenase from a PCB-degrading pseudomonad. Science. 1995 Nov 10;270(5238):976–980. doi: 10.1126/science.270.5238.976. [DOI] [PubMed] [Google Scholar]
  22. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  23. Harayama S., Rekik M., Bairoch A., Neidle E. L., Ornston L. N. Potential DNA slippage structures acquired during evolutionary divergence of Acinetobacter calcoaceticus chromosomal benABC and Pseudomonas putida TOL pWW0 plasmid xylXYZ, genes encoding benzoate dioxygenases. J Bacteriol. 1991 Dec;173(23):7540–7548. doi: 10.1128/jb.173.23.7540-7548.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Harayama S., Rekik M., Timmis K. N. Genetic analysis of a relaxed substrate specificity aromatic ring dioxygenase, toluate 1,2-dioxygenase, encoded by TOL plasmid pWW0 of Pseudomonas putida. Mol Gen Genet. 1986 Feb;202(2):226–234. doi: 10.1007/BF00331641. [DOI] [PubMed] [Google Scholar]
  25. Irie S., Doi S., Yorifuji T., Takagi M., Yano K. Nucleotide sequencing and characterization of the genes encoding benzene oxidation enzymes of Pseudomonas putida. J Bacteriol. 1987 Nov;169(11):5174–5179. doi: 10.1128/jb.169.11.5174-5179.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kobal V. M., Gibson D. T., Davis R. E., Garza A. X-ray determination of the absolute stereochemistry of the initial oxidation product formed from toluene by Pseudomonas puida 39-D. J Am Chem Soc. 1973 Jun 27;95(13):4420–4421. doi: 10.1021/ja00794a048. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Lee S. Y., Rasheed S. A simple procedure for maximum yield of high-quality plasmid DNA. Biotechniques. 1990 Dec;9(6):676–679. [PubMed] [Google Scholar]
  29. Locher H. H., Leisinger T., Cook A. M. 4-Sulphobenzoate 3,4-dioxygenase. Purification and properties of a desulphonative two-component enzyme system from Comamonas testosteroni T-2. Biochem J. 1991 Mar 15;274(Pt 3):833–842. doi: 10.1042/bj2740833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Markus A., Krekel D., Lingens F. Purification and some properties of component A of the 4-chlorophenylacetate 3,4-dioxygenase from Pseudomonas species strain CBS. J Biol Chem. 1986 Sep 25;261(27):12883–12888. [PubMed] [Google Scholar]
  31. Masai E., Yamada A., Healy J. M., Hatta T., Kimbara K., Fukuda M., Yano K. Characterization of biphenyl catabolic genes of gram-positive polychlorinated biphenyl degrader Rhodococcus sp. strain RHA1. Appl Environ Microbiol. 1995 Jun;61(6):2079–2085. doi: 10.1128/aem.61.6.2079-2085.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mason J. R., Cammack R. The electron-transport proteins of hydroxylating bacterial dioxygenases. Annu Rev Microbiol. 1992;46:277–305. doi: 10.1146/annurev.mi.46.100192.001425. [DOI] [PubMed] [Google Scholar]
  33. Nakatsu C. H., Straus N. A., Wyndham R. C. The nucleotide sequence of the Tn5271 3-chlorobenzoate 3,4-dioxygenase genes (cbaAB) unites the class IA oxygenases in a single lineage. Microbiology. 1995 Feb;141(Pt 2):485–495. doi: 10.1099/13500872-141-2-485. [DOI] [PubMed] [Google Scholar]
  34. Neidle E. L., Hartnett C., Ornston L. N., Bairoch A., Rekik M., Harayama S. Nucleotide sequences of the Acinetobacter calcoaceticus benABC genes for benzoate 1,2-dioxygenase reveal evolutionary relationships among multicomponent oxygenases. J Bacteriol. 1991 Sep;173(17):5385–5395. doi: 10.1128/jb.173.17.5385-5395.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Nordlund P., Sjöberg B. M., Eklund H. Three-dimensional structure of the free radical protein of ribonucleotide reductase. Nature. 1990 Jun 14;345(6276):593–598. doi: 10.1038/345593a0. [DOI] [PubMed] [Google Scholar]
  36. Ohlendorf D. H., Orville A. M., Lipscomb J. D. Structure of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa at 2.15 A resolution. J Mol Biol. 1994 Dec 16;244(5):586–608. doi: 10.1006/jmbi.1994.1754. [DOI] [PubMed] [Google Scholar]
  37. Pavel E. G., Martins L. J., Ellis W. R., Jr, Solomon E. I. Magnetic circular dichroism studies of exogenous ligand and substrate binding to the non-heme ferrous active site in phthalate dioxygenase. Chem Biol. 1994 Nov;1(3):173–183. doi: 10.1016/1074-5521(94)90007-8. [DOI] [PubMed] [Google Scholar]
  38. Pridmore R. D. New and versatile cloning vectors with kanamycin-resistance marker. Gene. 1987;56(2-3):309–312. doi: 10.1016/0378-1119(87)90149-1. [DOI] [PubMed] [Google Scholar]
  39. Rosenzweig A. C., Frederick C. A., Lippard S. J., Nordlund P. Crystal structure of a bacterial non-haem iron hydroxylase that catalyses the biological oxidation of methane. Nature. 1993 Dec 9;366(6455):537–543. doi: 10.1038/366537a0. [DOI] [PubMed] [Google Scholar]
  40. Senda T., Sugiyama K., Narita H., Yamamoto T., Kimbara K., Fukuda M., Sato M., Yano K., Mitsui Y. Three-dimensional structures of free form and two substrate complexes of an extradiol ring-cleavage type dioxygenase, the BphC enzyme from Pseudomonas sp. strain KKS102. J Mol Biol. 1996 Feb 9;255(5):735–752. doi: 10.1006/jmbi.1996.0060. [DOI] [PubMed] [Google Scholar]
  41. Shu L., Chiou Y. M., Orville A. M., Miller M. A., Lipscomb J. D., Que L., Jr X-ray absorption spectroscopic studies of the Fe(II) active site of catechol 2,3-dioxygenase. Implications for the extradiol cleavage mechanism. Biochemistry. 1995 May 23;34(20):6649–6659. doi: 10.1021/bi00020a010. [DOI] [PubMed] [Google Scholar]
  42. Simon M. J., Osslund T. D., Saunders R., Ensley B. D., Suggs S., Harcourt A., Suen W. C., Cruden D. L., Gibson D. T., Zylstra G. J. Sequences of genes encoding naphthalene dioxygenase in Pseudomonas putida strains G7 and NCIB 9816-4. Gene. 1993 May 15;127(1):31–37. doi: 10.1016/0378-1119(93)90613-8. [DOI] [PubMed] [Google Scholar]
  43. Stanier R. Y., Palleroni N. J., Doudoroff M. The aerobic pseudomonads: a taxonomic study. J Gen Microbiol. 1966 May;43(2):159–271. doi: 10.1099/00221287-43-2-159. [DOI] [PubMed] [Google Scholar]
  44. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  45. Subramanian V., Liu T. N., Yeh W. K., Gibson D. T. Toluene dioxygenase: purification of an iron-sulfur protein by affinity chromatography. Biochem Biophys Res Commun. 1979 Dec 14;91(3):1131–1139. doi: 10.1016/0006-291x(79)91998-3. [DOI] [PubMed] [Google Scholar]
  46. Subramanian V., Liu T. N., Yeh W. K., Narro M., Gibson D. T. Purification and properties of NADH-ferredoxinTOL reductase. A component of toluene dioxygenase from Pseudomonas putida. J Biol Chem. 1981 Mar 25;256(6):2723–2730. [PubMed] [Google Scholar]
  47. Subramanian V., Liu T. N., Yeh W. K., Serdar C. M., Wackett L. P., Gibson D. T. Purification and properties of ferredoxinTOL. A component of toluene dioxygenase from Pseudomonas putida F1. J Biol Chem. 1985 Feb 25;260(4):2355–2363. [PubMed] [Google Scholar]
  48. Suen W. C., Gibson D. T. Recombinant Escherichia coli strains synthesize active forms of naphthalene dioxygenase and its individual alpha and beta subunits. Gene. 1994 May 27;143(1):67–71. doi: 10.1016/0378-1119(94)90606-8. [DOI] [PubMed] [Google Scholar]
  49. Taira K., Hirose J., Hayashida S., Furukawa K. Analysis of bph operon from the polychlorinated biphenyl-degrading strain of Pseudomonas pseudoalcaligenes KF707. J Biol Chem. 1992 Mar 5;267(7):4844–4853. [PubMed] [Google Scholar]
  50. Takizawa N., Kaida N., Torigoe S., Moritani T., Sawada T., Satoh S., Kiyohara H. Identification and characterization of genes encoding polycyclic aromatic hydrocarbon dioxygenase and polycyclic aromatic hydrocarbon dihydrodiol dehydrogenase in Pseudomonas putida OUS82. J Bacteriol. 1994 Apr;176(8):2444–2449. doi: 10.1128/jb.176.8.2444-2449.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wackett L. P. Toluene dioxygenase from Pseudomonas putida F1. Methods Enzymol. 1990;188:39–45. doi: 10.1016/0076-6879(90)88010-8. [DOI] [PubMed] [Google Scholar]
  52. Wang Y., Garnon J., Labbé D., Bergeron H., Lau P. C. Sequence and expression of the bpdC1C2BADE genes involved in the initial steps of biphenyl/chlorobiphenyl degradation by Rhodococcus sp. M5. Gene. 1995 Oct 16;164(1):117–122. doi: 10.1016/0378-1119(95)00448-f. [DOI] [PubMed] [Google Scholar]
  53. Wende P., Bernhardt F. H., Pfleger K. Substrate-modulated reactions of putidamonooxin. The nature of the active oxygen species formed and its reaction mechanism. Eur J Biochem. 1989 Apr 15;181(1):189–197. doi: 10.1111/j.1432-1033.1989.tb14710.x. [DOI] [PubMed] [Google Scholar]
  54. Werlen C., Kohler H. P., van der Meer J. R. The broad substrate chlorobenzene dioxygenase and cis-chlorobenzene dihydrodiol dehydrogenase of Pseudomonas sp. strain P51 are linked evolutionarily to the enzymes for benzene and toluene degradation. J Biol Chem. 1996 Feb 23;271(8):4009–4016. doi: 10.1074/jbc.271.8.4009. [DOI] [PubMed] [Google Scholar]
  55. Yamaguchi M., Fujisawa H. Subunit structure of oxygenase component in benzoate-1,2-dioxygenase system from Pseudomonas arvilla C-1. J Biol Chem. 1982 Nov 10;257(21):12497–12502. [PubMed] [Google Scholar]
  56. Yeh W. K., Gibson D. T., Liu T. N. Toluene dioxygenase: a multicomponent enzyme system. Biochem Biophys Res Commun. 1977 Sep 9;78(1):401–410. doi: 10.1016/0006-291x(77)91268-2. [DOI] [PubMed] [Google Scholar]
  57. Zamanian M., Mason J. R. Benzene dioxygenase in Pseudomonas putida. Subunit composition and immuno-cross-reactivity with other aromatic dioxygenases. Biochem J. 1987 Jun 15;244(3):611–616. doi: 10.1042/bj2440611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Zylstra G. J., Gibson D. T. Aromatic hydrocarbon degradation: a molecular approach. Genet Eng (N Y) 1991;13:183–203. doi: 10.1007/978-1-4615-3760-1_8. [DOI] [PubMed] [Google Scholar]
  59. Zylstra G. J., Gibson D. T. Toluene degradation by Pseudomonas putida F1. Nucleotide sequence of the todC1C2BADE genes and their expression in Escherichia coli. J Biol Chem. 1989 Sep 5;264(25):14940–14946. [PubMed] [Google Scholar]

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

RESOURCES