Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Biodegradation of dioxins by recombinant Escherichia coli expressing rat CYP1A1 or its mutant

  • 186 Accesses

  • 11 Citations

Abstract

Among polychlorinated dibenzo-p-dioxins (PCDDs), 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TetraCDD) is the most toxic one. Recently, we reported that rat CYP1A1 mutant, F240A, expressed in yeast showed metabolic activity toward 2,3,7,8-TetraCDD. In this study, we successfully expressed N-terminal truncated P450s (Δ1A1 and ΔF240A) in Escherichia coli cells. Kinetic analysis using membrane fractions prepared from the recombinant E. coli cells revealed that ΔF240A has enzymatic properties similar to F240A expressed in yeast. The metabolism of PCDDs by recombinant E. coli cells expressing both ΔF240A and human NADPH-P450 reductase was also examined. When 2,3,7-TriCDD was added to the E.coli cell culture at a final concentration of 10 μM, approximately 90% of the 2,3,7-TriCDD was converted into multiple metabolites within 8 h. These results indicate the possible application of prokaryotic cells expressing ΔF240A to the bioremediation of PCDD-contaminated soil.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3

References

  1. Aoyama Y, Yoshida Y, Kubota S, Kumaoka H, Furumichi A (1978) NADPH-cytochrome P-450 reductase of yeast microsomes. Arch Biochem Biophys 185:362–369

  2. Armengaud J, Happe B, Timmis K (1998) Genetic analysis of dioxin dioxygenase of Sphingomonas sp. strain RW1: catabolic genes dispersed on the genome. J Bacteriol 180:3954–3966

  3. Barnes HJ, Arlotto MP, Waterman M (1991) Expression and enzymatic activity of recombinant cytochrome P450 17α-hydroxylation in Escherichia coli. Proc Natl Acad Sci U S A 88:5597–5601

  4. Bumpus JA, Tien M, Wright D, Aust SD (1985) Oxidation of persistent environmental pollutants by a white rot fungus. Science 228:1434–1436

  5. Bunge M, Adrian L, Kraus A, Opel M, Lorenz WG, Andreesen JR, Görisch H, Lechner U (2003) Reductive dehalogenation of chlorinated dioxins by an anaerobic bacterium. Nature 421:357–360

  6. Cosme J, Johnson EF (2000) Engineering microsomal cytochrome P450 2C5 to be a soluble, monomeric enzyme. Mutations that alter aggregation, phospholipid dependence of catalysis, and membrane binding. J Biol Chem 275:2545–2553

  7. Graham-Lorence S, Amarneh B, White RE (1995) A three-dimensional model of aromatase cytochrome P450. Protein Sci 4:1065–1080

  8. Guo Z, Gilliam EMJ, Ohmori S, Tukey RH, Guengerich FP (1994) Expression of modified human cytochrome P450 1A1 in Escherichia coli: effects of 5’ substitution, stabilization, purification, spectral characterization, and catalytic properties. Arch Biochem Biophys 312:436–446

  9. Enguita FJ, de la Fuente JL, Martin JF, Liras P (1996) An inducible expression system of histidine-tagged proteins in Streptomyces lividans for one-step purification by Ni2+ affinity chromatography. FEMS Microbiol Lett 137:151–156

  10. Fornwald JA, Donovan MJ, Geber R, Keller J, Taylor DP, Arcuri EJ, Brawner ME (1993) Soluble forms of the human T cell receptor CD4 are efficiently expressed by Streptomyces lividans. Biotechnology 11:1031–1036

  11. Inouye K, Shinkyo R, Takita T, Ohta M, Sakaki T (2002) Metabolism of polychlorinated dibenzo-p-dioxins (PCDDs) by human cytochrome P450-dependent monooxygenase systems. J Agric Food Chem 50:5496–5502

  12. Iwata H, Fujita K, Kushida H, Suzuki A, Konno Y, Nakamura K, Fujino A, Kamataki T (1998) High catalytic activity of human cytochrome P450 co-expressed with human NADPH-cytochrome P450 reductase in Escherichia coli. Biochem Pharmacol 55:1315–1325

  13. Jenkins CM, Waterman MR (1994) Flavodoxin and NADPH-flavodoxin reductase from Escherichia coli support bovine cytochrome P450c17 hydroxylase activities. J Biol Chem 269:27401–27408

  14. Kimura N, Urushigawa Y (2001) Metabolism of dibenzo-p-dioxin and chlorinated dibenzo-p-dioxin by a gram-positive bacterium, Rhodococcus opacus SAO101. J Biosci Bioeng 92:138–143

  15. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275

  16. Mast N, Andersson U, Nakayama K, Bijorkhem I, Pikuleva A (2004) Expression of human cytochrome P450 46A1 in Escherichia coli: effects of N- and C-terminal modifications. Arch Biochem Biophys 428:99–108

  17. Murtazina D, Puchkaev AV, Schein CH, Oezguen N, Braun W, Nanavati A, Pikuleva IA (2002) Membrane–protein interactions contribute to efficient 27-hydroxylation of cholesterol by mitochondrial cytochrome P450 27A1. J Biol Chem 277:37582–37589

  18. Nakashima N, Tamura T (2004a) A novel system of expressing recombinant proteins over a wide temperature range from 4 to 35 degrees C. Biotechnol Bioeng 86:136–148

  19. Nakashima N, Tamura T (2004b) Isolation and characterization of a rolling-circle-type plasmids from Rhodococcus erythropolis and application of the plasmid to multiple-recombinant protein expression. Appl Environ Microbiol 70:1181–1189

  20. Nojiri H, Nam JW, Kosaka M, Morii K, Takemura IT, Furihata K, Yamane H, Omori T (1999) Diverse oxygenations catalyzed by carbazole 1,9a-dioxygenase from Pseudomonas sp. Strain CA10. J Bacteriol 181:3105–3113

  21. Ohta Y, Sakaki T, Yabusaki Y, Ohkawa H, Kawato S (1994) Rotation and membrane topology of genetically expressed methylcolanthrene-inducible cytochrome P-4501A1 lacking the N-terminal hydrophobic segment in yeast microsomes. J Biol Chem 269:15597–15600

  22. Olins PO, Rangwala SH (1989) A novel sequence element derived from Bacteriophage T7 mRNA acts as an enhancer of translation of the lacZ gene in Escherichia coli. J Biol Chem 264:16973–1697

  23. Omura T, Sato R (1964) The carbon monoxide-binding pigment of liver microsomes: II. Solubilization, purification, and properties. J Biol Chem 239:2379–238

  24. Orihara K, Yamazaki T, Shinkyo R, Sakaki T, Inouye K, Tsukamoto A, Sugiura J, Shishido K (2005) Rat cytochrome P450-mediated transformation of dichlorodibenzo-p-dioxins by recombinant white-rot basidiomycete Coriolus hirsutus. Appl Microbiol Biotechnol 69(1):22–28

  25. Parikh A, Guengerich FP (1997) Drug metabolism by Escherichia coli expressing human cytochromes P450. Nat Biotechnol 15:784–788

  26. Sato SI, Nam JW, Kasuga K, Nojiri H, Yamane H, Omori T (1997) Identification and characterization of genes encoding carbazol 1,9a-dioxygenase in Pseudomonas sp. Strain CA10. J Bacteriol 179:4850–4858

  27. Sakaki T, Soga A, Yabusaki Y, Ohkawa H (1984) Characterization of three forms of cytochrome P-450 isolated from liver microsomes of rats treated with 3-methylcholamthrene. J Biochem 96:117–126

  28. Sakaki T, Kominami S, Takemori S, Ohkawa H, Akiyoshi-Shibata M, Yabusaki Y (1994) Kinetic studies on a genetically engineered fused enzyme between rat cytochrome P4501A1 and yeast NADPH-P450 reductase. Biochemistry 33:4933–4939

  29. Sakaki T, Shinkyo R, Takita T, Ohta M, Inouye K (2002) Biodegradation of polychlorinated dibenzo-p-dioxins by recombinant yeast expressing rat CYP1A subfamily. Arch Biochem Biophys 401:91–98

  30. Shinkyo R, Sakaki T, Ohta M, Inouye K (2003a) Metabolic pathways of dioxin by CYP1A1: species difference between rat and human CYP1A subfamily in the metabolism of dioxins. Arch Biochem Biophys 409:180–187

  31. Shinkyo R, Sakaki T, Takita T, Ohta M, Inouye K (2003b) Generation of 2,3,7,8-TCDD-metabolizing enzyme by modifying rat CYP1A1 through site-directed mutagenesis. Biochem Biophys Res Commun 308:511–517

  32. Spratt JM, Britton WJ, Triccas JA (2003) Identification of strong promoter elements of Mycobacterium smegmatis and their utility for foreign gene expression in mycobacteria. FEMS Microbiol Lett 224:139–142

  33. Sulistyaningdyah WT, Ogawa J, Li QS, Shinkyo R, Sakaki T, Inouye K, Schmid RD, Shimizu S (2004) Metabolism of polychlorinated dibenzo-p-dioxins by cytochrome P450 BM-3 and its mutant. Biotechnol Lett 26:1857–1860

  34. Vail RB, Homann MJ, Hanna I, Zaks A (2005) Preparative synthesis of drug metabolites using human cytochrome P450s 3A4, 2C9, and 1A2 with NADPH-P450 reductase expressed in Escherichia coli. J Ind Microbiol Biotechnol 32:67–74

  35. Williams PA, Comse J, Sridhar V, Johnson EF, McRee DE (2000) Mammalian microsomal cytochrome P450 monooxygenase: structural adaptations for membrane binding and functional diversity. Mol Cell 5:121–131

Download references

Author information

Correspondence to Toshiyuki Sakaki.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Shinkyo, R., Kamakura, M., Ikushiro, S. et al. Biodegradation of dioxins by recombinant Escherichia coli expressing rat CYP1A1 or its mutant. Appl Microbiol Biotechnol 72, 584–590 (2006). https://doi.org/10.1007/s00253-005-0286-1

Download citation

Keywords

  • Membrane Fraction
  • Dioxin
  • Coli Cell
  • Prokaryotic Cell
  • P450 Reductase