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. 1996 Oct;178(19):5699–5705. doi: 10.1128/jb.178.19.5699-5705.1996

Gene overexpression, purification, and identification of a desulfurization enzyme from Rhodococcus sp. strain IGTS8 as a sulfide/sulfoxide monooxygenase.

B Lei 1, S C Tu 1
PMCID: PMC178409  PMID: 8824615

Abstract

The oxidation of dibenzothiophene to dibenzothiophene sulfone has been linked to the enzyme encoded by the sox/dszC gene from Rhodococcus sp. strain IGTS8 (S. A. Denome, C. Oldfield, L. J. Nash, and K. D. Young, J. Bacteriol. 176:6707-6717, 1994; C. S. Piddington, B. R. Kovacevich, and J. Rambosek, Appl. Environ. Microbiol. 61:468-475, 1995). However, this enzyme has not been characterized, and the type of its catalytic activity remains unclassified. In this work, the sox/dszC gene was overexpressed in Escherichia coli, a procedure for the purification of the expressed enzyme was developed, and the properties of and the reactions catalyzed by the purified enzyme were characterized. This enzyme binds one flavin mononucleotide (Kd, 7 micrometers) or reduced flavin mononucleotide (FMNH2) (Kd < 10(-8) M) per 90,200-Da homodimer, and FMNH2 is an essential cosubstrate for its activity. Patterns of product formation were examined under different FMNH2 availabilities, and results indicate that this enzyme catalyzes a stepwise conversion of dibenzothiophene to the corresponding sulfoxide and subsequently to the sulfone. On the basis of isotope labeling patterns with H2(18)O and 18O2, dibenzothiophene sulfoxide and sulfone obtained their oxygen atom(s) from molecular oxygen rather than water in their formation from dibenzothiophene. The enzyme also utilizes benzyl sulfide and benzyl sulfoxide as substrates. Hence, it is identified as a sulfide/sulfoxide monooxygenase. This monooxygenase is similar to the microsomal flavin-containing monooxygenase but is unique among microbial flavomonooxygenases in its ability to catalyze two consecutive monooxygenation reactions.

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Selected References

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  1. Denome S. A., Oldfield C., Nash L. J., Young K. D. Characterization of the desulfurization genes from Rhodococcus sp. strain IGTS8. J Bacteriol. 1994 Nov;176(21):6707–6716. doi: 10.1128/jb.176.21.6707-6716.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. GIBSON Q. H., HASTINGS J. W. The oxidation of reduced flavin mononucleotide by molecular oxygen. Biochem J. 1962 May;83:368–377. doi: 10.1042/bj0830368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Gallagher J. R., Olson E. S., Stanley D. C. Microbial desulfurization of dibenzothiophene: a sulfur-specific pathway. FEMS Microbiol Lett. 1993 Feb 15;107(1):31–35. doi: 10.1016/0378-1097(93)90349-7. [DOI] [PubMed] [Google Scholar]
  4. HUMMEL J. P., DREYER W. J. Measurement of protein-binding phenomena by gel filtration. Biochim Biophys Acta. 1962 Oct 8;63:530–532. doi: 10.1016/0006-3002(62)90124-5. [DOI] [PubMed] [Google Scholar]
  5. Izumi Y., Ohshiro T., Ogino H., Hine Y., Shimao M. Selective Desulfurization of Dibenzothiophene by Rhodococcus erythropolis D-1. Appl Environ Microbiol. 1994 Jan;60(1):223–226. doi: 10.1128/aem.60.1.223-226.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Jakoby W. B., Ziegler D. M. The enzymes of detoxication. J Biol Chem. 1990 Dec 5;265(34):20715–20718. [PubMed] [Google Scholar]
  7. 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]
  8. Laborde A. L., Gibson D. T. Metabolism of dibenzothiophene by a Beijerinckia species. Appl Environ Microbiol. 1977 Dec;34(6):783–790. doi: 10.1128/aem.34.6.783-790.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Lei B. F., Becvar J. E. A new reducing agent of flavins and its application to the assay of bacterial luciferase. Photochem Photobiol. 1991 Sep;54(3):473–476. doi: 10.1111/j.1751-1097.1991.tb02043.x. [DOI] [PubMed] [Google Scholar]
  11. Lei B., Cho K. W., Tu S. C. Mechanism of aldehyde inhibition of Vibrio harveyi luciferase. Identification of two aldehyde sites and relationship between aldehyde and flavin binding. J Biol Chem. 1994 Feb 25;269(8):5612–5618. [PubMed] [Google Scholar]
  12. Lei B., Liu M., Huang S., Tu S. C. Vibrio harveyi NADPH-flavin oxidoreductase: cloning, sequencing and overexpression of the gene and purification and characterization of the cloned enzyme. J Bacteriol. 1994 Jun;176(12):3552–3558. doi: 10.1128/jb.176.12.3552-3558.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. MASSEY V., SWOBODA B. E. THE FLAVIN COMPOSITION OF PIG HEART MUSCLE PREPARATIONS. Biochem Z. 1963;338:474–484. [PubMed] [Google Scholar]
  14. Monticello D. J., Bakker D., Finnerty W. R. Plasmid-mediated degradation of dibenzothiophene by Pseudomonas species. Appl Environ Microbiol. 1985 Apr;49(4):756–760. doi: 10.1128/aem.49.4.756-760.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Omori T., Monna L., Saiki Y., Kodama T. Desulfurization of dibenzothiophene by Corynebacterium sp. strain SY1. Appl Environ Microbiol. 1992 Mar;58(3):911–915. doi: 10.1128/aem.58.3.911-915.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Piddington C. S., Kovacevich B. R., Rambosek J. Sequence and molecular characterization of a DNA region encoding the dibenzothiophene desulfurization operon of Rhodococcus sp. strain IGTS8. Appl Environ Microbiol. 1995 Feb;61(2):468–475. doi: 10.1128/aem.61.2.468-475.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  18. Taylor D. G., Trudgill P. W. Camphor revisited: studies of 2,5-diketocamphane 1,2-monooxygenase from Pseudomonas putida ATCC 17453. J Bacteriol. 1986 Feb;165(2):489–497. doi: 10.1128/jb.165.2.489-497.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Tu S. C., Mager H. I. Biochemistry of bacterial bioluminescence. Photochem Photobiol. 1995 Oct;62(4):615–624. doi: 10.1111/j.1751-1097.1995.tb08708.x. [DOI] [PubMed] [Google Scholar]
  20. Zenno S., Saigo K., Kanoh H., Inouye S. Identification of the gene encoding the major NAD(P)H-flavin oxidoreductase of the bioluminescent bacterium Vibrio fischeri ATCC 7744. J Bacteriol. 1994 Jun;176(12):3536–3543. doi: 10.1128/jb.176.12.3536-3543.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

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