Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Aug;175(16):5159–5167. doi: 10.1128/jb.175.16.5159-5167.1993

Thioredoxin-thioredoxin reductase system of Streptomyces clavuligerus: sequences, expression, and organization of the genes.

G Cohen 1, M Yanko 1, M Mislovati 1, A Argaman 1, R Schreiber 1, Y Av-Gay 1, Y Aharonowitz 1
PMCID: PMC204983  PMID: 8349555

Abstract

The genes that encode thioredoxin and thioredoxin reductase of Streptomyces clavuligerus were cloned, and their DNA sequences were determined. Previously, we showed that S. clavuligerus possesses a disulfide reductase with broad substrate specificity that biochemically resembles the thioredoxin oxidoreductase system and may play a role in the biosynthesis of beta-lactam antibiotics. It consists consists of two components, a 70-kDa NADPH-dependent flavoprotein disulfide reductase with two identical subunits and a 12-kDa heat-stable protein general disulfide reductant. In this study, we found, by comparative analysis of their predicted amino acid sequences, that the 35-kDa protein is in fact thioredoxin reductase; it shares 48.7% amino acid sequence identity with Escherichia coli thioredoxin reductase, the 12-kDa protein is thioredoxin, and it shares 28 to 56% amino acid sequence identity with other thioredoxins. The streptomycete thioredoxin reductase has the identical cysteine redox-active region--Cys-Ala-Thr-Cys--and essentially the same flavin adenine dinucleotide- and NADPH dinucleotide-binding sites as E. coli thioredoxin reductase and is partially able to accept E. coli thioredoxin as a substrate. The streptomycete thioredoxin has the same cysteine redox-active segment--Trp-Cys-Gly-Pro-Cys--that is present in virtually all eucaryotic and procaryotic thioredoxins. However, in vivo it is unable to donate electrons to E. coli methionine sulfoxide reductase and does not serve as a substrate in vitro for E. coli thioredoxin reductase. The S. clavuligerus thioredoxin (trxA) and thioredoxin reductase (trxB) genes are organized in a cluster. They are transcribed in the same direction and separated by 33 nucleotides. In contrast, the trxA and trxB genes of E. coli, the only other organism in which both genes have been characterized, are physically widely separated.

Full text

PDF
5159

Images in this article

5163Image
on p.5163

Selected References

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

  1. Aboagye-Kwarteng T., Smith K., Fairlamb A. H. Molecular characterization of the trypanothione reductase gene from Crithidia fasciculata and Trypanosoma brucei: comparison with other flavoprotein disulphide oxidoreductases with respect to substrate specificity and catalytic mechanism. Mol Microbiol. 1992 Nov;6(21):3089–3099. doi: 10.1111/j.1365-2958.1992.tb01766.x. [DOI] [PubMed] [Google Scholar]
  2. Aharonowitz Y., Av-Gay Y., Schreiber R., Cohen G. Characterization of a broad-range disulfide reductase from Streptomyces clavuligerus and its possible role in beta-lactam antibiotic biosynthesis. J Bacteriol. 1993 Feb;175(3):623–629. doi: 10.1128/jb.175.3.623-629.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bibb M. J., Findlay P. R., Johnson M. W. The relationship between base composition and codon usage in bacterial genes and its use for the simple and reliable identification of protein-coding sequences. Gene. 1984 Oct;30(1-3):157–166. doi: 10.1016/0378-1119(84)90116-1. [DOI] [PubMed] [Google Scholar]
  4. Brown N. L., Ford S. J., Pridmore R. D., Fritzinger D. C. Nucleotide sequence of a gene from the Pseudomonas transposon Tn501 encoding mercuric reductase. Biochemistry. 1983 Aug 16;22(17):4089–4095. doi: 10.1021/bi00286a015. [DOI] [PubMed] [Google Scholar]
  5. Chen N. Y., Zhang J. J., Paulus H. Chromosomal location of the Bacillus subtilis aspartokinase II gene and nucleotide sequence of the adjacent genes homologous to uvrC and trx of Escherichia coli. J Gen Microbiol. 1989 Nov;135(11):2931–2940. doi: 10.1099/00221287-135-11-2931. [DOI] [PubMed] [Google Scholar]
  6. Decottignies P., Schmitter J. M., Dutka S., Jacquot J. P., Miginiac-Maslow M. Characterization and primary structure of a second thioredoxin from the green alga, Chlamydomonas reinhardtii. Eur J Biochem. 1991 Jun 1;198(2):505–512. doi: 10.1111/j.1432-1033.1991.tb16043.x. [DOI] [PubMed] [Google Scholar]
  7. Gan Z. R. Yeast thioredoxin genes. J Biol Chem. 1991 Jan 25;266(3):1692–1696. [PubMed] [Google Scholar]
  8. Greer S., Perham R. N. Glutathione reductase from Escherichia coli: cloning and sequence analysis of the gene and relationship to other flavoprotein disulfide oxidoreductases. Biochemistry. 1986 May 6;25(9):2736–2742. doi: 10.1021/bi00357a069. [DOI] [PubMed] [Google Scholar]
  9. Haller B. L., Fuchs J. A. Mapping of trxB, a mutation responsible for reduced thioredoxin reductase activity. J Bacteriol. 1984 Sep;159(3):1060–1062. doi: 10.1128/jb.159.3.1060-1062.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Holmgren A. Bovine thioredoxin system. Purification of thioredoxin reductase from calf liver and thymus and studies of its function in disulfide reduction. J Biol Chem. 1977 Jul 10;252(13):4600–4606. [PubMed] [Google Scholar]
  11. Holmgren A. Thioredoxin and glutaredoxin systems. J Biol Chem. 1989 Aug 25;264(24):13963–13966. [PubMed] [Google Scholar]
  12. Johnson R. S., Biemann K. The primary structure of thioredoxin from Chromatium vinosum determined by high-performance tandem mass spectrometry. Biochemistry. 1987 Mar 10;26(5):1209–1214. doi: 10.1021/bi00379a001. [DOI] [PubMed] [Google Scholar]
  13. Jones S. W., Luk K. C. Isolation of a chicken thioredoxin cDNA clone. Thioredoxin mRNA is differentially expressed in normal and Rous sarcoma virus-transformed chicken embryo fibroblasts. J Biol Chem. 1988 Jul 15;263(20):9607–9611. [PubMed] [Google Scholar]
  14. Karplus P. A., Schulz G. E. Refined structure of glutathione reductase at 1.54 A resolution. J Mol Biol. 1987 Jun 5;195(3):701–729. doi: 10.1016/0022-2836(87)90191-4. [DOI] [PubMed] [Google Scholar]
  15. Kuriyan J., Kong X. P., Krishna T. S., Sweet R. M., Murgolo N. J., Field H., Cerami A., Henderson G. B. X-ray structure of trypanothione reductase from Crithidia fasciculata at 2.4-A resolution. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8764–8768. doi: 10.1073/pnas.88.19.8764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kuriyan J., Krishna T. S., Wong L., Guenther B., Pahler A., Williams C. H., Jr, Model P. Convergent evolution of similar function in two structurally divergent enzymes. Nature. 1991 Jul 11;352(6331):172–174. doi: 10.1038/352172a0. [DOI] [PubMed] [Google Scholar]
  17. Laddaga R. A., Chu L., Misra T. K., Silver S. Nucleotide sequence and expression of the mercurial-resistance operon from Staphylococcus aureus plasmid pI258. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5106–5110. doi: 10.1073/pnas.84.15.5106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Le Marechal P., Hoang B. M., Schmitter J. M., Van Dorsselaer A., Decottignies P. Purification, properties and primary structure of thioredoxin from Aspergillus nidulans. Eur J Biochem. 1992 Dec 1;210(2):421–429. doi: 10.1111/j.1432-1033.1992.tb17437.x. [DOI] [PubMed] [Google Scholar]
  19. Lim C. J., Geraghty D., Fuchs J. A. Cloning and nucleotide sequence of the trxA gene of Escherichia coli K-12. J Bacteriol. 1985 Jul;163(1):311–316. doi: 10.1128/jb.163.1.311-316.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lim C. J., Gleason F. K., Fuchs J. A. Cloning, expression, and characterization of the Anabaena thioredoxin gene in Escherichia coli. J Bacteriol. 1986 Dec;168(3):1258–1264. doi: 10.1128/jb.168.3.1258-1264.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lipman D. J., Pearson W. R. Rapid and sensitive protein similarity searches. Science. 1985 Mar 22;227(4693):1435–1441. doi: 10.1126/science.2983426. [DOI] [PubMed] [Google Scholar]
  22. Luthman M., Holmgren A. Rat liver thioredoxin and thioredoxin reductase: purification and characterization. Biochemistry. 1982 Dec 21;21(26):6628–6633. doi: 10.1021/bi00269a003. [DOI] [PubMed] [Google Scholar]
  23. Mark D. F., Chase J. W., Richardson C. C. Genetic mapping of trxA, a gene affecting thioredoxin in Escherichia coli K12. Mol Gen Genet. 1977 Oct 20;155(2):145–152. doi: 10.1007/BF00393153. [DOI] [PubMed] [Google Scholar]
  24. Mattevi A., Schierbeek A. J., Hol W. G. Refined crystal structure of lipoamide dehydrogenase from Azotobacter vinelandii at 2.2 A resolution. A comparison with the structure of glutathione reductase. J Mol Biol. 1991 Aug 20;220(4):975–994. doi: 10.1016/0022-2836(91)90367-f. [DOI] [PubMed] [Google Scholar]
  25. Meng M., Hogenkamp H. P. Purification, characterization, and amino acid sequence of thioredoxin from Corynebacterium nephridii. J Biol Chem. 1981 Sep 10;256(17):9174–9182. [PubMed] [Google Scholar]
  26. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  27. Mizusawa S., Ward D. F. A bacteriophage lambda vector for cloning with BamHI and Sau3A. Gene. 1982 Dec;20(3):317–322. doi: 10.1016/0378-1119(82)90200-1. [DOI] [PubMed] [Google Scholar]
  28. Newton G. L., Fahey R. C., Cohen G., Aharonowitz Y. Low-molecular-weight thiols in streptomycetes and their potential role as antioxidants. J Bacteriol. 1993 May;175(9):2734–2742. doi: 10.1128/jb.175.9.2734-2742.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. O'Donnell M. E., Williams C. H., Jr Reaction of both active site thiols of reduced thioredoxin reductase with N-ethylmaleimide. Biochemistry. 1985 Dec 17;24(26):7617–7621. doi: 10.1021/bi00347a018. [DOI] [PubMed] [Google Scholar]
  30. Perry D., Abraham E. P., Baldwin J. E. Factors affecting the isopenicillin N synthetase reaction. Biochem J. 1988 Oct 1;255(1):345–351. [PMC free article] [PubMed] [Google Scholar]
  31. Russel M., Model P. Characterization of the cloned fip gene and its product. J Bacteriol. 1984 Feb;157(2):526–532. doi: 10.1128/jb.157.2.526-532.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Russel M., Model P. Sequence of thioredoxin reductase from Escherichia coli. Relationship to other flavoprotein disulfide oxidoreductases. J Biol Chem. 1988 Jun 25;263(18):9015–9019. [PubMed] [Google Scholar]
  33. Russel M., Model P. The role of thioredoxin in filamentous phage assembly. Construction, isolation, and characterization of mutant thioredoxins. J Biol Chem. 1986 Nov 15;261(32):14997–15005. [PubMed] [Google Scholar]
  34. Schiering N., Kabsch W., Moore M. J., Distefano M. D., Walsh C. T., Pai E. F. Structure of the detoxification catalyst mercuric ion reductase from Bacillus sp. strain RC607. Nature. 1991 Jul 11;352(6331):168–172. doi: 10.1038/352168a0. [DOI] [PubMed] [Google Scholar]
  35. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  36. Shiffman D., Mevarech M., Jensen S. E., Cohen G., Aharonowitz Y. Cloning and comparative sequence analysis of the gene coding for isopenicillin N synthase in Streptomyces. Mol Gen Genet. 1988 Nov;214(3):562–569. doi: 10.1007/BF00330495. [DOI] [PubMed] [Google Scholar]
  37. Stephens P. E., Lewis H. M., Darlison M. G., Guest J. R. Nucleotide sequence of the lipoamide dehydrogenase gene of Escherichia coli K12. Eur J Biochem. 1983 Oct 3;135(3):519–527. doi: 10.1111/j.1432-1033.1983.tb07683.x. [DOI] [PubMed] [Google Scholar]
  38. Tartaglia L. A., Storz G., Brodsky M. H., Lai A., Ames B. N. Alkyl hydroperoxide reductase from Salmonella typhimurium. Sequence and homology to thioredoxin reductase and other flavoprotein disulfide oxidoreductases. J Biol Chem. 1990 Jun 25;265(18):10535–10540. [PubMed] [Google Scholar]
  39. Tonissen K. F., Robins A. J., Wells J. R. Nucleotide sequence of a cDNA encoding rat thioredoxin. Nucleic Acids Res. 1989 May 25;17(10):3973–3973. doi: 10.1093/nar/17.10.3973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wierenga R. K., Hol W. G. Predicted nucleotide-binding properties of p21 protein and its cancer-associated variant. Nature. 1983 Apr 28;302(5911):842–844. doi: 10.1038/302842a0. [DOI] [PubMed] [Google Scholar]
  41. Wollman E. E., d'Auriol L., Rimsky L., Shaw A., Jacquot J. P., Wingfield P., Graber P., Dessarps F., Robin P., Galibert F. Cloning and expression of a cDNA for human thioredoxin. J Biol Chem. 1988 Oct 25;263(30):15506–15512. [PubMed] [Google Scholar]

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

RESOURCES