• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. Sep 1996; 178(17): 5243–5248.
PMCID: PMC178323

Characterization of methyltransferase and hydroxylase genes involved in the biosynthesis of the immunosuppressants FK506 and FK520.

Abstract

FK506 and FK520 are 23-membered macrocyclic polyketides with potent immunosuppressive and antifungal activities. The gene encoding 31-O-demethyl-FK506 methyltransferase, fkbM, was isolated from Streptomyces sp. strains MA6858 and MA6548, two FK506 producers, and Streptomyces hygroscopicus subsp. ascomyceticus, an FK520 producer. The nucleotide sequence of the fkbM gene revealed an open reading frame encoding a polypeptide of 260 amino acids. Disruption of fkbM in Streptomyces sp. strain MA6548 yielded a mutant that produced 31-O-demethyl-FK506, confirming the involvement of the isolated genes in the biosynthesis of FK506 and FK520. Heterologous expression of fkbM in Streptomyces lividans established that fkbM encodes an O-methyltransferase catalyzing the methylation of the C-31 hydroxyl group of 31-O-demethyl-FK506 and FK520. A second open reading frame, fkbD, was found upstream of fkbM in all three aforementioned species and was predicted to encode a protein of 388 residues that showed a strong resemblance to cytochrome P-450 hydroxylases. Disruption of fkbD had a polar effect on the synthesis of the downstream fkbM gene product and resulted in the formation of 9-deoxo-31-O-demethyl-FK506. This established the product of fkbD as the cytochrome P-450 9-deoxo-FK506 hydroxylase, which is responsible for hydroxylation at position C-9 of the FK506 and FK520 macrolactone ring.

Full Text

The Full Text of this article is available as a PDF (750K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Bibb MJ, Cohen SN. Gene expression in Streptomyces: construction and application of promoter-probe plasmid vectors in Streptomyces lividans. Mol Gen Genet. 1982;187(2):265–277. [PubMed]
  • Bibb MJ, Findlay PR, Johnson MW. 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. [PubMed]
  • Decker H, Motamedi H, Hutchinson CR. Nucleotide sequences and heterologous expression of tcmG and tcmP, biosynthetic genes for tetracenomycin C in Streptomyces glaucescens. J Bacteriol. 1993 Jun;175(12):3876–3886. [PMC free article] [PubMed]
  • Devereux J, Haeberli P, Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. [PMC free article] [PubMed]
  • Fischer RT, Trzaskos JM, Magolda RL, Ko SS, Brosz CS, Larsen B. Lanosterol 14 alpha-methyl demethylase. Isolation and characterization of the third metabolically generated oxidative demethylation intermediate. J Biol Chem. 1991 Apr 5;266(10):6124–6132. [PubMed]
  • Garrity GM, Heimbuch BK, Motamedi H, Shafiee A. Genetic relationships among actinomycetes that produce the immunosuppressant macrolides FK506, FK520/FK523 and rapamycin. J Ind Microbiol. 1993 Jan;12(1):42–47. [PubMed]
  • Gewain KM, Occi JL, Foor F, MacNeil DJ. Vectors for generating nested deletions and facilitating subcloning G+C-rich DNA between Escherichia coli and Streptomyces sp. Gene. 1992 Sep 21;119(1):149–150. [PubMed]
  • Hatanaka H, Kino T, Miyata S, Inamura N, Kuroda A, Goto T, Tanaka H, Okuhara M. FR-900520 and FR-900523, novel immunosuppressants isolated from a Streptomyces. II. Fermentation, isolation and physico-chemical and biological characteristics. J Antibiot (Tokyo) 1988 Nov;41(11):1592–1601. [PubMed]
  • Ingrosso D, Fowler AV, Bleibaum J, Clarke S. Sequence of the D-aspartyl/L-isoaspartyl protein methyltransferase from human erythrocytes. Common sequence motifs for protein, DNA, RNA, and small molecule S-adenosylmethionine-dependent methyltransferases. J Biol Chem. 1989 Nov 25;264(33):20131–20139. [PubMed]
  • Ishizaki T, Hirayama N, Shinkawa H, Nimi O, Murooka Y. Nucleotide sequence of the gene for cholesterol oxidase from a Streptomyces sp. J Bacteriol. 1989 Jan;171(1):596–601. [PMC free article] [PubMed]
  • Kino T, Hatanaka H, Miyata S, Inamura N, Nishiyama M, Yajima T, Goto T, Okuhara M, Kohsaka M, Aoki H, et al. FK-506, a novel immunosuppressant isolated from a Streptomyces. II. Immunosuppressive effect of FK-506 in vitro. J Antibiot (Tokyo) 1987 Sep;40(9):1256–1265. [PubMed]
  • Molnár I, Aparicio JF, Haydock SF, Khaw LE, Schwecke T, König A, Staunton J, Leadlay PF. Organisation of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: analysis of genes flanking the polyketide synthase. Gene. 1996 Feb 22;169(1):1–7. [PubMed]
  • Motamedi H, Hutchinson CR. Cloning and heterologous expression of a gene cluster for the biosynthesis of tetracenomycin C, the anthracycline antitumor antibiotic of Streptomyces glaucescens. Proc Natl Acad Sci U S A. 1987 Jul;84(13):4445–4449. [PMC free article] [PubMed]
  • Motamedi H, Shafiee A, Cai SJ. Integrative vectors for heterologous gene expression in Streptomyces spp. Gene. 1995 Jul 4;160(1):25–31. [PubMed]
  • Nielsen JB, Hsu MJ, Byrne KM, Kaplan L. Biosynthesis of the immunosuppressant immunomycin: the enzymology of pipecolate incorporation. Biochemistry. 1991 Jun 11;30(23):5789–5796. [PubMed]
  • Nishida H, Sakakibara T, Aoki F, Saito T, Ichikawa K, Inagaki T, Kojima Y, Yamauchi Y, Huang LH, Guadliana MA, et al. Generation of novel rapamycin structures by microbial manipulations. J Antibiot (Tokyo) 1995 Jul;48(7):657–666. [PubMed]
  • Omer CA, Lenstra R, Litle PJ, Dean C, Tepperman JM, Leto KJ, Romesser JA, O'Keefe DP. Genes for two herbicide-inducible cytochromes P-450 from Streptomyces griseolus. J Bacteriol. 1990 Jun;172(6):3335–3345. [PMC free article] [PubMed]
  • Parsons WH, Sigal NH, Wyvratt MJ. FK-506--a novel immunosuppressant. Ann N Y Acad Sci. 1993 Jun 23;685:22–36. [PubMed]
  • Rosenberg M, Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed]
  • Schwecke T, Aparicio JF, Molnár I, König A, Khaw LE, Haydock SF, Oliynyk M, Caffrey P, Cortés J, Lester JB, et al. The biosynthetic gene cluster for the polyketide immunosuppressant rapamycin. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7839–7843. [PMC free article] [PubMed]
  • Shafiee A, Motamedi H, Chen T. Enzymology of FK-506 biosynthesis. Purification and characterization of 31-O-desmethylFK-506 O:methyltransferase from Streptomyces sp. MA6858. Eur J Biochem. 1994 Oct 15;225(2):755–764. [PubMed]
  • Summers RG, Wendt-Pienkowski E, Motamedi H, Hutchinson CR. Nucleotide sequence of the tcmII-tcmIV region of the tetracenomycin C biosynthetic gene cluster of Streptomyces glaucescens and evidence that the tcmN gene encodes a multifunctional cyclase-dehydratase-O-methyl transferase. J Bacteriol. 1992 Mar;174(6):1810–1820. [PMC free article] [PubMed]
  • Trower MK, Lenstra R, Omer C, Buchholz SE, Sariaslani FS. Cloning, nucleotide sequence determination and expression of the genes encoding cytochrome P-450soy (soyC) and ferredoxinsoy (soyB) from Streptomyces griseus. Mol Microbiol. 1992 Aug;6(15):2125–2134. [PubMed]
  • Zalkin H, Ebbole DJ. Organization and regulation of genes encoding biosynthetic enzymes in Bacillus subtilis. J Biol Chem. 1988 Feb 5;263(4):1595–1598. [PubMed]

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

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links