Nucleotide sequence of the gene for cholesterol oxidase from a Streptomyces sp

Ishizaki, Takayuki; Hirayama, Noriaki; Shinkawa, Hidenori; Nimi, Osamu; Murooka, Yoshikatsu

doi:10.1128/jb.171.1.596-601.1989

Cited by 91 publications

(39 citation statements)

References 31 publications

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“…These are the properties of translationally coupled genes found in prokaryotes (30). The predicted amino acid sequence of fkbD revealed strong homology to cytochrome P-450 hydroxylases, especially to those from streptomycetes (12,14,21,29). Di- Isolation and sequence analysis of fkbM and fkbD homologs.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 1996

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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.The polyketide, immunosuppressant compound FK506 ( Fig. 1) (13) is a 23-membered macrolide with potent antifungal activity produced by several Streptomyces species. FK506 is approximately 100-fold more potent than the structurally unrelated immunosuppressive compound cyclosporin A. Both drugs are important therapeutic agents for the prevention of graft rejection following organ and bone marrow transplantations and for the treatment of autoimmune diseases (22). FK520 (also known as immunomycin and ascomycin) is another immunosuppressive compound similar to FK506 (Fig. 1) in which the allyl group is replaced by an ethyl group at position C-21 of the macrolactone ring (9). Both the antifungal and the immunosuppressive activities of FK520 are approximately one-half of those exhibited by FK506 (9).Through precursor incorporation experiments, Byrne et al. (3) demonstrated that the polyketide portion of FK506 and FK520 is derived, for the most part, from acetate and propionate. Those authors also established the origin of the pipecolate and the cyclohexyl rings to be lysine and shikimic acid, respectively, and demonstrated that the source of the methyl portion of the methoxyl groups at C-13, C-15, and C-31 of FK520 (Fig. 1) is L-methionine.The enzymology of FK506 biosynthesis has also been explored to some extent. The pipecolate-activating enzyme which presumably incorporates pipecolate into the completed polyketide chain has been characterized previously (19). Both 31-O-demethyl-FK520 methyltransferase and 31-O-demethyl-FK506 methyltransferase (FKMT) have been isolated from the producing strains (3, 27). These two enzymes can use each other's substrate interchangeably and methylate the C-31 OH and not the C-13 or C-15 OH group (27).Here, we report the isolation and molecular characterization of two genes involved in the biosynthesis of FK506. One gene, fkbM, encodes FKMT, and the other, fkbD, encodes a cytochrome P-450 9-deoxo-FK506 hydroxylase that catalyzes hydroxylation at C-9. MATERIALS AND METHODSStandard recombinant DNA techniques were performed as described by Sambrook et al. (24).Probe design. N-terminal amino acid sequencing of FKMT from Streptomyces sp. strain MA6858 (27) gave a 39-mer with the sequence SDVVETLRLPNGA TVAHVNAGEAQFLYREIFTDRCYLRH. This peptide sequence was then used to design two nonoverlapping degenerate oligonucleotide probes, P1 and P2, in which inosine was incorporated at the third position of highly degenerate codons (2). P1 corresponded to ...

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Section: Resultsmentioning

confidence: 99%

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

et al. 1996

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“…strains SA-COO (19) and A19249 (5). These Streptomyces cholesterol oxidases were originally designated ChoA and ChoM (respectively) but now both are found in the protein databases with the name ChoD.…”

Section: Discussionmentioning

confidence: 99%

“…Supporting this notion, the sequences of the cholesterol oxidase genes cloned to date, choA and choM from two Streptomyces spp. isolates (5,19) and choB from B. sterolicum (35), show a high degree of similarity at both the nucleotide and amino acid levels. To determine whether R. equi contains chorelated sequences, Southern blots of genomic DNA digestions from three strains of this species (103 Ϫ , ATCC 6939, and MAD) ( Table 1) Ϫ .…”

Section: Identification Of the R Equi Cholesterol Oxidase Gene Choementioning

confidence: 99%

Identification and Mutagenesis by Allelic Exchange ofchoE, Encoding a Cholesterol Oxidase from the Intracellular PathogenRhodococcus equi

et al. 2001

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The virulence mechanisms of the facultative intracellular parasite Rhodococcus equi remain largely unknown. Among the candidate virulence factors of this pathogenic actinomycete is a secreted cholesterol oxidase, a putative membrane-damaging toxin. We identified and characterized the gene encoding this enzyme, the choE monocistron. Its protein product, ChoE, is homologous to other secreted cholesterol oxidases identified in Brevibacterium sterolicum and Streptomyces spp. ChoE also exhibits significant similarities to putative cholesterol oxidases encoded by Mycobacterium tuberculosis and Mycobacterium leprae. Genetic tools for use with R. equi are poorly developed. Here we describe the first targeted mutagenesis system available for this bacterium. It is based on a suicide plasmid, a selectable marker (the aacC4 apramycin resistance gene from Salmonella), and homologous recombination. The choE allele was disrupted by insertion of the aacC4 gene, cloned in pUC19 and introduced by electroporation in R. equi. choE recombinants were isolated at frequencies between 10 ؊2 and 10 ؊3 . Twelve percent of the recombinants were double-crossover choE mutants. The choE mutation was associated with loss of cooperative (CAMP-like) hemolysis with sphingomyelinase-producing bacteria (Listeria ivanovii). Functional complementation was achieved by expression of choE from pVK173-T, a pAL5000 derivative conferring hygromycin resistance. Our data demonstrate that ChoE is an important cytolytic factor for R. equi. The highly efficient targeted mutagenesis procedure that we used to generate choE isogenic mutants will be a valuable tool for the molecular analysis of R. equi virulence.

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“…Comparison of the nt and aa sequences to those of the cholesterol oxidase gene (choA) of Streptomyces sp. SA-C00 10 ) showed significant similarity of 64% and 58%, respectively.…”

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confidence: 99%

Hyperexpression and Analysis ofchoBEncoding Cholesterol Oxidase ofBrevibacterium sterolicuminEscherichia coliandStreptomyces lividans.

Ohta

Fujishiro

Yamaguchi

et al. 1992

Bioscience, Biotechnology, and Biochemistry

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Nucleotide sequence of the gene for cholesterol oxidase from a Streptomyces sp

Cited by 91 publications

References 31 publications

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

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

Identification and Mutagenesis by Allelic Exchange ofchoE, Encoding a Cholesterol Oxidase from the Intracellular PathogenRhodococcus equi

Hyperexpression and Analysis ofchoBEncoding Cholesterol Oxidase ofBrevibacterium sterolicuminEscherichia coliandStreptomyces lividans.

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