Functional Analyses of Oxygenases in Jadomycin Biosynthesis and Identification of JadH as a Bifunctional Oxygenase/Dehydrase

A novel angucycline metabolite, 2,3-dehydro-UWM6, was identified in a jadH mutant of Streptomyces venezuelae ISP5230. Both UWM6 and 2,3-dehydro-UWM6 could be converted to jadomycin A or B by a ketosynthase α (jadA) mutant of S. venezuelae. These angucycline intermediates were also converted to jadomycin A by transformant of the heterologous host Streptomyces lividans expressing the jadFGH oxygenases in vivo and by its cell-free extracts in vitro; thus the three gene products JadFGH are implicated in catalysis of the post-polyketide synthase biosynthetic reactions converting UWM6 to jadomycin aglycone. Genetic and biochemical analyses indicate that JadH possesses dehydrase activity, not previously associated with polyketide-modifying oxygenase. Since the formation of aromatic polyketides often requires multiple dehydration steps, bifunctionality of oxygenases modifying aromatic polyketides may be a general phenomenon. A novel angucycline metabolite, 2,3-dehydro-UWM6, was identified in a jadH mutant of Streptomyces venezuelae ISP5230. Both UWM6 and 2,3-dehydro-UWM6 could be converted to jadomycin A or B by a ketosynthase α (jadA) mutant of S. venezuelae. These angucycline intermediates were also converted to jadomycin A by transformant of the heterologous host Streptomyces lividans expressing the jadFGH oxygenases in vivo and by its cell-free extracts in vitro; thus the three gene products JadFGH are implicated in catalysis of the post-polyketide synthase biosynthetic reactions converting UWM6 to jadomycin aglycone. Genetic and biochemical analyses indicate that JadH possesses dehydrase activity, not previously associated with polyketide-modifying oxygenase. Since the formation of aromatic polyketides often requires multiple dehydration steps, bifunctionality of oxygenases modifying aromatic polyketides may be a general phenomenon. Polyketides, a large structurally diverse group of secondary metabolites, are produced in bacteria, fungi and plants by polyketide synthase (PKS) 1The abbreviations used are: PKS, polyketide synthase; LC-MS, liquid chromatography-mass spectroscopy. complexes (1Hopwood D.A. Chem. Rev. 1997; 97: 2465-2497Crossref PubMed Scopus (629) Google Scholar, 2Shen B. Top. Curr. Chem. 2000; 209: 1-51Crossref Google Scholar). The remarkable diversity of polyketides is to a great extent due to post-PKS modifications that determine the ultimate structures of polyketide metabolites formed. Prominent among the tailoring enzymes responsible for post-PKS modifications are oxidoreductases and group transferases (3Rawlings B.J. Nat. Prod. Rep. 1999; 16: 425-484Crossref PubMed Scopus (155) Google Scholar, 4Hutchinson C.R. Fujii I. Annu. Rev. Microbiol. 1995; 49: 201-238Crossref PubMed Scopus (210) Google Scholar, 5Rix U. Fischer C. Remsing L.L. Rohr J. Nat. Prod. Rep. 2002; 19: 542-580Crossref PubMed Scopus (227) Google Scholar). The atypical angucycline antibiotic jadomycin B (1) and its aglycone jadomycin A (2) are produced in cultures of Streptomyces venezuelae ISP5230 grown under stress conditions in a medium containing isoleucine (6Han L. Yang K.Q. Ramalingam E. Mosher R.H. Vining L.C. Microbiology. 1994; 140: 3379-3389Crossref PubMed Scopus (81) Google Scholar, 7Yang K.Q. Han L. Ayer S.W. Vining L.C. Microbiology. 1996; 142: 123-132Crossref PubMed Scopus (55) Google Scholar). The angucycline origin of jadomycins was verified by accumulation of a well known antibiotic rabelomycin (3) in the jadF disruption mutant VS655 (7Yang K.Q. Han L. Ayer S.W. Vining L.C. Microbiology. 1996; 142: 123-132Crossref PubMed Scopus (55) Google Scholar) and by production of UWM6 (4) in Streptomyces lividans transformed with the jad PKS gene cluster (i.e. jadABCDEIJ) (8Kulowski K. Pienkowski E.W. Han L. Yang K.Q. Vining L.C. Hutchinson C.R. J. Am. Chem. Soc. 1999; 121: 1786-1794Crossref Scopus (71) Google Scholar). One unique feature of the jadomycin family is its nitrogen-containing pentacyclic benz[b]oxazolophenanthridine ring, formed via a hitherto uncharacterized oxidative cleavage between C-5 and C-6 of ring B in an angucyclic polyketide intermediate. The phenanthridine ring system is plausibly derived by condensation of an amino acid with the postulated acid/aldehyde intermediate (5), formed during angucyclic ring opening (7Yang K.Q. Han L. Ayer S.W. Vining L.C. Microbiology. 1996; 142: 123-132Crossref PubMed Scopus (55) Google Scholar, 9Ayer S.W. McInnes A.G. Thibault P. Walter J.A. Doull J.L. Parnell T. Vining L.C. Tetrahedron Lett. 1991; 32: 6301-6304Crossref Scopus (70) Google Scholar). Strengthening this possibility is the formation of jadomycin analogues when different amino acids are supplied as precursors (10Doull J.L. Singh A.K. Hoare M. Ayer S.W. J. Ind. Microbiol. 1994; 13: 120-125Crossref PubMed Scopus (112) Google Scholar, 11Rix U. Zheng J.T. Remsing L.L. Greenwell L. Yang K.Q. Rohr J. J. Am. Chem. Soc. 2004; 126: 4496-4497Crossref PubMed Scopus (71) Google Scholar). Until now, the only oxygenase gene identified in the jad biosynthetic cluster (Fig. 1a)is jadF, which is implicated in oxidative ring cleavage and displays strong sequence homologies with FAD- and NADPH-dependent monooxygenases (7Yang K.Q. Han L. Ayer S.W. Vining L.C. Microbiology. 1996; 142: 123-132Crossref PubMed Scopus (55) Google Scholar). Sequence analyses suggest that the two genes, jadG and jadH, immediately downstream of jadF also encode oxygenases. JadG resembles anthrone oxygenases such as ActVA-orf6 and TcmH, while JadH strongly resembles JadF in its amino acid sequence. Consequently, JadF and JadH are potential candidates for participation in oxidative ring B cleavage (7Yang K.Q. Han L. Ayer S.W. Vining L.C. Microbiology. 1996; 142: 123-132Crossref PubMed Scopus (55) Google Scholar, 12, McVey, J. (1998) Characterization of the Downstream Genes for Jadomycin B Biosynthesis in Streptomyces venezuelae ISP5230. M.Sc. dissertation, Dalhousie University, CanadaGoogle Scholar). In this work, we further investigated the functions of JadF, JadG and JadH by product profile analyses of their mutants, enzymatic assay and in vivo/in vitro bioconversion experiments. We demonstrate the requirement for the co-presence of JadF, JadG and JadH to completely convert UWM6 to jadomycin A and established JadH as a bifunctional oxygenase/dehydrase. S. venezuelae ISP5230 and the derived strains VS655 (jadF mutant) and VS662a (jadR2 mutant) have been described previously (7Yang K.Q. Han L. Ayer S.W. Vining L.C. Microbiology. 1996; 142: 123-132Crossref PubMed Scopus (55) Google Scholar, 13Yang K.Q. Han L. Vining L.C. J. Bacteriol. 1995; 177: 6111-6117Crossref PubMed Google Scholar). Escherichia coli ET12567 has been described by MacNeil et al. (14MacNeil D.J. Gewain K.M. Rudy C.L. Dezeny G. Gibbons P.H. MacNeil T. Gene (Amst.). 1992; 111: 61-68Crossref PubMed Scopus (602) Google Scholar); other E. coli strains were from commercial sources; S. lividans TK24 was described by Hopwood et al. (15Hopwood D.A. Kieser T. Wright H.M. Bibb M.J. J. Gen. Microbiol. 1982; 129: 2257-2269Google Scholar). Plasmid pWHM1238, described in Kulowski et al. (1999), was kindly provided by Dr. Ben Shen (8Kulowski K. Pienkowski E.W. Han L. Yang K.Q. Vining L.C. Hutchinson C.R. J. Am. Chem. Soc. 1999; 121: 1786-1794Crossref Scopus (71) Google Scholar); plasmid pUWL201, described in Doumith et al. (2000), was kindly provided by Dr. Udo Wehmeier (16Doumith M. Weingarten P. Wehmeier U.F. Salah-Bey K. Benhamou B. Capdevila C. Michel J.M. Piepersberg W. Raynal M.C. Mol. Gen. Genet. 2000; 264: 477-485Crossref PubMed Scopus (90) Google Scholar). Ultrafiltration centrifugation tubes (Centriplus YM series) were purchased from Millipore. Restriction enzymes, T4 DNA ligase, and Pfu DNA polymerase were purchased from Promega or Takara. Competent E. coli cells were prepared and transformed by standard procedures (17Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory, Cold Spring Laboratory, NY2001Google Scholar). Plasmid DNA was isolated from E. coli by the alkaline method (17Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory, Cold Spring Laboratory, NY2001Google Scholar). Cultures of S. venezuelae strains used for DNA extraction were grown in MYME medium (13Yang K.Q. Han L. Vining L.C. J. Bacteriol. 1995; 177: 6111-6117Crossref PubMed Google Scholar) at 30 °C for 36 h; genomic DNA was isolated as described by Kieser et al. (18Kieser T. Bibb M.J. Buttner M.J. Chater K.F. Hopwood D.A. Practical Streptomyces Genetics. The John Innes Foundation, Norwich, UK2000Google Scholar). DNA was manipulated by standard procedures (17Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory, Cold Spring Laboratory, NY2001Google Scholar). Protoplasts of S. venezuelae were prepared and transformed as described previously (7Yang K.Q. Han L. Ayer S.W. Vining L.C. Microbiology. 1996; 142: 123-132Crossref PubMed Scopus (55) Google Scholar). pJV69A was constructed by inserting a 7.2-kb XhoI fragment (with intact jadDFGHK and partial jadL) into pBluescript II KS(+). The jadFGH portion of pJV69A was re-sequenced; its revised sequence was deposited in GenBank™ (accession number AY773079). Related proteins were searched with BLASTP (www.ncbi.nlm.nih.gov), and selected sequences were aligned with ClustalX (ftp-igbmc.u-strasbg.fr/pub/ClustalX/) (19Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35620) Google Scholar). Disruption of jadH—To facilitate inactivation of this gene, a 6.0-kb BamHI fragment of S. venezuelae DNA containing jadFGHK was cloned in pHJL400, furnishing pJV77A. Construction of the jadH disruption plasmid involved removing an EcoRI/MluI fragment from pJV77A. The remaining fragment, consisting of the vector plus a 3.8-kb BamHI/MluI insert, was blunt-ended by treatment with the Klenow fragment of DNA polymerase I and religated to generate pJV91. Digestion of pJV91 with NcoI (internal to jadH) and ligation of the linear product to an apramycin resistance gene with NcoI ends yielded the two recombinant plasmids, pJV92A and pJV92B, which carried the apramycin resistance gene in opposite orientations. When both plasmids were introduced into S. venezuelae, only pJV92B gave transformants (VS667). Selection for an apramycin-resistant and thiostrepton-sensitive phenotype gave the jadH mutant VS668 (Fig. 2). In-frame Deletion of jadA—Two 1.5-kb fragments flanking jadA were obtained by PCR using primer pairs P1 and P2. For the fragment upstream of jadA, P1F (5′-CCCAAGCTTGCAGTGCCTGGCCGACCA-3′, HindIII) and P1R (5′-GGAATTCCATATGTCACGCGTTCGCCTCCCA-3′, NdeI) were used; for the fragment downstream of jadA, P2F (5′-GGAATTCCATATGAGCGCGTCCGTGGTG-3′, NdeI) and P2R (5′-CGGAATTCAGGCGGCGGCGACGGC-3′, EcoRI) were used. After digestion with appropriate enzymes, the two fragments were inserted into HindIII/EcoRI-digested pHJL400 to generate pHK400A. The latter was used to transform protoplasts of VS662a. Transformants were propagated on MYM (maltase, yeast extract, and malt extract; Ref. 6Han L. Yang K.Q. Ramalingam E. Mosher R.H. Vining L.C. Microbiology. 1994; 140: 3379-3389Crossref PubMed Scopus (81) Google Scholar) agar without thiostrepton selection for three generations; colonies from spores collected after non-selective propagation were picked for sensitivity to thiostrepton and examined for loss of jadomycin B production in cultures grown under conditions supporting biosynthesis of the antibiotic. The mutant was that the loss of jadomycin production in with of jadA was investigated by PCR using and (Fig. For with in jadH, the genomic DNA was completely with by and to a The was a fragment from pJV69A with by the method (Fig. 2). The genomic DNA from jadA was with XhoI and a fragment from was as a of with DNA fragments on the was by the method using procedures described by (Fig. cultures of VS668 grown in liquid medium containing apramycin as described by Doull et al. (10Doull J.L. Singh A.K. Hoare M. Ayer S.W. J. Ind. Microbiol. 1994; 13: 120-125Crossref PubMed Scopus (112) Google Scholar) were with After of the by liquid the product was isolated and its was by as described U. C. Remsing L.L. Greenwell Vining L.C. Yang K. Rohr J. PubMed Scopus Google Scholar). The of (7Yang K.Q. Han L. Ayer S.W. Vining L.C. Microbiology. 1996; 142: 123-132Crossref PubMed Scopus (55) Google Scholar) was by and the fragment was into the of to plasmid in which the of jadF is opposite to that of was with and to the insert, which was cloned into vector (16Doumith M. Weingarten P. Wehmeier U.F. Salah-Bey K. Benhamou B. Capdevila C. Michel J.M. Piepersberg W. Raynal M.C. Mol. Gen. Genet. 2000; 264: 477-485Crossref PubMed Scopus (90) Google Scholar) to and from were with a fragment of pJV69A to generate in which jadFGH is downstream of the of of into S. lividans TK24 by standard procedures gave the transformant (18Kieser T. Bibb M.J. Buttner M.J. Chater K.F. Hopwood D.A. Practical Streptomyces Genetics. The John Innes Foundation, Norwich, UK2000Google Scholar). was isolated from a of VS655 as described previously (7Yang K.Q. Han L. Ayer S.W. Vining L.C. Microbiology. 1996; 142: 123-132Crossref PubMed Scopus (55) Google Scholar). was isolated from S. venezuelae mutant VS668 as described S. lividans TK24 was transformed with cultures of the transformant grown as described UWM6 was obtained (8Kulowski K. Pienkowski E.W. Han L. Yang K.Q. Vining L.C. Hutchinson C.R. J. Am. Chem. Soc. 1999; 121: 1786-1794Crossref Scopus (71) Google Scholar); UWM6 was and was converted to extracts used as the for bioconversion and were by to of jadA mutant was for jadomycin production by the described by Doull et al. (10Doull J.L. Singh A.K. Hoare M. Ayer S.W. J. Ind. Microbiol. 1994; 13: 120-125Crossref PubMed Scopus (112) Google Scholar) that the medium was with 2,3-dehydro-UWM6, and were to the cultures at the after were with After the cultures were with and the extracts were in The were by and liquid chromatography-mass cultures of jadFGH in and were grown at 30 for in (18Kieser T. Bibb M.J. Buttner M.J. Chater K.F. Hopwood D.A. Practical Streptomyces Genetics. The John Innes Foundation, Norwich, UK2000Google Scholar) with thiostrepton was collected by centrifugation and to medium with UWM6 or The cultures were for and with Cultures of to as was in with thiostrepton at 30 for After the was and with were collected by centrifugation and with a containing and the were in of and on for 30 were by for and was by centrifugation at °C for The was by a centrifugation and to was by method PubMed Scopus Google Scholar). containing of cell-free extracts and or and isoleucine were with at 30 °C for The reactions were by of of and the was with of was prepared with the as and used as Plasmid was used as for the jadF plasmid Plasmid constructed by inserting a fragment from pJV69A to pBluescript II was used as for the jadH plasmids The fragments containing jadF or jadH were by PCR using Pfu DNA For the (jadF and (jadF EcoRI) were used; for the and EcoRI) were used. The jadF and jadH were inserted into the which the and an sequence. of the was by DNA and of E. coli transformed with and were at °C in medium with and When cultures grown to was to a of was at °C for to were by centrifugation The were in and on for was to the and the was by centrifugation The proteins was on a of previously with The was with of proteins were with proteins were by and their were by the method PubMed Scopus Google Scholar). The of by that two proteins of and been produced (Fig. Both of the two proteins were also identified by digestion and of the fragments by with the sequence of the containing oxygenase and were at 30 °C for were by of and with of the were in the was used as described (7Yang K.Q. Han L. Ayer S.W. Vining L.C. Microbiology. 1996; 142: 123-132Crossref PubMed Scopus (55) Google Scholar). A and 2,3-dehydro-UWM6, and rabelomycin were by on a system with a and an The with acid and with acid The of B from to between and at for and to between and For an system was the described for and for the using with a of and a to The from in The amino acid sequences of JadF and JadH are and of other aromatic with JadF and with (7Yang K.Q. Han L. Ayer S.W. Vining L.C. Microbiology. 1996; 142: 123-132Crossref PubMed Scopus (55) Google Scholar, 12, McVey, J. (1998) Characterization of the Downstream Genes for Jadomycin B Biosynthesis in Streptomyces venezuelae ISP5230. M.Sc. dissertation, Dalhousie University, CanadaGoogle Scholar) and oxygenases implicated in aromatic polyketide ring cleavage For postulated to be a L. E. U. G. C. Rohr J. J.A. Chem. 1999; PubMed Scopus Google Scholar, J.A. J. Bacteriol. PubMed Scopus Google Scholar) responsible for the ring cleavage in the acid polyketide with JadF and with The oxygenase with JadF and with is to in the gene cluster for biosynthesis of the acid polyketide M. Rohr J. J.A. C. Chem. 2004; PubMed Google and the oxygenase to JadF and to is postulated to ring cleavage in the J. Chem. 2002; PubMed Scopus Google Scholar). in FAD- and monooxygenases are in JadF and a by a with the of P. J. Mol. PubMed Scopus Google Scholar, 2000; PubMed Scopus Google a responsible for the G. G. P. B. J. Mol. PubMed Scopus Google Scholar) and a involved in both and (Fig. 1997; PubMed Scopus Google Scholar). The sequence of JadG is with a constructed from two acids and a of sequence and an via gene The two are to anthrone oxygenases such as and amino acid Hopwood D.A. J. Bacteriol. 1997; PubMed Google Scholar, G. L. F. G. C. B. J. PubMed Scopus Google Scholar) and and amino acid B. Hutchinson C.R. 32: PubMed Scopus (71) Google that JadG in jadomycin of the at in S. venezuelae genomic DNA with the at in DNA from the apramycin-resistant and thiostrepton-sensitive transformant were with disruption of jadH in VS668 (Fig. of the transformant under conditions jadomycin production by the in of a with and and of the that was 2,3-dehydro-UWM6 U. C. Remsing L.L. Greenwell Vining L.C. Yang K. Rohr J. PubMed Scopus Google Scholar). facilitate the of appropriate for bioconversion a jadA mutant was constructed by a fragment from the jadA sequence in a in which the of jadomycin biosynthesis has been by inserting an apramycin resistance into the gene (13Yang K.Q. Han L. Vining L.C. J. Bacteriol. 1995; 177: 6111-6117Crossref PubMed Google Scholar). the mutant from the in jadomycin B without and in the of the when with Since S. venezuelae strains derived from VS662a be the in bioconversion VS662a protoplasts transformed with were for a The strains selected were propagated for three on MYM thiostrepton-sensitive strains examined under conditions supporting jadomycin B produced this while the PCR of the jadA (Fig. The of jadA was also verified by (Fig. One of the was and jadomycin B production by in the after a of that in was when the was to The may be by a partial of the jadA by ketosynthase α gene in S. venezuelae ISP5230. 2,3-dehydro-UWM6, and rabelomycin were as for bioconversion by with the of mutant) and of the for of the that 2,3-dehydro-UWM6 and two to jadomycin B and jadomycin in a to that in S. venezuelae ISP5230 of jadomycin A was that of jadomycin The of the by that 2,3-dehydro-UWM6 was converted to of UWM6 with the rabelomycin and of jadomycins A and B. The between jadomycins A and B was also to that in S. venezuelae ISP5230. The of this bioconversion be due to the of the which is converted to rabelomycin by dehydration and (8Kulowski K. Pienkowski E.W. Han L. Yang K.Q. Vining L.C. Hutchinson C.R. J. Am. Chem. Soc. 1999; 121: 1786-1794Crossref Scopus (71) Google Scholar). jadomycins were when rabelomycin was the the of rabelomycin in vivo and in A and A and A and vitro in a the functions of UWM6 and 2,3-dehydro-UWM6 were used as for by When UWM6 was with a of its h; a to jadomycin A and was by to have the a to rabelomycin was also In cultures of UWM6 also of jadomycin was converted to In 2,3-dehydro-UWM6 was in cultures of while in gave jadomycin as and of the and was when the of the were to and the was When at this UWM6 was converted to a to jadomycin A was also In the of 2,3-dehydro-UWM6, and to jadomycin A and were after at 30 Both UWM6 and 2,3-dehydro-UWM6 were in h; and the of jadomycin A converted from UWM6 was only of that from In the with cell-free extract, the of UWM6 and 2,3-dehydro-UWM6 was not and jadomycin A was not The by vivo and in indicate that post-PKS of UWM6 to jadomycin A is by the co-presence of JadF, JadG and JadH not the possibility of a from the the of JadF and which were to be bifunctional the two enzymes were in E. coli and for under conditions 30 and 2,3-dehydro-UWM6 were used as potential and of the by gave the JadF not UWM6 or for both UWM6 and 2,3-dehydro-UWM6 and and were converted to rabelomycin and In without enzymes, the The were not by the or The of or of also jadA ketosynthase in mutant of S. UWM6 and 2,3-dehydro-UWM6 were identified as intermediates in jadomycin In the biosynthesis of jadomycin the from UWM6 requires two and an at and cleavage and of ring B with of an amino acid to generate the ring of UWM6 to jadomycin A by vivo/in that the reactions in this are by functions provided on JadF, and The group in rabelomycin when jadF is while the in 2,3-dehydro-UWM6 when jadH is that both JadF and also dehydrase JadF and JadH The JadH dehydration was verified by in vitro enzymatic UWM6 and 2,3-dehydro-UWM6 were at to rabelomycin and by has been is a involved in the biosynthesis of the in S. Mol. PubMed Scopus Google Scholar). JadH is the of a bifunctional in polyketide that UWM6 is converted to jadomycin A by and the between JadF and JadF is also to be a bifunctional oxygenase/dehydrase. JadF convert UWM6 in potential of JadF by we prepared JadF by removing the with and investigated the of JadF with the potential as used for was after at 30 °C for and that the JadF may be due to the dehydration of JadF an appropriate with in of this is and rabelomycin was also in the jadG mutant U. C. Remsing L.L. Greenwell Vining L.C. Yang K. Rohr J. PubMed Scopus Google the dehydration of JadF on its with is a in angucycline and other aromatic polyketide biosynthetic in and Chem. Rev. 1997; 97: PubMed Scopus Google Scholar, S. C. Rohr J. J.M. J. Chem. 1994; Scopus Google Scholar, L. S. B. Hopwood D.A. Microbiol. Lett. 1999; PubMed Google Scholar, B. U. Remsing L.L. T. F. C. J.A. Rohr J. Chem. 2004; PubMed Scopus Google Scholar). The between the oxygenase enzymes involved in and to indicate that bifunctionality is a general of aromatic polyketide-modifying oxygenases. Sequence analyses and of JadG in other PKS gene JadG as an anthrone the jadG inactivation mutant three U. C. Remsing L.L. Greenwell Vining L.C. Yang K. Rohr J. PubMed Scopus Google and VS668 yielded 2,3-dehydro-UWM6, an angucyclic without of the (Fig. also that be when UWM6 or 2,3-dehydro-UWM6 was converted by we the possibility that JadG the after (8Kulowski K. Pienkowski E.W. Han L. Yang K.Q. Vining L.C. Hutchinson C.R. J. Am. Chem. Soc. 1999; 121: 1786-1794Crossref Scopus (71) Google by were also in products II and of was formed by the JadG Hopwood D.A. C. PubMed Scopus Google Scholar, Hopwood D.A. C. 1995; PubMed Scopus Google Scholar). In vivo and in vitro bioconversion with that ring B cleavage when JadF, and JadH are Both JadF and JadH to a responsible for ring cleavage in JadF JadH are the to ring B cleavage in The requirement of JadG for ring B cleavage is strongly by jadG inactivation ring B intact and biochemical are to ring B and JadG is not to the responsible for this oxidative ring cleavage (Fig. The of Chem. Rev. 1997; 97: PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar) and C. F. Rohr J. J. Am. Chem. Soc. PubMed Scopus Google Scholar) ring B cleavage to that for The associated enzymes for may with with and with the enzymes for are with with and with the biosynthetic gene cluster possesses an oxygenase gene a to both JadF and JadH may for the ring B after cleavage in In the was to be a intermediate to a ring product J. Am. Chem. Soc. Scopus (55) Google Scholar) (Fig. In was to be the 2,3-dehydro-UWM6 as well as a novel were to be These were obtained after inactivation of and The two enzymes were to in a to the cleavage T. Fischer C. C. Rohr J. J. Am. Chem. Soc. 2004; 126: PubMed Scopus Google Scholar). of the biosynthesis of polyketides may in the ring cleavage reactions and in In by post-PKS reactions to the jadomycin we demonstrate JadF, and JadH the post-PKS modifications from UWM6 to jadomycin A and identified the post-PKS by bifunctional oxygenase/dehydrase.

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