title: Spliceostatin hemiketal biosynthesis in Burkholderia spp. is catalyzed by an iron/α-ketoglutarate-dependent dioxygenase.
abstract: Spliceostatins are potent spliceosome inhibitors biosynthesized by a hybrid nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) system of the trans-acyl transferase (AT) type. Burkholderia sp. FERM BP-3421 produces hemiketal spliceostatins, such as FR901464, as well as analogs containing a terminal carboxylic acid. We provide genetic and biochemical evidence for hemiketal biosynthesis by oxidative decarboxylation rather than the previously hypothesized Baeyer-Villiger oxidative release postulated to be catalyzed by a flavin-dependent monooxygenase (FMO) activity internal to the last module of the PKS. Inactivation of Fe(II)/α-ketoglutarate-dependent dioxygenase gene fr9P led to loss of hemiketal congeners, whereas the mutant was still able to produce all major carboxylic acid-type compounds. FMO mutants, on the other hand, produced both hemiketal and carboxylic acid analogs containing an exocyclic methylene instead of an epoxide, indicating that the FMO is involved in epoxidation rather than Baeyer-Villiger oxidation. Moreover, recombinant Fr9P enzyme was shown to catalyze hydroxylation to form β-hydroxy acids, which upon decarboxylation led to hemiketal FR901464. Finally, a third oxygenase activity encoded in the biosynthetic gene cluster, the cytochrome P450 monooxygenase Fr9R, was assigned as a 4-hydroxylase based on gene inactivation results. Identification and deletion of the gene involved in hemiketal formation allowed us to generate a strain--the dioxygenase fr9P(-) mutant--that accumulates only the carboxylic acid-type spliceostatins, which are as potent as the hemiketal analogs, when derivatized to increase cell permeability, but are chemically more stable.
authors.affiliation: Natural Products Laboratory, Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development, Groton, CT 06340 alessandra.eustaquio@pfizer.com.
authors.forename: Alessandra S
authors.initials: AS
authors.lastname: Eustáquio
authors.affiliation: Natural Products Laboratory, Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development, Groton, CT 06340.
authors.forename: Jeffrey E
authors.initials: JE
authors.lastname: Janso
authors.affiliation: Natural Products Laboratory, Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development, Groton, CT 06340.
authors.forename: Anokha S
authors.initials: AS
authors.lastname: Ratnayake
authors.affiliation: Natural Products Laboratory, Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development, Groton, CT 06340.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: O'Donnell
authors.affiliation: Natural Products Laboratory, Worldwide Medicinal Chemistry, Pfizer Worldwide Research and Development, Groton, CT 06340.
authors.forename: Frank E
authors.initials: FE
authors.lastname: Koehn
mesh_terms: D055162:Biocatalysis; D019117:Burkholderia / Q000201:enzymology / Q000378:metabolism*; D049308:Dioxygenases / Q000378:metabolism*; D007501:Iron / Q000378:metabolism*; D008969:Molecular Sequence Data
chemical_list: D007501:Iron; D049308:Dioxygenases
keywords: bacteria; natural product; secondary metabolite; thailanstatin
references.citation: Biochemistry. 2011 Mar 15;50(10):1739-54
references.pmid: 21250661
references.citation: Org Lett. 2013 Jul 19;15(14):3610-3
references.pmid: 23822896
references.citation: J Am Chem Soc. 2007 Mar 7;129(9):2648-59
references.pmid: 17279752
references.citation: Nat Prod Rep. 2013 Jan;30(1):175-200
references.pmid: 23051833
references.citation: ACS Chem Biol. 2014 Mar 21;9(3):643-8
references.pmid: 24377313
references.citation: J Am Chem Soc. 2014 Mar 26;136(12):4488-91
references.pmid: 24617828
references.citation: Nat Prod Rep. 2010 Jul;27(7):996-1047
references.pmid: 20464003
references.citation: J Med Chem. 2013 Sep 12;56(17):6576-82
references.pmid: 23919277
references.citation: Chem Rev. 2004 Sep;104(9):3947-80
references.pmid: 15352783
references.citation: Appl Environ Microbiol. 2012 Mar;78(6):2034-8
references.pmid: 22247174
references.citation: J Antibiot (Tokyo). 1996 Dec;49(12):1196-203
references.pmid: 9031664
references.citation: J Nat Prod. 2013 Apr 26;76(4):685-93
references.pmid: 23517093
references.citation: J Antibiot (Tokyo). 2004 Mar;57(3):188-96
references.pmid: 15152804
references.citation: J Med Chem. 2013 Sep 12;56(17):6573-5
references.pmid: 23981063
references.citation: Cancer Sci. 2012 Sep;103(9):1611-6
references.pmid: 22691055
references.citation: Angew Chem Int Ed Engl. 2013 Dec 9;52(50):13215-8
references.pmid: 24307486
references.citation: J Antibiot (Tokyo). 1996 Dec;49(12):1204-11
references.pmid: 9031665
references.citation: J Am Chem Soc. 2005 Dec 21;127(50):17594-5
references.pmid: 16351075
references.citation: Biosci Biotechnol Biochem. 2008 Nov;72(11):2946-52
references.pmid: 18997414
references.citation: J Am Chem Soc. 2001 Oct 17;123(41):9974-83
references.pmid: 11592876
references.citation: Angew Chem Int Ed Engl. 2013 Jan 21;52(4):1143-7
references.pmid: 23212972
references.citation: J Antibiot (Tokyo). 2004 Mar;57(3):180-7
references.pmid: 15152803
references.citation: J Am Chem Soc. 2012 Nov 7;134(44):18181-4
references.pmid: 23102024
references.citation: Org Lett. 2013 Oct 4;15(19):5088-91
references.pmid: 24050251
references.citation: J Biol Chem. 2014 Jan 24;289(4):1938-47
references.pmid: 24302718
references.citation: J Antibiot (Tokyo). 1994 Dec;47(12):1395-401
references.pmid: 7844034
references.citation: Clin Cancer Res. 2013 Nov 15;19(22):6296-304
references.pmid: 23983259
references.citation: J Antibiot (Tokyo). 1997 Jan;50(1):96-9
references.pmid: 9066774
references.citation: J Antibiot (Tokyo). 1992 Jun;45(6):914-21
references.pmid: 1500359
references.citation: Nat Chem Biol. 2007 Sep;3(9):570-5
references.pmid: 17643112
references.citation: Nat Chem Biol. 2007 Sep;3(9):576-83
references.pmid: 17643111
references.citation: ACS Chem Biol. 2011 Mar 18;6(3):229-33
references.pmid: 21138297
references.citation: Nat Prod Rep. 2000 Aug;17(4):367-83
references.pmid: 11014338
references.citation: Curr Opin Chem Biol. 2009 Apr;13(2):135-43
references.pmid: 19217343
references.citation: Proc Natl Acad Sci U S A. 2002 Oct 29;99(22):14002-7
references.pmid: 12381784
references.citation: J Antibiot (Tokyo). 2013 Sep;66(9):555-8
references.pmid: 23652603
references.citation: Biosci Biotechnol Biochem. 2004 Oct;68(10):2178-82
references.pmid: 15502365
references.citation: BMC Microbiol. 2008 Apr 02;8:54
references.pmid: 18384685
references.citation: J Am Chem Soc. 2011 Mar 2;133(8):2452-62
references.pmid: 21291275
references.citation: Nat Rev Drug Discov. 2012 Nov;11(11):847-59
references.pmid: 23123942
references.citation: Nat Prod Rep. 2010 Feb;27(2):255-78
references.pmid: 20111804
references.citation: Chem Biol. 2008 Sep 22;15(9):950-9
references.pmid: 18804032
references.citation: Gene. 1998 May 28;212(1):77-86
references.pmid: 9661666
references.citation: Chembiochem. 2010 Jul 26;11(11):1574-82
references.pmid: 20589823
references.citation: Gene. 1995 May 26;158(1):15-22
references.pmid: 7789804
references.citation: J Biol Chem. 1997 Sep 12;272(37):23031-6
references.pmid: 9287300
references.citation: Nat Struct Biol. 2000 Feb;7(2):127-33
references.pmid: 10655615