title: A key cytochrome P450 hydroxylase in pradimicin biosynthesis.
abstract: Pradimicins A-C (1-3) are a group of antifungal and antiviral polyketides from Actinomadura hibisca. The sugar moieties in pradimicins are required for their biological activities. Consequently, the 5-OH that is used for glycosylation plays a critical role in pradimicin biosynthesis. A cytochrome P450 monooxygenase gene, pdmJ, was amplified from the genomic DNA of A. hibisca and expressed in Escherichia coli BL21(DE3). PdmJ introduced a hydroxyl group to G-2A (4), a key pradimicin biosynthetic intermediate, at C-5 to form JX134 (5). A d-Ala-containing pradimicin analog, JX137a (6) was tested as an alternative substrate, but no product was detected by LC-MS, indicating that PdmJ has strict substrate specificity. Kinetic studies revealed a typical substrate inhibition of PdmJ activity. The optimal substrate concentration for the highest velocity is 115μM under the test conditions. Moreover, the conversion rate of 4 to 5 was reduced by the presence of 6, likely due to competitive inhibition. Coexpression of PdmJ and a glucose 1-dehydrogenase in E. coli BL21(DE3) provides an efficient method to produce the important intermediate 5 from 4.
authors.affiliation: Department of Biological Engineering, Utah State University, 4105 Old Main Hill, Logan, UT 84322, United States.
authors.forename: Kandy L
authors.initials: KL
authors.lastname: Napan
authors.forename: Jia
authors.initials: J
authors.lastname: Zeng
authors.forename: Jon Y
authors.initials: JY
authors.lastname: Takemoto
authors.forename: Jixun
authors.initials: J
authors.lastname: Zhan
mesh_terms: D000595:Amino Acid Sequence; D018943:Anthracyclines / Q000737:chemistry*; D002626:Chemistry, Pharmaceutical / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid / Q000379:methods; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods; D008956:Models, Chemical; D008969:Molecular Sequence Data; D061065:Polyketides / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D000095643:Pradimicins and Benanomicins
chemical_list: D018943:Anthracyclines; D003577:Cytochrome P-450 Enzyme System; D061065:Polyketides; C058036:pradimicin A; D000095643:Pradimicins and Benanomicins
references.citation: J Ind Microbiol Biotechnol. 2011 May;38(5):633-41
references.pmid: 20721599
references.citation: Chem Biol. 1998 Nov;5(11):661-7
references.pmid: 9831532
references.citation: Proc Natl Acad Sci U S A. 1999 Mar 2;96(5):1846-51
references.pmid: 10051557
references.citation: Appl Microbiol Biotechnol. 2008 Feb;78(1):55-65
references.pmid: 18057930
references.citation: J Biol Chem. 1964 Jul;239:2370-8
references.pmid: 14209971
references.citation: J Microbiol Biotechnol. 2007 May;17(5):830-9
references.pmid: 18051306
references.citation: J Antibiot (Tokyo). 1990 Jul;43(7):755-62
references.pmid: 2387769
references.citation: J Antibiot (Tokyo). 1993 Mar;46(3):398-405
references.pmid: 8478258
references.citation: Proteins. 1994 Oct;20(2):197-201
references.pmid: 7846029
references.citation: Nucleic Acids Res. 2010 Jan;38(Database issue):D161-6
references.pmid: 19858104
references.citation: Gene. 2010 Aug 1;461(1-2):5-14
references.pmid: 20399259
references.citation: Glycobiology. 1998 Apr;8(4):407-14
references.pmid: 9499388
references.citation: J Antibiot (Tokyo). 1990 Jul;43(7):771-7
references.pmid: 2387770
references.citation: Drug Metab Dispos. 2001 Apr;29(4 Pt 1):368-74
references.pmid: 11259318
references.citation: J Antibiot (Tokyo). 1993 Mar;46(3):406-11
references.pmid: 8267791
references.citation: Annu Rev Pharmacol Toxicol. 2005;45:291-310
references.pmid: 15832445
references.citation: Eur J Clin Microbiol Infect Dis. 1997 Jan;16(1):93-7
references.pmid: 9063679
references.citation: Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5597-601
references.pmid: 1829523
references.citation: J Virol. 2007 Jan;81(1):362-73
references.pmid: 17050611
references.citation: Chembiochem. 2008 Jul 21;9(11):1710-5
references.pmid: 18604835
references.citation: Appl Microbiol Biotechnol. 2008 Jun;79(4):555-62
references.pmid: 18437375
references.citation: J Antibiot (Tokyo). 1993 Mar;46(3):387-97
references.pmid: 8478257
references.citation: J Antibiot (Tokyo). 1993 Mar;46(3):507-10
references.pmid: 8478269
references.citation: Chembiochem. 2009 Jun 15;10(9):1447-52
references.pmid: 19431159