title: The determinants of activity and specificity in actinorhodin type II polyketide ketoreductase.
abstract: In the actinorhodin type II polyketide synthase, the first polyketide modification is a regiospecific C9-carbonyl reduction, catalyzed by the ketoreductase (actKR). Our previous studies identified the actKR 94-PGG-96 motif as a determinant of stereospecificity. The molecular basis for reduction regiospecificity is, however, not well understood. In this study, we examined the activities of 20 actKR mutants through a combination of kinetic studies, PKS reconstitution, and structural analyses. Residues have been identified that are necessary for substrate interaction, and these observations have suggested a structural model for this reaction. Polyketides dock at the KR surface and are steered into the enzyme pocket where C7-C12 cyclization is mediated by the KR before C9-ketoreduction can occur. These molecular features can potentially serve as engineering targets for the biosynthesis of novel, reduced polyketides.
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA 92697, USA.
authors.forename: Pouya
authors.initials: P
authors.lastname: Javidpour
authors.forename: Joel
authors.initials: J
authors.lastname: Bruegger
authors.forename: Supawadee
authors.initials: S
authors.lastname: Srithahan
authors.forename: Tyler P
authors.initials: TP
authors.lastname: Korman
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Crump
authors.forename: John
authors.initials: J
authors.lastname: Crosby
authors.forename: Michael D
authors.initials: MD
authors.lastname: Burkart
authors.forename: Shiou-Chuan
authors.initials: SC
authors.lastname: Tsai
mesh_terms: D000213:Acyl Carrier Protein / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000880:Anthraquinones / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D003500:Cyclization; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D061065:Polyketides / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D044024:Tetralones / Q000378:metabolism; D000095622:Benzoisochromanequinones
chemical_list: D000213:Acyl Carrier Protein; D000429:Alcohol Oxidoreductases; D000880:Anthraquinones; D001426:Bacterial Proteins; D009249:NADP; D061065:Polyketides; D044024:Tetralones; C019106:tetralol; C087192:polyketide synthase ketoreductase; C013390:actinorhodin; D000095622:Benzoisochromanequinones
references.citation: Nature. 1984 May 31-Jun 6;309(5967):462-4
references.pmid: 6328317
references.citation: Chem Biol. 1999 Apr;6(4):189-95
references.pmid: 10099131
references.citation: Nat Prod Rep. 2010 Feb;27(2):238-54
references.pmid: 20111803
references.citation: Science. 1998 Oct 2;282(5386):63-8
references.pmid: 9756477
references.citation: Chem Biol Interact. 2003 Feb 1;143-144:247-53
references.pmid: 12604210
references.citation: Chem Biol. 2005 Oct;12(10):1145-53
references.pmid: 16242657
references.citation: J Am Chem Soc. 2008 Sep 3;130(35):11598-9
references.pmid: 18693734
references.citation: J Am Chem Soc. 2003 Nov 26;125(47):14307-12
references.pmid: 14624579
references.citation: Biochemistry. 2003 Apr 29;42(16):4648-57
references.pmid: 12705828
references.citation: Org Lett. 2007 Jan 18;9(2):335-8
references.pmid: 17217298
references.citation: Biochemistry. 2006 Nov 28;45(47):14085-93
references.pmid: 17115703
references.citation: Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11542-6
references.pmid: 7972098
references.citation: Biochemistry. 2002 Feb 26;41(8):2719-26
references.pmid: 11851419
references.citation: Curr Opin Chem Biol. 2003 Apr;7(2):285-95
references.pmid: 12714063
references.citation: J Bacteriol. 1993 Apr;175(8):2197-204
references.pmid: 8468280
references.citation: Biochemistry. 2004 Nov 23;43(46):14529-38
references.pmid: 15544323
references.citation: Science. 1993 Dec 3;262(5139):1546-50
references.pmid: 8248802
references.citation: Nat Struct Mol Biol. 2004 Sep;11(9):888-93
references.pmid: 15286722
references.citation: Chem Biol. 2007 Aug;14(8):898-908
references.pmid: 17719489
references.citation: Chem Biol. 1998 Dec;5(12):699-711
references.pmid: 9862793
references.citation: Chem Rev. 1997 Nov 10;97(7):2465-2498
references.pmid: 11851466
references.citation: Chem Biol. 2001 Apr;8(4):329-40
references.pmid: 11325589
references.citation: Proteins. 2003 Sep 1;52(4):609-23
references.pmid: 12910460
references.citation: Org Lett. 2009 Jan 15;11(2):305-8
references.pmid: 19128188
references.citation: Structure. 2006 Apr;14(4):737-48
references.pmid: 16564177
references.citation: Chem Biol. 1999 Sep;6(9):607-15
references.pmid: 10467128
references.citation: Chem Biol. 2006 Mar;13(3):277-85
references.pmid: 16638533
references.citation: Bioorg Med Chem. 1998 Aug;6(8):1171-7
references.pmid: 9784859
references.citation: Nat Rev Microbiol. 2010 Dec;8(12):879-89
references.pmid: 21079635
references.citation: Chem Biol. 2006 Mar;13(3):287-96
references.pmid: 16638534
references.citation: Eur J Biochem. 1971 Sep 24;22(2):203-12
references.pmid: 4398961
references.citation: J Am Chem Soc. 2003 Oct 22;125(42):12708-9
references.pmid: 14558809
references.citation: Curr Top Med Chem. 2009;9(17):1958-610
references.pmid: 19903160
references.citation: Org Lett. 2007 Nov 22;9(24):4951-4
references.pmid: 17973397
references.citation: Acc Chem Res. 2009 May 19;42(5):631-9
references.pmid: 19292437
references.citation: Biochemistry. 2008 Feb 19;47(7):1837-47
references.pmid: 18205400
references.citation: Biochemistry. 2011 May 31;50(21):4638-49
references.pmid: 21506596