Interrogating the molecular basis for multiple macrolactone ring formation by the pikromycin polyketide synthase.
The pikromycin polyketide synthase (PKS) is unique in its ability to generate both 12 and 14 membered ring macrolactones. As such, dissection of the molecular basis for controlling metabolic diversity in this system remains an important objective for understanding modular PKS function and expanding chemical diversity. Here, we describe a series of experiments designed to probe the importance of the protein-protein interaction that occurs between the final two monomodules, PikAIII (module 5) and PikAIV (module 6), for the production of the 12 membered ring macrolactone 10-deoxymethynolide. The results obtained from these in vitro studies demonstrate that PikAIII and PikAIV generate the 12 membered ring macrocycle most efficiently when engaged in their native protein-protein interaction. Accordingly, the data are consistent with PikAIV adopting an alternative conformation that enables the terminal thioesterase domain to directly off-load the PikAIII-bound hexaketide intermediate for macrocyclization.
Kittendorf
Jeffrey D
JD
University of Michigan Life Sciences Institute, Department of Medicinal Chemistry, 210 Washtenaw Avenue, Ann Arbor, MI 48109, USA.
Beck
Brian J
BJ
Buchholz
Tonia J
TJ
Seufert
Wolfgang
W
Sherman
David H
DH
D001483:Base Sequence; D020134:Catalytic Domain; D003500:Cyclization; D017931:DNA Primers; D004950:Esterases / Q000378:metabolism; D007783:Lactones / Q000737:chemistry*; D018942:Macrolides / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D048630:Polyketide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D007783:Lactones; D018942:Macrolides; D048630:Polyketide Synthases; D004950:Esterases; C003145:picromycin
Nature. 2000 Feb 3;403(6769):571-5
10676969
ACS Chem Biol. 2006 Sep 19;1(8):505-9
17168537
Gene. 2000 Mar 7;245(1):203-11
10713461
Biochemistry. 2001 Feb 27;40(8):2326-31
11327852
Chem Biol. 2001 May;8(5):475-85
11358694
J Am Chem Soc. 2001 Jul 11;123(27):6465-74
11439032
J Nat Prod. 2001 Nov;64(11):1447-50
11720530
J Biol Chem. 2002 Apr 12;277(15):13082-90
11809757
Chem Biol. 2002 May;9(5):575-83
12031664
Protein Expr Purif. 2002 Jun;25(1):1-7
12071692
Protein Expr Purif. 2002 Jun;25(1):8-15
12071693
Biochemistry. 2002 Oct 22;41(42):12590-7
12379101
J Am Chem Soc. 2003 Apr 23;125(16):4682-3
12696866
Chem Biol. 2003 Aug;10(8):723-31
12954331
J Am Chem Soc. 2003 Oct 15;125(41):12551-7
14531700
Chem Commun (Camb). 2003 Nov 21;(22):2780-2
14651102
Chem Biol. 2003 Dec;10(12):1225-32
14700630
Chem Biol. 2004 Jan;11(1):87-97
15112998
Nature. 1990 Nov 8;348(6297):176-8
2234082
Science. 1991 May 3;252(5006):675-9
2024119
Chem Biol. 1996 Nov;3(11):923-36
8939709
Proc Natl Acad Sci U S A. 1998 Oct 13;95(21):12111-6
9770448
Chem Biol. 1999 Feb;6(2):117-25
10021418
Chem Biol. 2005 Feb;12(2):249-56
15734652
J Am Chem Soc. 2005 Jun 15;127(23):8441-52
15941278
J Am Chem Soc. 2005 Jun 29;127(25):8910-1
15969542
Angew Chem Int Ed Engl. 2005 Nov 25;44(46):7557-60
16247819
Nat Rev Microbiol. 2005 Dec;3(12):925-36
16322741
Science. 2006 Mar 3;311(5765):1258-62
16513975
Chembiochem. 2006 Mar;7(3):485-94
16470766
Structure. 2006 Apr;14(4):737-48
16564177
J Nat Prod. 2006 May;69(5):847-9
16724858
Proc Natl Acad Sci U S A. 2006 Jul 25;103(30):11124-9
16844787
Curr Opin Biotechnol. 2006 Dec;17(6):597-605
17046237
Curr Opin Chem Biol. 2000 Feb;4(1):22-7
10679375