Wu Jiaquan, Zaleski Toby J, Valenzano Chiara, Khosla Chaitan, Cane David E
Department of Chemistry, Box H, Brown University, Providence, Rhode Island 02912-9108, USA.
J Am Chem Soc. 2005 Dec 14;127(49):17393-404. doi: 10.1021/ja055672+.
Picromycin/methymycin synthase (PICS) is a modular polyketide synthase (PKS) that is responsible for the biosynthesis of both 10-deoxymethynolide (1) and narbonolide (2), the parent 12- and 14-membered aglycone precursors of the macrolide antibiotics methymycin and picromycin, respectively. PICS module 2 is a dehydratase (DH)-containing module that catalyzes the formation of the unsaturated triketide intermediate using malonyl-CoA as the chain extension substrate. Recombinant PICS module 2+TE, with the PICS thioesterase domain appended to the C-terminus to allow release of polyketide products, was expressed in Escherichia coli. Purified PICS module 2+TE converted malonyl-CoA and 4, the N-acetylcysteamine thioester of (2S,3R)-2-methyl-3-hydroxypentanoic acid, to a 1:2 mixture of the triketide acid (4S,5R)-4-methyl-5-hydroxy-2-heptenoic acid (5) and (3S,4S,5R)-3,5-dihydroxy-4-methyl-n-heptanoic acid-delta-lactone (10) with a combined kcat of 0.6 min(-1). The triketide lactone 10 is formed by thioesterase-catalyzed cyclization of the corresponding d-3-hydroxyacyl-SACP intermediate, a reaction which competes with dehydration catalyzed by the dehydratase domain. PICS module 2+TE showed a strong preference for the syn-diketide-SNAC 4, with a 20-fold greater kcat/K(m) than the anti-(2S,3S)-diketide-SNAC 14, and a 40-fold advantage over the syn-(2R,3S)-diketide-SNAC 13. PICS module 2(DH(0))+TE, with an inactivated DH domain, produced exclusively 10, while three PICS module 2(KR(0))+TE mutants, with inactivated KR domains, produced exclusively or predominantly the unreduced triketide ketolactone, (4S,5R)-3-oxo-4-methyl-5-hydroxy-n-heptanoic acid-delta-lactone (7). These studies establish for the first time the structure and stereochemistry of the intermediates of a polyketide chain elongation cycle catalyzed by a DH-containing module, while confirming the importance of key active site residues in both KR and DH domains.
苦霉素/甲基霉素合酶（PICS）是一种模块化聚酮合酶（PKS），负责10-脱氧甲基内酯（1）和纳波内酯（2）的生物合成，它们分别是大环内酯抗生素甲基霉素和苦霉素的母体12元和14元苷元前体。PICS模块2是一个含有脱水酶（DH）的模块，它以丙二酰辅酶A作为链延伸底物催化不饱和三酮中间体的形成。重组PICS模块2+TE在大肠杆菌中表达，该模块在C端附加了PICS硫酯酶结构域以允许聚酮产物的释放。纯化后的PICS模块2+TE将丙二酰辅酶A和（2S,3R）-2-甲基-3-羟基戊酸的N-乙酰半胱氨酸硫酯（4）转化为三酮酸（4S,5R）-4-甲基-5-羟基-2-庚烯酸（5）和（3S,4S,5R）-3,5-二羟基-4-甲基-n-庚酸-δ-内酯（10）的1:2混合物，总催化常数kcat为0.6 min⁻¹。三酮内酯10是由硫酯酶催化相应的d-3-羟基酰基-SACP中间体环化形成的，该反应与脱水酶结构域催化的脱水反应相互竞争。PICS模块2+TE对顺式二酮-SNAC 4有强烈偏好，其kcat/Km比反式-（2S,3S）-二酮-SNAC 14大20倍，比顺式-（2R,3S）-二酮-SNAC 13有40倍优势。带有失活DH结构域的PICS模块2（DH(0)）+TE仅产生10，而三个带有失活KR结构域的PICS模块2（KR(0)）+TE突变体仅产生或主要产生未还原的三酮酮内酯，即（4S,5R）-3-氧代-4-甲基-5-羟基-n-庚酸-δ-内酯（7）。这些研究首次确定了由含DH模块催化的聚酮链延伸循环中间体的结构和立体化学，同时证实了KR和DH结构域中关键活性位点残基的重要性。
