Kim Beom Seok, Cropp T Ashton, Beck Brian J, Sherman David H, Reynolds Kevin A
Department of Medicinal Chemistry and Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond 23219, USA.
J Biol Chem. 2002 Dec 13;277(50):48028-34. doi: 10.1074/jbc.M207770200. Epub 2002 Oct 3.
The pikromycin biosynthetic gene cluster contains the pikAV gene encoding a type II thioesterase (TEII). TEII is not responsible for polyketide termination and cyclization, and its biosynthetic role has been unclear. During polyketide biosynthesis, extender units such as methylmalonyl acyl carrier protein (ACP) may prematurely decarboxylate to generate the corresponding acyl-ACP, which cannot be used as a substrate in the condensing reaction by the corresponding ketosynthase domain, rendering the polyketide synthase module inactive. It has been proposed that TEII may serve as an "editing" enzyme and reactivate these modules by removing acyl moieties attached to ACP domains. Using a purified recombinant TEII we have tested this hypothesis by using in vitro enzyme assays and a range of acyl-ACP, malonyl-ACP, and methylmalonyl-ACP substrates derived from either PikAIII or the loading didomain of DEBS1 (6-deoxyerythronolide B synthase; AT(L)-ACP(L)). The pikromycin TEII exhibited high K(m) values (>100 microm) with all substrates and no apparent ACP specificity, catalyzing cleavage of methylmalonyl-ACP from both AT(L)-ACP(L) (k(cat)/K(m) 3.3 +/- 1.1 m(-1) s(-1)) and PikAIII (k(cat)/K(m) 2.9 +/- 0.9 m(-1) s(-1)). The TEII exhibited some acyl-group specificity, catalyzing hydrolysis of propionyl (k(cat)/K(m) 15.8 +/- 1.8 m(-1) s(-1)) and butyryl (k(cat)/K(m) 17.5 +/- 2.1 m(-1) s(-1)) derivatives of AT(L)-ACP(L) faster than acetyl (k(cat)/K(m) 4.9 +/- 0.7 m(-1) s(-1)), malonyl (k(cat)/K(m) 3.9 +/- 0.5 m(-1) s(-1)), or methylmalonyl derivatives. PikAIV containing a TEI domain catalyzed cleavage of propionyl derivative of AT(L)-ACP(L) at a dramatically lower rate than TEII. These results provide the first unequivocal in vitro evidence that TEII can hydrolyze acyl-ACP thioesters and a model for the action of TEII in which the enzyme remains primarily dissociated from the polyketide synthase, preferentially removing aberrant acyl-ACP species with long half-lives. The lack of rigorous substrate specificity for TEII may explain the surprising observation that high level expression of the protein in Streptomyces venezuelae leads to significant (>50%) titer decreases.
苦霉素生物合成基因簇包含编码II型硫酯酶（TEII）的pikAV基因。TEII不负责聚酮化合物的终止和环化，其生物合成作用一直不清楚。在聚酮化合物生物合成过程中，诸如甲基丙二酰酰基载体蛋白（ACP）等延伸单元可能会过早脱羧，生成相应的酰基-ACP，而该酰基-ACP不能作为相应酮合成酶结构域缩合反应的底物，从而使聚酮合成酶模块失活。有人提出，TEII可能作为一种“编辑”酶，通过去除连接在ACP结构域上的酰基部分来重新激活这些模块。我们使用纯化的重组TEII，通过体外酶分析以及一系列源自PikAIII或DEBS1（6-脱氧红霉内酯B合成酶；AT(L)-ACP(L)）的装载双结构域的酰基-ACP、丙二酰-ACP和甲基丙二酰-ACP底物，对这一假设进行了测试。苦霉素TEII对所有底物均表现出高K(m)值（>100微摩尔），且没有明显的ACP特异性，能催化从AT(L)-ACP(L)（k(cat)/K(m) 3.3±1.1 m(-1) s(-1)）和PikAIII（k(cat)/K(m) 2.9±0.9 m(-1) s(-1)）中切割甲基丙二酰-ACP。TEII表现出一定的酰基特异性，催化AT(L)-ACP(L)的丙酰基（k(cat)/K(m) 15.8±1.8 m(-1) s(-1)）和丁酰基（k(cat)/K(m) 17.5±2.1 m(-1) s(-1)）衍生物的水解速度快于乙酰基（k(cat)/K(m) 4.9±0.7 m(-1) s(-1)）、丙二酰基（k(cat)/K(m) 3.9±0.5 m(-1) s(-1)）或甲基丙二酰基衍生物。含有TEI结构域的PikAIV催化AT(L)-ACP(L)的丙酰基衍生物的切割速度比TEII低得多。这些结果提供了首个明确的体外证据，表明TEII可以水解酰基-ACP硫酯，并提出了TEII的作用模型，即该酶主要与聚酮合成酶解离，优先去除半衰期长的异常酰基-ACP种类。TEII缺乏严格的底物特异性，这可能解释了在委内瑞拉链霉菌中该蛋白的高水平表达导致效价显著降低（>50%）这一令人惊讶的现象。
