Alcohol stereochemistry in polyketide backbones is controlled by the β-ketoreductase domains of modular polyketide synthases
Alcohol stereochemistry in polyketide backbones is controlled by the β-ketoreductase domains of modular polyketide synthases
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DOI：
10.1021/ja973913a
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发表时间：
1998-03-18
影响因子：
15
通讯作者：
Khosla, C
中科院分区：
文献类型：
--
作者：
Kao, CM;McPherson, M;Khosla, C
模块化聚酮酶（PKS; MW> 300 000）催化聚酮天然产物的生物合成。1，2这些酶部分通过其模块化活性位点组织来编程聚酮化合物生物合成中的一系列复杂反应步骤，这激发了人们对通过PKS基因的合理和组合工程来产生新分子的兴趣。2虽然功能获得、替换和失活实验已经揭示了PKS活性位点的显著广泛的底物特异性，3-15这些多功能酶的立体化学控制的结构基础仍然知之甚少。在单功能醇脱氢酶中，羰基还原的立体化学控制已被广泛研究。16，17这些酶（每个亚基的分子量约为35，000）催化醛和酮还原为相应的醇（以及逆反应），使用NADH或NADPH作为辅因子。在酮还原过程中，特定的氢化物离子（pro-R或pro-S）从辅因子转移到羰基底物的一面，生成D或L醇。晶体结构表明，这种立体化学控制是通过辅因子和底物的定向结合来实现的，因此氢化物离子总是从每个分子的同一面去除和添加。醇脱氢酶和模块化PKS之间的立体化学控制机制的相似性是不明显的先验，由于后者的酶的多结构域组织。在模块化PKS中，立体化学可能由β-酮还原酶（KR）结构域以类似于单功能脱氢酶的方式控制，因为KR以定向方式结合NADPH和聚酮化合物中间体，从而决定β-羟基立体化学。然而，典型的还原聚酮化合物含有D和L立体化学的羟基，因此需要通过相关KR结构域对单个β-（酮酰基）-ACP中间体进行替代的结合模式。例如，三酮化合物内酯1（图1），6-脱氧腺苷酸B合酶（DEBS）的双模块衍生物的产物，15，18含有L-（3S）和D-（5 R）构型的羟基。为了研究还原聚酮化合物中羟基立体化学的控制，我们在DEBS的三模块衍生物中进行了几次KR结构域置换。9我们的研究结果表明，β-羟基立体化学控制是单个KR结构域的固有特性，并且与β-（酮酰基）-ACP底物的取代模式无关。为了检查β-酮酰基硫酯还原的立体化学，我们构建了天蓝色链霉菌CH 999/pCK 139的三种衍生物（图1）。质粒pKOS 011 -56含有DEBS模块5的KR结构域代替天然KR 2。在质粒pKA 0392和pKA 0404中，KR 2分别被雷帕霉素合酶（RAPS）20模块2和4的KR结构域取代。RAPS KR 2片段还含有pupidine失活的脱氢酶（“null DH”）。22将每个质粒导入S. coelicolor CH 999，23，24，并分析所得菌株的聚酮化合物生产。25
Modular polyketide synthases (PKSs; MW> 300 000) catalyze the biosynthesis of polyketide natural products. 1, 2 These enzymes program the complex series of reaction steps in polyketide biosynthesis in part by their modular active site organization, which has stimulated much interest in generating new molecules through the rational and combinatorial engineering of PKS genes. 2 While gain-of-function, replacement, and inactivation experiments have revealed the remarkably broad substrate specificity of PKS active sites, 3-15 the structural basis for stereochemical control by these multifunctional enzymes remains poorly understood. In the monofunctional alcohol dehydrogenases, stereochemical control of carbonyl reduction has been extensively studied. 16, 17 These enzymes (MW≈ 35 000 per subunit) catalyze the reduction of aldehydes and ketones to the corresponding alcohols (as well as the reverse reaction), using NADH or NADPH as cofactors. During ketoreduction, a specific hydride ion (pro-R or pro-S) is transferred from the cofactor to one face of the carbonyl substrate, generating a D or L alcohol. Crystal structures have shown that this stereochemical control is achieved through oriented binding of both the cofactor and substrate, so that the hydride ion is always removed from and added to the same face of each molecule. Similarities in stereochemical control mechanisms between the alcohol dehydrogenases and modular PKSs are not obvious a priori, due to the multidomain organization of the latter enzymes. In modular PKSs, stereochemistry might be controlled by the β-ketoreductase (KR) domains in a manner analogous to the monofunctional dehydrogenases, in that the KR binds NADPH and a polyketide intermediate in an oriented fashion and thus dictates β-hydroxyl stereochemistry. However, a typical reduced polyketide contains hydroxyl groups of both D and L stereochemistry, thereby requiring alternative modes of binding for the individual β-(ketoacyl)-ACP intermediates by the relevant KR domain. For example, the triketide lactone 1 (Figure 1), the product of a bimodular derivative of the 6-deoxyerythronolide B synthase (DEBS), 15, 18 contains hydroxyl groups in both the L-(3S) and D-(5R) configurations. 10 To study the control of hydroxyl stereochemistry in reduced polyketides, we have carried out several KR domain replacements in a three-module derivative of the DEBS. 9 Our results demonstrate that β-hydroxyl stereochemical control is an intrinsic property of individual KR domains and is independent of the substitution pattern of β-(ketoacyl)-ACP substrates.To examine the stereochemistry of β-ketoacyl thioester reduction, we constructed three derivatives of Streptomyces coelicolor CH999/pCK139 (Figure 1). Plasmid pKOS011-56 contains the KR domain of DEBS module 5 in place of the native KR2. 19 In plasmids pKAO392 and pKAO404, KR2 is replaced by the KR domains of the rapamycin synthase (RAPS) 20 modules 2 and 4, respectively. 21 The RAPS KR2 segment also contains a putatively inactive dehydratase (“null DH”). 22 Each plasmid was introduced into S. coelicolor CH999, 23, 24 and the resulting strains analyzed for polyketide production. 25