Comparative analysis of the substrate specificity of trans- versus cis-acyltransferases of assembly line polyketide synthases.
Comparative analysis of the substrate specificity of trans- versus cis-acyltransferases of assembly line polyketide synthases.
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DOI：
10.1021/bi5004316
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发表时间：
2014-06-17
期刊：
影响因子：
2.9
通讯作者：
Khosla C
中科院分区：
文献类型：
--
作者：
Dunn BJ;Watts KR;Robbins T;Cane DE;Khosla C
酰基转移酶（AT）结构域由于在聚酮生物合成过程中选择延伸单元中的关键作用而成为重要的工程靶标。装配线聚酮酶（PKS）的子集由离散的反式作用AT提供服务。理论上，这些反式AT可以补充失活的顺式AT，促进引入非同源的延伸单元。这种方法需要更好地了解天然存在的trans-AT的底物特异性和催化机制。我们动力学分析了反式AT的disorazole和kirromycin酶，并比较了他们的代表性顺式AT的6-脱氧腺苷酸B合酶（DEBS）。在转酰化过程中，地索拉唑AT对丙二酰辅酶A的支持超过甲基丙二酰辅酶A的40000倍，而红霉素AT对乙基丙二酰辅酶A的支持超过甲基丙二酰辅酶A的20倍。相反，地索拉唑AT对酰基载体蛋白（ACP）底物的特异性比其对应的Kirromycin更广。ACP的存在对羧基酰辅酶A底物的顺式-AT结构域的特异性（kcat/KM）影响不大，但对反式-AT的相应特异性参数有显着影响，表明这些酶不严格按典型的乒乓机制起作用。为了研究在完整PKSs的背景下分离AT的动力学分析的相关性，我们补充了一个体外AT无效DEBS装配线与反式AT。而地索拉唑AT有效地补充突变体PKS在亚化学计量的蛋白质比例，kirromycin AT是相当少的有效性。我们的研究结果表明，羧基酰辅酶A和ACP特异性的知识是至关重要的反式AT与突变体PKS的组合，以产生新的聚酮化合物的选择。
Due to their pivotal role in extender unit selection during polyketide biosynthesis, acyltransferase (AT) domains are important engineering targets. A subset of assembly line polyketide synthases (PKSs) are serviced by discrete, trans-acting ATs. Theoretically, these trans-ATs can complement an inactivated cis-AT, promoting introduction of a noncognate extender unit. This approach requires a better understanding of the substrate specificity and catalytic mechanism of naturally occurring trans-ATs. We kinetically analyzed trans-ATs from the disorazole and kirromycin synthases and compared them to a representative cis-AT from the 6-deoxyerythronolide B synthase (DEBS). During transacylation, the disorazole AT favored malonyl-CoA over methylmalonyl-CoA by >40000-fold, whereas the kirromycin AT favored ethylmalonyl-CoA over methylmalonyl-CoA by 20-fold. Conversely, the disorazole AT had broader specificity than its kirromycin counterpart for acyl carrier protein (ACP) substrates. The presence of the ACP had little effect on the specificity (kcat/KM) of the cis-AT domain for carboxyacyl-CoA substrates but had a marked influence on the corresponding specificity parameters for the trans-ATs, suggesting that these enzymes do not act strictly by a canonical ping-pong mechanism. To investigate the relevance of the kinetic analysis of isolated ATs in the context of intact PKSs, we complemented an in vitro AT-null DEBS assembly line with either trans-AT. Whereas the disorazole AT efficiently complemented the mutant PKS at substoichiometric protein ratios, the kirromycin AT was considerably less effective. Our findings suggest that knowledge of both carboxyacyl-CoA and ACP specificity is critical to the choice of a trans-AT in combination with a mutant PKS to generate novel polyketides.
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影响因子：
--
作者：
Jensen K;Niederkrüger H;Zimmermann K;Vagstad AL;Moldenhauer J;Brendel N;Frank S;Pöplau P;Kohlhaas C;Townsend CA;Oldiges M;Hertweck C;Piel J
通讯作者：
Piel J
影响因子：
--
作者：
Tang, Yinyan;Chen, Alice Y.;Khosla, Chaitan
通讯作者：
Khosla, Chaitan
影响因子：
15
作者：
Kumar, P;Koppisch, AT;Khoslaa, C
通讯作者：
Khoslaa, C
影响因子：
--
作者：
Bonnett SA;Rath CM;Shareef AR;Joels JR;Chemler JA;Håkansson K;Reynolds K;Sherman DH
通讯作者：
Sherman DH
影响因子：
2.9
作者：
Gokhale, RS;Lau, J;Khosla, C
通讯作者：
Khosla, C