Ma Suzanne M, Tang Yi
Department of Chemical and Biomolecular Engineering, University of California-Los Angeles, 420 Westwood Plaza, Los Angeles, CA 90095, USA.
FEBS J. 2007 Jun;274(11):2854-64. doi: 10.1111/j.1742-4658.2007.05818.x. Epub 2007 Apr 27.
The biosynthesis of lovastatin in Aspergillus terreus requires two megasynthases. The lovastatin nonaketide synthase, LovB, synthesizes the intermediate dihydromonacolin L using nine malonyl-coenzyme A molecules, and is a reducing, iterative type I polyketide synthase. The iterative type I polyketide synthase is mechanistically different from bacterial type I polyketide synthases and animal fatty acid synthases. We have cloned the minimal polyketide synthase domains of LovB as standalone proteins and assayed their activities and substrate specificities. The didomain proteins ketosynthase-malonyl-coenzyme A:acyl carrier protein acyltransferase (KS-MAT) and acyl carrier protein-condensation (ACP-CON) domain were expressed solubly in Escherichia coli. The monodomains MAT, ACP and CON were also obtained as soluble proteins. The MAT domain can be readily labeled by [1,2-(14)C]malonyl-coenzyme A and can transfer the acyl group to both the cognate LovB ACP and heterologous ACPs from bacterial type I and type II polyketide synthases. Using the LovB ACP-CON didomain as an acyl acceptor, LovB MAT transferred malonyl and acetyl groups with k(cat)/K(m) values of 0.62 min(-1).mum(-1) and 0.032 min(-1).mum(-1), respectively. The LovB MAT domain was able to substitute the Streptomyces coelicolor FabD in supporting product turnover in a bacterial type II minimal polyketide synthase assay. The activity of the KS domain was assayed independently using a KS-MAT (S656A) mutant in which the MAT domain was inactivated. The KS domain displayed no activity towards acetyl groups, but was able to recognize malonyl groups in the absence of cerulenin. The relevance of these finding to the priming mechanism of fungal polyketide synthase is discussed.
土曲霉中洛伐他汀的生物合成需要两种大型合成酶。洛伐他汀非aketide合酶LovB利用九个丙二酰辅酶A分子合成中间体二氢莫纳可林L，是一种还原型迭代I型聚酮合酶。迭代I型聚酮合酶在机制上不同于细菌I型聚酮合酶和动物脂肪酸合酶。我们已将LovB的最小聚酮合酶结构域克隆为独立蛋白，并测定了它们的活性和底物特异性。双结构域蛋白酮合成酶-丙二酰辅酶A：酰基载体蛋白酰基转移酶（KS-MAT）和酰基载体蛋白-缩合（ACP-CON）结构域在大肠杆菌中可溶表达。单结构域MAT、ACP和CON也作为可溶性蛋白获得。MAT结构域可被[1,2-(14)C]丙二酰辅酶A轻松标记，并能将酰基转移至同源的LovB ACP以及来自细菌I型和II型聚酮合酶的异源ACP。以LovB ACP-CON双结构域作为酰基受体，LovB MAT转移丙二酰基和乙酰基的k(cat)/K(m)值分别为0.62 min(-1).μm(-1)和0.032 min(-1).μm(-1)。在细菌II型最小聚酮合酶测定中，LovB MAT结构域能够替代天蓝色链霉菌FabD来支持产物周转。使用KS-MAT（S656A）突变体独立测定KS结构域的活性，其中MAT结构域已失活。KS结构域对乙酰基无活性，但在没有浅蓝菌素的情况下能够识别丙二酰基。讨论了这些发现与真菌聚酮合酶引发机制的相关性。
