Directed evolution and structural characterization of a simvastatin synthase.
Directed evolution and structural characterization of a simvastatin synthase.
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辛伐他汀合酶的定向进化和结构表征。
DOI：
10.1016/j.chembiol.2009.09.017
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发表时间：
2009-10-30
影响因子：
--
通讯作者：
Tang Y
中科院分区：
文献类型：
--
作者：
Gao X;Xie X;Pashkov I;Sawaya MR;Laidman J;Zhang W;Cacho R;Yeates TO;Tang Y
来自天然产物生物合成途径的酶是创造定制生物催化剂以生产半合成药物化合物的有吸引力的候选者。LovD是一种酰基转移酶，可将无活性的莫那可林J酸（MJA）转化为降低胆固醇的洛伐他汀。LovD还可以使用MJA和人工合成的α-二甲基丁基硫酯合成畅销药物辛伐他汀，尽管作为生物催化剂的性能不理想。本研究采用定向进化的方法改进了LovD半合成辛伐他汀的性质。获得了催化效率、溶解度和热稳定性均有所提高的突变体，其中最佳突变体在大肠杆菌生物催化平台上的催化效率提高了约11倍。为了了解LovD酶学的结构基础，我们测定了7个x射线晶体结构，包括亲本LovD、改良突变体G5和与配体共结晶的G5。结构之间的比较表明，有益的突变使G5的结构稳定在一个更紧凑的构象中，有利于催化。
Enzymes from natural product biosynthetic pathways are attractive candidates for creating tailored biocatalysts to produce semisynthetic pharmaceutical compounds. LovD is an acyltransferase that converts the inactive monacolin J acid (MJA) into the cholesterol-lowering lovastatin. LovD can also synthesize the blockbuster drug simvastatin using MJA and a synthetic α-dimethylbutyryl thioester, albeit with suboptimal properties as a biocatalyst. Here we used directed evolution to improve the properties of LovD towards semisynthesis of simvastatin. Mutants with improved catalytic efficiency, solubility and thermal stability were obtained, with the best mutant displaying an ~11-fold increase in an Escherichia coli based biocatalytic platform. To understand the structural basis of LovD enzymology, seven X-ray crystal structures were determined, including the parent LovD, an improved mutant G5, and G5 co-crystallized with ligands. Comparisons between the structures reveal that beneficial mutations stabilize the structure of G5 in a more compact conformation that is favorable for catalysis.
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影响因子：
5.7
作者：
Heikinheimo, P;Goldman, A;Ollis, DL
通讯作者：
Ollis, DL
DOI：
10.1073/pnas.0504033102
发表时间：
2005-06-28
影响因子：
11.1
作者：
Hsu, CC;Hong, ZY;Wong, CH
通讯作者：
Wong, CH
影响因子：
2.2
作者：
Kumar, MS;Kumar, PM;Sadhukhan, AK
通讯作者：
Sadhukhan, AK
影响因子：
56.9
作者：
Kennedy, J;Auclair, K;Hutchinson, CR
通讯作者：
Hutchinson, CR
影响因子：
6.8
作者：
Nardini, M;Dijkstra, BW
通讯作者：
Dijkstra, BW