Computation-Aided Engineering of Cytochrome P450 for the Production of Pravastatin.
Computation-Aided Engineering of Cytochrome P450 for the Production of Pravastatin.
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DOI：
10.1021/acscatal.2c03974
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发表时间：
2022-12-16
期刊：
影响因子：
12.9
通讯作者：
Munro, Andrew W.
中科院分区：
文献类型：
--
作者：
Ashworth, Mark A.;Bombino, Elvira;de Jong, Rene M.;Wijma, Hein J.;Janssen, Dick B.;McLean, Kirsty J.;Munro, Andrew W.
关键词：
CYP 105 AS 1是来自东方拟无枝酸菌的细胞色素P450，其催化甲羟氨苄青霉素单氧化为6-表普伐他汀。对于降胆固醇药物普伐他汀的发酵生产，需要反转酶的立体选择性，这已经通过易错PCR诱变和筛选部分实现。在目前的研究中，我们报告进一步优化的立体选择性的计算辅助方法。使用Rosetta的CoupledMoves方案，设计突变体的虚拟文库以在前普伐他汀方向上结合制密菌素。通过检查发生的频率的有益的取代和合理的检查它们的相互作用，一小组的8个突变体预测显示出所需的选择性，这些变体进行了实验测试。最好的CYP 105 AS 1变体给出了>99%的立体选择性羟化制甲羟氨苄青霉素为普伐他汀，完全消除了不需要的6-表-普伐他汀非对映体。通过50 × 100 ps和5 × 22 ns的超短分子动力学模拟，对酶-底物复合物进行了研究，发现近攻击构象的出现频率与实验观察到的立体选择性一致。这些结果表明，计算方法和合理的检查相结合，可以提高CYP 105 AS 1的立体选择性超过所获得的定向进化。此外，这项工作为已知结构的酶的特异性工程设计提供了一个通用的计算机框架。
CYP105AS1 is a cytochrome P450 from Amycolatopsis orientalis that catalyzes monooxygenation of compactin to 6-epi-pravastatin. For fermentative production of the cholesterol-lowering drug pravastatin, the stereoselectivity of the enzyme needs to be inverted, which has been partially achieved by error-prone PCR mutagenesis and screening. In the current study, we report further optimization of the stereoselectivity by a computationally aided approach. Using the CoupledMoves protocol of Rosetta, a virtual library of mutants was designed to bind compactin in a pro-pravastatin orientation. By examining the frequency of occurrence of beneficial substitutions and rational inspection of their interactions, a small set of eight mutants was predicted to show the desired selectivity and these variants were tested experimentally. The best CYP105AS1 variant gave >99% stereoselective hydroxylation of compactin to pravastatin, with complete elimination of the unwanted 6-epi-pravastatin diastereomer. The enzyme–substrate complexes were also examined by ultrashort molecular dynamics simulations of 50 × 100 ps and 5 × 22 ns, which revealed that the frequency of occurrence of near-attack conformations agreed with the experimentally observed stereoselectivity. These results show that a combination of computational methods and rational inspection could improve CYP105AS1 stereoselectivity beyond what was obtained by directed evolution. Moreover, the work lays out a general in silico framework for specificity engineering of enzymes of known structure.
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FireProt：热稳定多点突变体的基于能量和进化的计算设计。
影响因子：
4.3
作者：
Bednar D;Beerens K;Sebestova E;Bendl J;Khare S;Chaloupkova R;Prokop Z;Brezovsky J;Baker D;Damborsky J
通讯作者：
Damborsky J
影响因子：
21.8
作者：
Dodani SC;Kiss G;Cahn JK;Su Y;Pande VS;Arnold FH
通讯作者：
Arnold FH
DOI：
10.1002/cbic.202000735
发表时间：
2021-03-16
期刊：
Chembiochem : a European journal of chemical biology
影响因子：
--
作者：
Bracco P;Wijma HJ;Nicolai B;Buitrago JAR;Klünemann T;Vila A;Schrepfer P;Blankenfeldt W;Janssen DB;Schallmey A
通讯作者：
Schallmey A
DOI：
10.1073/pnas.251555398
发表时间：
2001-12-04
影响因子：
11.1
作者：
Bolon, DN;Mayo, SL
通讯作者：
Mayo, SL
影响因子：
2.9
作者：
BERRY, EA;TRUMPOWER, BL
通讯作者：
TRUMPOWER, BL