Computation-Aided Engineering of Cytochrome P450 for the Production of Pravastatin

Abstract

CYP105AS1 is a cytochrome P450 from Amycolatopsis orientalis that catalyzes monooxygenation of compactin to 6-epipravastatin. 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 propravastatin 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.

Multidisciplinary approaches for enzyme biocatalysis in pharmaceuticals: protein engineering, computational biology, and nanoarchitectonics

“…These advances are not only driven by biology, but are heavily reliant on computational biology. [103][104][105][106][107][108][109] The marriage of computational modeling and biological understanding empowers researchers to predict, manipulate, and optimize enzyme behavior at a molecular level. Furthermore, the integration of nanoarchitectonics into enzyme biocatalysis brings novel strategies for enzyme immobilization, encapsulation, and deployment within custom-designed nanomaterials.…”

“…Further exemplification of enzyme engineering can be found in Table 3, where additional instances are provided. 52,103,[165][166][167][168][169][170][171][172][173][174][175][176][177][178][179][180] As the pharmaceutical industry seeks sustainable, efficient, and precise solutions, computational biology emerges as a transformative tool, bridging the gap between theory and experiment and propelling innovation in drug development and production.…”

Rational design of enzyme activity and enantioselectivity

“…It turns out that 28 of 33 active variants showed the enantioselectivity that they were designed for ( Wijma et al, 2015 ). Recently, a similar approach was applied to improve stereoselectivity of CYP105AS1, a cytochrome P450 from Amycolatopsis orientalis ( Ashworth et al, 2022 ) . Previous directed evolution has generated mutants of CYP105AS1 that can catalyze the hydroxylation of substrate compactin to yield pravastatin, an LDL cholesterol-lowering drug, but with modest selectivity of an ee value lower than 90%.…”

“…Finally, based on Rosetta calculation, docking results and the visual inspection, mutations at positions F76, P80, T95, V180, and T286 were selected for introduction at the template. The best double mutant T95F/V180M gave perfect production of the desired epimer with selectivity >99% ( Ashworth et al, 2022 ). Computational design based on calculation of binding energy was also applied to design enzyme variants with improved diastereoselectivity.…”

Regio‐ and stereoselective steroid hydroxylation by CYP109A2 from Bacillus megaterium explored by X‐ray crystallography and computational modeling

“…For each of the 13 poses, five independently seeded MD simulations of 20 ns were run giving a total of 100 ns of simulation time per pose. The last 10 ns of each trajectory was analyzed for the frequency of occurrence of strict NACs, following published procedures [35,39]. Because NAC frequencies were analyzed on the fly, every 100 fs, the reported fractions are the average of 500 000 observations (= 5 9 10 000 000/100).…”