H 2 O 2 -Driven Hydroxylation of Steroids Catalyzed by Cytochrome P450 CYP105D18: Exploration of the Substrate Access Channel

Autor: Bashu Dev Pardhe, Kyoung Pyo Kwon, Jong Kook Park, Jun Hyuck Lee, Tae-Jin Oh
Rok vydání: 2023
Předmět:
Zdroj: Appl Environ Microbiol
ISSN: 1098-5336
0099-2240
Popis: CYP105D18 supports H(2)O(2) as an oxygen surrogate for catalysis well and shows high H(2)O(2) resistance capacity. We report the hydroxylation of different steroids using H(2)O(2) as a cosubstrate. Testosterone was regiospecifically hydroxylated to 2β-hydroxytestosterone. Based on the experimental data and molecular docking, we predicted that hydroxylation of methyl testosterone and nandrolone would occur at position 2 in the A-ring, while hydroxylation of androstenedione and adrenosterone was predicted to occur in the B-ring. Further, structure-guided rational design of the substrate access channel was performed with the mutagenesis of residues S63, R82, and F184. Among the mutants, S63A showed a marked decrease in product formation, while F184A showed a significant increase in product formation in testosterone, nandrolone, methyl testosterone, androstenedione, and adrenosterone. The catalytic efficiency (k(cat)/K(m)) toward testosterone was increased 1.36-fold in the F184A mutant over that in the wild-type enzyme. These findings might facilitate the potential use of CYP105D18 and further engineering to establish the basis of biotechnological applications. IMPORTANCE The structural modification of steroids is a challenging chemical reaction. Modifying the core ring and the side chain improves the biological activity of steroids. In particular, bacterial cytochrome P450s are used as promiscuous enzymes for the activation of nonreactive carbons of steroids. In the present work, we reported the H(2)O(2)-mediated hydroxylation of steroids by CYP105D18, which also overcomes the use of expensive cofactors. Further, exploring the substrate access channel and modifying the bulky amino acid F184A increase substrate conversion while modifying the substrate recognizing amino acid S63 markedly decreases product formation. Exploring the substrate access channel and the rational design of CYP105D18 can improve the substrate conversion, which facilitates the engineering of P450s for industrial application.
Databáze: OpenAIRE
Externí odkaz: