Regio- and stereo-selective 1ß-hydroxylation of lithocholic acid by cytochrome P450 BM3 mutants.
Biotechnol Bioeng
; 120(8): 2230-2241, 2023 08.
Article
em En
| MEDLINE
| ID: mdl-37424513
ABSTRACT
Regio- and stereo-selective hydroxylation of bile acids is a valuable reaction but often lacks suitable catalysts. In the research, semi-rational design in protein engineering techniques had been applied on cytochrome P450 monooxygenase CYP102A1 (P450 BM3) from Bacillus megaterium, and a mutation library had been set up for the 1ß-hydroxylation of lithocholic acid (LCA) to produce 1ß-OH-LCA. After four rounds of mutagenesis, a key residue at W72 was identified to regulate the regio- and stereo-selectivity at C1 of LCA. A quadruple variant (G87A/W72T/A74L/L181M) was identified to reach 99.4% selectivity of 1ß-hydroxylation and substrate conversion of 68.1% resulting in a 21.5-fold higher level of 1ß-OH-LCA production than the template LG-23. Molecular docking indicated that introducing hydrogen bonds at W72 was responsible for enhancing selectivity and catalytic activity, which gave some insights into the structure-based understanding of Csp3 -H activation by the developed P450 BM3 mutants.
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MEDLINE
Assunto principal:
Bacillus megaterium
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Ácido Litocólico
Idioma:
En
Ano de publicação:
2023
Tipo de documento:
Article