Steroids hydroxylation catalyzed by the monooxygenase mutant 139-3 from Bacillus megaterium BM3

• Main Authors: Xing Liu (Author), Jian-qiang Kong (Author)
• Format: Book
• Published: Elsevier, 2017-07-01T00:00:00Z.
• Subjects: article
• Online Access: Connect to this object online.
• Notes: 2211-3835; 2211-3843; 10.1016/j.apsb.2017.04.006
• Keywords: Cytochrome P450; BM3; 139-3; Steroids hydroxylation; 1α-OH-androstenedione; Biocatalyst; Therapeutics. Pharmacology; RM1-950
• Source: Acta Pharmaceutica Sinica B, Vol 7, Iss 4, Pp 510-516 (2017)

The search of new substrates with pharmaceutical and industrial potential for biocatalysts including cytochrome P450 enzymes is always challenging. Cytochrome P450 BM3 mutant 139-3, a versatile biocatalyst, exhibited hydroxylation activities towards fatty acids and alkanes. However, there were limited reports about its hydroxylation activity towards steroids. Herein, an Escherichia coli-based whole-cell extract containing the recombinant 139-3 protein was used as the biocatalyst to screen 13 steroids. Results revealed that 139-3 was able to specifically hydroxylate androstenedione (1) at 1α-position, generating a hydroxylated steroid 1α-OH-androstenedione (1a). To investigate whether C-1α hydroxylation catalyzed by BM3 mutant 139-3 could be industrially used, an optimization of catalyzing conditions was performed. Accordingly, the BM3 mutant 139-3 enzyme was observed to display maximum activity at 37 °C, under pH 7.0 for 4 h, with 37% transformation rate. Moreover, four 139-3 variants were generated by random mutagenesis with the aim of improving its activity and expanding substrate scope. Surprisingly, these mutants, sharing a common mutated site R379S, lost their activities towards androstenedione (1). These data clearly indicated that arginine residue located at site 379 played key role in the hydroxylation activities of 139-3. Overall, these new findings broadened the substrate scope of 139-3 enzyme, thereby expanding its potential applications as a biocatalyst on steroids hydroxylation in pharmaceutical industry.