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

Abstract

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.

Enhanced cell surface hydrophobicity favors the 9α‐hydroxylation of androstenedione by resting Rhodococcus sp. cells

“…In this context, possible explanation of our observation may come from the metabolic and physiological responses of Rhodococcus cells to the presence of hydrocarbons [20]. Results obtained in our study are also in accordance with the recently published evidences for the positive correlation between the ability of some bacteria for both, assimilation of n-alkanes and performing steroid transformation reactions [21,22].…”

Isolation and characterization of a multifunctional flavonoid glycosyltransferase from Ornithogalum caudatum with glycosidase activity

“…The glycosylated products were monitored by HPLC chromatography ( Supplementary Table S3). The exact structure of these glycosylated products was further elucidated by combinational usage of HPLC-UV, HPLC-MS and NMR, as described previously 23,37,40,42 .…”

The enzymatic biosynthesis of acylated steroidal glycosides and their cytotoxic activity

“…However, the glycosylation activity of OsSGT1 towards 17βhydroxyl group would be lost if additional hydroxyl group at 2β-(2β-OH-testosterone, 13), 15β-(15β-OH-testosterone, 14), 16β-(16β-OH-testosterone, 15), or 16α-position (16α-OH-testosterone, 16), even a methyl group at C17-position (methyltestosterone and its derivatives, 17-20) was attached to testosterone (8), generating not any glycosylated products. Moreover, OsSGT1 has no activity towards other compounds, including steroids without 3β-and 17β-hydroxyl groups (11-12 and 21-24), flavonoids (25-31), alkaloids (32)(33)(34)(35)(36)(37)(38), triterpenoids (39-42), phenolic acids (43-47) and coumarins (48-49) as shown in Supplementary Information Fig. S4.…”

“…1 and Supplementary Information Fig. S4 include diverse structures like steroids , flavonoids (25-31), alkaloids (32)(33)(34)(35)(36)(37)(38), triterpenoids (39-42), phenolic acids (43-47) and coumarins (48-49) are used as the sugar acceptors for OsSGT1-catalyzed glycosylation reactions ( Fig. 1 and Supplementary Information Fig.…”

“…The reaction mixture was incubated at 50 1C for 1 h. The formation of glycosylated products was Table 6 The cytotoxic activities of monoacylates against human tumor cell lines. unambiguously determined by a combination of HPLC-UV, HPLC-MS and NMR as described previously 35,36 . The determination conditions for HPLC-UV were summarized in Supplementary Information Table S3.…”