Abstract
M01A82W, M11A82W and M01A82WS72I are three cytochrome P450 BM3 (CYP102A1) variants. They can catalyze the hydroxylation of testosterone (TES) and norethisterone at different positions, thereby making them promising biocatalysts for steroid hydroxylation. With the aim of obtaining more hydroxylated steroid precursors it is necessary to probe the steroidal substrate diversity of these BM3 variants. Here, three purified BM3 variants were first incubated with eight steroids, including testosterone (TES), methyltestosterone (MT), cholesterol, β-sitosterol, dehydroepiandrosterone (DHEA), diosgenin, pregnenolone and ergosterol. The results indicated that the two 3-keto-Δ4-steroids TES and MT can be hydroxylated at various positions by the three BM3 mutants, respectively. On the contrary, the three enzymes displayed no any activity toward the remaining six 3-hydroxy-Δ5-steroids. This result indicates that the BM3 mutants prefer 3-keto-Δ4-steroids as hydroxylation substrates. To further verify this notion, five other substrates, including two 3-hydroxy-Δ5-steroids and three 3-keto-Δ4-steroids, were carefully selected to incubate with the three BM3 variants. The results indicated the three 3-keto-Δ4-steroids can be metabolized to form hydroxysteroids by the three BM3 variants. On the other hand, the two 3-hydroxy-Δ5-steroids cannot be hydroxylated at any position by the BM3 mutants. These results further support the above conclusion, therefore demonstrating the 3-keto-Δ4–steroid substrate preference of BM3 mutants, and laying a foundation for microbial production of more hydroxylated steroid intermediates using BM3 variants.
Highlights
The hydroxylation of steroids is an important enzymatic reaction in steroid metabolism and the resultant hydroxylated steroids can be used as the key-intermediates for the biosynthesis of steroid drugs with diverse therapeutic purposes [1,2,3,4,5]
The hydroxylation ofto eight steroids by three purified variants was substrate specificity of three BM3 mutants M01A82W, M11A82W and M01A82WS72I, which were results indicated that only 3-keto-∆ -steroids can be metabolized to hydroxylated metabolites
M01A82WS72I, which had been which shown toadisplay foundation for a structure-activity relationship of bacterial regio- and stereoselective hydroxylation activitystudy towards a few P450 steroid probes [6,7]. We revealed that these P450 BM3 mutants preferred 3-keto-∆4 -steroids as substrates
Summary
The hydroxylation of steroids is an important enzymatic reaction in steroid metabolism and the resultant hydroxylated steroids can be used as the key-intermediates for the biosynthesis of steroid drugs with diverse therapeutic purposes [1,2,3,4,5]. It is necessary to probe the steroidal substrate diversity of were shown previously to hydroxylate TES and norethisterone at various positions, was explored. Variants was substrate specificity of three BM3 mutants M01A82W, M11A82W and M01A82WS72I, which were results indicated that only 3-keto-∆ -steroids can be metabolized to hydroxylated metabolites. Detailed confirming a substrate preference of BM3 mutants for 3-keto-∆4 -steroids These results broaden the analysis showed only the three 3-keto-Δ4-steroids could be metabolized into different hydroxylated known steroidal substrate promiscuity of BM3 variants, thereby expanding their synthetic utility as metabolites, confirming a substrate preference of BM3 mutants for 3-keto-Δ4-steroids. The known steroidal substrate promiscuity of BM3 variants, thereby expanding their synthetic utility as biological catalysts
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