Probing Steroidal Substrate Specificity of Cytochrome P450 BM3 Variants.
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.
State Key Laboratory of Bioactive Substance and Function of Natural Medicines &amp; Ministry of Health Key Laboratory of Biosynthesis of Natural Products, Institute of Materia Medica, Chinese Academy of Medical Sciences &amp; Peking Union Medical College, Beijing 100050, China. seraphlx@imm.ac.cn.
Xing
X
Liu
State Key Laboratory of Bioactive Substance and Function of Natural Medicines &amp; Ministry of Health Key Laboratory of Biosynthesis of Natural Products, Institute of Materia Medica, Chinese Academy of Medical Sciences &amp; Peking Union Medical College, Beijing 100050, China. wangzhibiao@live.cn.
Zhi-Biao
ZB
Wang
State Key Laboratory of Bioactive Substance and Function of Natural Medicines &amp; Ministry of Health Key Laboratory of Biosynthesis of Natural Products, Institute of Materia Medica, Chinese Academy of Medical Sciences &amp; Peking Union Medical College, Beijing 100050, China. wangyanan@imm.ac.cn.
Ya-Nan
YN
Wang
State Key Laboratory of Bioactive Substance and Function of Natural Medicines &amp; Ministry of Health Key Laboratory of Biosynthesis of Natural Products, Institute of Materia Medica, Chinese Academy of Medical Sciences &amp; Peking Union Medical College, Beijing 100050, China. jianqiangk@imm.ac.cn.
Jian-Qiang
JQ
Kong
D001419:Bacteria / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000072461:Cytochrome P450 Family 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004798:Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006900:Hydroxylation; D009154:Mutation; D009640:Norethindrone / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D013256:Steroids / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013739:Testosterone / Q000737:chemistry / Q000378:metabolism
D004798:Enzymes; D013256:Steroids; D013739:Testosterone; D000072461:Cytochrome P450 Family 1; D009640:Norethindrone
3-hydroxy-Δ5-steroids; 3-keto-Δ4-steroids; BM3 variants; hydroxylation; substrate specificity