Steroid Hydroxylation by Mutant Cytochrome P450 BM3-LG23 Using Two Expression Chassis

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Cytochrome P450 BM3 is a highly active enzyme from *Priestia megaterium*.
• Mutant variants can exhibit altered catalytic activities.
• Heterologous expression allows for studying enzyme function in different hosts.

Purpose of the Study:

• To express and characterize the CYP102A1-LG23 mutant variant of cytochrome P450 BM3.
• To investigate its in vivo activity on C19 steroids.
• To evaluate *Mycolicibacterium smegmatis* as a host for heterologous gene expression.

Main Methods:

• Gene expression of *cyp102a1-LG23* in *Escherichia coli* and *Mycolicibacterium smegmatis*.
• In vivo enzymatic assays using androstenedione (AD), androstadienedione (ADD), testosterone (TS), and dehydroepiandrosterone (DHEA).
• Structural confirmation of steroid products using mass spectrometry and 1H NMR spectroscopy.

Main Results:

• The mutant enzyme catalyzed 7β-hydroxylation of AD and TS.
• Novel mono- and dihydroxylation of ADD and DHEA were observed, including 7β-, 6β-, 11α-hydroxylation of ADD and 7α/7β-hydroxylation and 7α,15α-dihydroxylation of DHEA.
• Successful gene expression and enzyme activity were demonstrated in *Mycolicibacterium smegmatis*.

Conclusions:

• The mutant cytochrome P450 BM3-LG23 exhibits unusual hydroxylation capabilities on C19 steroids.
• *Mycolicibacterium smegmatis* is a suitable host for heterologous expression of P450 enzymes.
• The findings open prospects for biotechnological production of hydroxysteroids.