title: Cloning, expression and characterization of CYP102D1, a self-sufficient P450 monooxygenase from Streptomyces avermitilis.
abstract: Among 33 cytochrome P450s (CYPs) of Streptomyces avermitilis, CYP102D1 encoded by the sav575 gene is naturally a unique and self-sufficient CYP. Since the native cyp102D1 gene could not be expressed well in Escherichia coli, its expression was attempted using codon-optimized synthetic DNA. The gene was successfully overexpressed and the recombinant CYP102D1 was functionally active, showing a Soret peak at 450 nm in the reduced CO difference spectrum. FMN/FAD isolated from the reductase domain showed the same fluorescence in thin layer chromatography separation as the authentic standards. Characterization of the substrate specificity of CYP102D1 based on NADPH oxidation rate revealed that it catalysed the oxidation of saturated and unsaturated fatty acids with very good regioselectivity, similar to other CYP102A families depending on NADPH supply. In particular, CYP102D1 catalysed the rapid oxidation of myristoleic acid with a k(cat)/K(m) value of 453.4 ± 181.5 μM(-1)·min(-1). Homology models of CYP102D1 based on other members of the CYP102A family allowed us to alter substrate specificity to aromatic compounds such as daidzein. Interestingly, replacement of F96V/M246I in the active site increased catalytic activity for daidzein with a k(cat)/K(m) value of 100.9 ± 10.4 μM(-1)·min(-1) (15-fold).
authors.affiliation: School of Chemical and Biological Engineering, Institute of Bioengineering, Seoul National University, Seoul, South Korea.
authors.forename: Kwon-Young
authors.initials: KY
authors.lastname: Choi
authors.forename: EunOk
authors.initials: E
authors.lastname: Jung
authors.forename: Da-Hye
authors.initials: DH
authors.lastname: Jung
authors.forename: Bishnu Prasad
authors.initials: BP
authors.lastname: Pandey
authors.forename: Hyungdon
authors.initials: H
authors.lastname: Yun
authors.forename: Hyung-yun
authors.initials: HY
authors.lastname: Park
authors.forename: Romas J
authors.initials: RJ
authors.lastname: Kazlauskas
authors.forename: Byung-Gee
authors.initials: BG
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism; D005229:Fatty Acids, Monounsaturated / Q000378:metabolism; D007529:Isoflavones / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D013302:Streptomyces / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D005227:Fatty Acids; D005229:Fatty Acids, Monounsaturated; D007529:Isoflavones; D009249:NADP; C004742:daidzein; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; C054211:9-tetradecenoic acid