Cloning, expression and characterization of CYP102D1, a self-sufficient P450 monooxygenase from Streptomyces avermitilis

Title
Cloning, expression and characterization of CYP102D1, a self-sufficient P450 monooxygenase from Streptomyces avermitilis
Author(s)
최권영[최권영]정은옥[정은옥]정다혜[정다혜]비쉬누 판데이[비쉬누 판데이]윤형돈박현윤[박현윤]로마스 카즈라우스카스[로마스 카즈라우스카스]김병기[김병기]
Keywords
BACILLUS-MEGATERIUM; FLAVOCYTOCHROME P450BM3; CYTOCHROME-P450 MONOOXYGENASES; LIVER MICROSOMES; ENZYME; HYDROXYLATION; P450CYP102A2; DERIVATIVES; SELECTIVITY; OXIDATION
Issue Date
201205
Publisher
WILEY-BLACKWELL
Citation
FEBS JOURNAL, v.279, no.9, pp.1650 - 1662
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 kcat/Km value of 453.4 +/- 181.5 mu m-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 kcat/Km value of 100.9 +/- 10.4 mu m-1.min-1 (15-fold).
URI
http://hdl.handle.net/YU.REPOSITORY/28262http://dx.doi.org/10.1111/j.1742-4658.2011.08462.x
ISSN
1742-464X
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