Cloning, expression and characterisation of CYP102A2, a self-sufficient P450 monooxygenase from Bacillus subtilis

Applied Microbiology and Biotechnology
Michael BuddeVlada B Urlacher

Abstract

The gene encoding CYP102A2, a novel P450 monooxygenase from Bacillus subtilis, was cloned and expressed in Escherichia coli. The recombinant enzyme formed was purified by immobilised metal chelate affinity chromatography (IMAC) and characterised. CYP102A2 is a 119-kDa self-sufficient monooxygenase, consisting of an FMN/FAD-containing reductase domain and a heme domain. The deduced amino acid sequence of CYP102A2 exhibits a high level of identity with the amino acid sequences of CYP102A1 from B. megaterium (59%) and CYP102A3 from B. subtilis (60%). In reduced, CO-bound form, the enzyme shows a typical Soret band at 449 nm. It catalyses the oxidation of even- and odd-chain saturated and unsaturated fatty acids. In all reactions investigated, the products were the respective omega-3, omega-2 and omega-1 hydroxylated fatty acids. Activity was highest towards oleic acid (K(M)=17.36+/-1.4 microM, k(cat)=2,244+/-72 min(-1)) and linoleic acid (K(M)=12.25+/-1.8 microM, k(cat)=1,950+/-84 min(-1)). Comparison of a CYP102A2 homology model with the CYP102A1 crystal structure revealed significant differences in the substrate access channels, which might explain the differences in the catalytic properties of these two enzymes.

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Citations

May 17, 2008·Applied Microbiology and Biotechnology·Matthias DietrichVlada B Urlacher
Feb 24, 2010·Applied Microbiology and Biotechnology·Toshiki Furuya, Kuniki Kino
Feb 27, 2010·Applied Microbiology and Biotechnology·Marco GirhardVlada B Urlacher
Mar 16, 2013·Applied Microbiology and Biotechnology·Yujin Cao, Xiao Zhang
Jun 16, 2012·Microbial Cell Factories·Kwon-Young ChoiByung-Gee Kim
Aug 15, 2014·Applied Microbiology and Biotechnology·Jin H BaeByung-Gee Kim
Oct 30, 2007·Progress in Lipid Research·B L HilkerD Hildebrand
Apr 24, 2007·Journal of Applied Microbiology·B S KimJ S Ahn
Sep 19, 2006·Biochimica Et Biophysica Acta·Frank HannemannRita Bernhardt
Jul 19, 2013·Biotechnology Advances·Kyoung-Rok Kim, Deok-Kun Oh
Jun 16, 2016·Progress in Lipid Research·Katja ZornUwe T Bornscheuer
Jan 20, 2018·Annual Review of Food Science and Technology·Uwe T Bornscheuer
Mar 7, 2018·The Protein Journal·Swi See AngMariam-Aisha Fatima
Mar 28, 2006·Chembiochem : a European Journal of Chemical Biology·Michael BuddeVlada B Urlacher
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