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

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 chelat 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 Bacillus megaterium (59%) and CYP102A3 from Bacillus subtilis (60%). In reduced, CO-bound form, the enzyme shows a typical Soret band at 450 nm. It catalyses the oxidation of even- and odd-chain saturated and unsaturated fatty acids. In all reactions investigated, the products were the respective ù-3, ù-2 and ù-1 hydroxylated fatty acids. Activity was highest towards oleic and linoleic acid (KM=17.4 ± 1.4 ìM, kcat= 2244 ± 72 min-1), linoleic acid (KM=12.25 ± 1.8 ìM, kcat= 1950 ± 84 min-1). Comparison of CYP102A2 homology model to CYP102A1 crystal structure revealed significant differences in the substrate access channels, which might explain the differences in catalytic properties of these two enzymes.