title: 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 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.
authors.affiliation: Institute of Technical Biochemistry, Stuttgart University, Allmandring 31, 70569 Stuttgart, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Budde
authors.forename: S C
authors.initials: SC
authors.lastname: Maurer
authors.forename: R D
authors.initials: RD
authors.lastname: Schmid
authors.forename: V B
authors.initials: VB
authors.lastname: Urlacher
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism; D015870:Gene Expression; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D005227:Fatty Acids; D003577:Cytochrome P-450 Enzyme System