title: Expression, purification and characterization of two Clostridium acetobutylicum flavodoxins: potential electron transfer partners for CYP152A2.
abstract: Two flavodoxin genes from Clostridium acetobutylicum, CacFld1 (CAC0587) and CacFld2 (CAC3417), were expressed in Escherichia coli and investigated for their ability to support activity of CYP152A2, a fatty acid hydroxylase from C. acetobutylicum. E. coli flavodoxin reductase (FdR) was used as a redox partner, since flavodoxin reductase CacFdR (CAC0196) from C. acetobutylicum could not be purified in a functional form. CacFld1 was shown to accept electrons from FdR and transfer them to CYP152A2. Since H₂O₂ was generated by uncoupling at different stages of the reconstituted electron transfer chain, catalase was used as H₂O₂ scavenger in order to exclude peroxygenation by CYP152A2. The reconstituted P450 system with CacFld1 and FdR oxidized myristic acid with a K(M) of 137 μM and a k(cat) of 36 min⁻¹. Furthermore, the hydroxylase activity of CYP152A2 towards myristic acid with CacFld1 was 17-fold higher than without CacFld1. Along with CYP152A2 and a physiological flavodoxin reductase, CacFld1 is therefore likely to be involved in oxygen detoxification in C. acetobutylicum. Flavodoxin CacFld2 did not accept electrons from NADPH-reduced FdR, though it cannot be excluded as a candidate redox partner for CYP152A2 in the presence of an appropriate physiological reductase.
authors.affiliation: Institute of Technical Biochemistry, Universität Stuttgart, 70569 Stuttgart, Germany.
authors.forename: Sumire Honda
authors.initials: SH
authors.lastname: Malca
authors.forename: Marco
authors.initials: M
authors.lastname: Girhard
authors.forename: Stefanie
authors.initials: S
authors.lastname: Schuster
authors.forename: Peter
authors.initials: P
authors.lastname: Dürre
authors.forename: Vlada B
authors.initials: VB
authors.lastname: Urlacher
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D046969:Clostridium acetobutylicum / Q000235:genetics / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004579:Electron Transport; D004926:Escherichia coli / Q000235:genetics; D005418:Flavodoxin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006418:Heme / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D006900:Hydroxylation; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D019814:Myristic Acid / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D010084:Oxidation-Reduction; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
chemical_list: D001426:Bacterial Proteins; D005418:Flavodoxin; D019814:Myristic Acid; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D006861:Hydrogen Peroxide; D007501:Iron; D009247:NADH, NADPH Oxidoreductases; C116041:flavodoxin NADPH oxidoreductase