Expression, purification and characterization of two Clostridium acetobutylicum flavodoxins: potential electron transfer partners for CYP152A2.
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.
Malca
Sumire Honda
SH
Institute of Technical Biochemistry, Universität Stuttgart, 70569 Stuttgart, Germany.
Girhard
Marco
M
Schuster
Stefanie
S
Dürre
Peter
P
Urlacher
Vlada B
VB
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
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