tailieunhanh - Báo cáo khoa học: Kinetic and binding studies with purified recombinant proteins ferredoxin reductase, ferredoxin and cytochrome P450 comprising the morpholine mono-oxygenase from Mycobacteriumsp. strain HE5

The P450morsystem fromMycobacteriumsp. strain HE5, supposed to cata-lyse the hydroxylation of different N-heterocycles, is composed of three components: ferredoxin reductase (FdRmor), Fe3S4 ferredoxin (Fd mor) and cytochrome P450 (P450mor). In this study, we purified Fdmorand P450mor as recombinant proteins as well as FdRmor, which has been isolated previ-ously. Kinetic investigations of the redox couple FdRmor⁄Fdmorrevealed a 30-fold preference for the NADH-dependent reduction of nitroblue tetrazo-lium (NBT) and an absolute requirement for Fd morin this reaction, com-pared with the NADH-dependent reduction of cytochromec. . | iFEBS Journal Kinetic and binding studies with purified recombinant proteins ferredoxin reductase ferredoxin and cytochrome P450 comprising the morpholine mono-oxygenase from Mycobacterium sp. strain HE5 Bernhard Sielaff and Jan R. Andreesen Institut fur Mikrobiologie Martin-Luther-Universitat Halle Germany Keywords cytochrome P450 ferredoxin ferredoxin reductase morpholine mono-oxygenase Mycobacterium Correspondence J. R. Andreesen Institut fur Mikrobiologie Martin-Luther-Universitat Halle Halle Germany Fax 49 345 552 7010 Tel 49 345 552 6350 E-mail . Website mibio Received 17 November 2004 revised 13 December 2004 accepted 24 December 2004 doi The P450mor system from Mycobacterium sp. strain HE5 supposed to catalyse the hydroxylation of different N-heterocycles is composed of three components ferredoxin reductase FdRmor Fe3S4 ferredoxin Fdmor and cytochrome P450 P450mor . In this study we purified Fdmor and P450mor as recombinant proteins as well as FdRmor which has been isolated previously. Kinetic investigations of the redox couple FdRmor Fdmor revealed a 30-fold preference for the NADH-dependent reduction of nitroblue tetrazo-lium NBT and an absolute requirement for Fdmor in this reaction compared with the NADH-dependent reduction of cytochrome c. The quite low Km nM of FdRmor for Fdmor measured with NBT as the electron acceptor indicated high specificity. The addition of sequences providing His-tags to the N- or C-terminus of Fdmor did not significantly alter kinetic parameters but led to competitive background activities of these fusion proteins. Production of P450mor as an N-terminal His-tag fusion protein enabled the purification of this protein in its spectral active form which has previously not been possible for wild-type P450mor. The proposed substrates morpholine piperidine or pyrrolidine failed to produce substrate-binding spectra of P450mor under