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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 j.andreesen@mikrobiologie. uni-halle.de Website www.biologie.uni-halle.de mibio Received 17 November 2004 revised 13 December 2004 accepted 24 December 2004 doi 10.1111 j.1742-4658.2005.04550.x 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 5.3 0.3 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
