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Kinetic and binding studies with purified recombinant proteins ferredoxin reductase, ferredoxin and cytochrome P450 comprising the morpholine mono‐oxygenase from Mycobacterium sp. strain HE5
Author(s) -
Sielaff Bernhard,
Andreesen Jan R.
Publication year - 2005
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2005.04550.x
Subject(s) - ferredoxin , morpholine , chemistry , reductase , stereochemistry , biochemistry , hydroxylation , fusion protein , recombinant dna , enzyme , organic chemistry , gene
The P450 mor system from Mycobacterium sp. strain HE5, supposed to catalyse the hydroxylation of different N‐heterocycles, is composed of three components: ferredoxin reductase (FdR mor ), Fe 3 S 4 ferredoxin (Fd mor ) and cytochrome P450 (P450 mor ). In this study, we purified Fd mor and P450 mor as recombinant proteins as well as FdR mor , which has been isolated previously. Kinetic investigations of the redox couple FdR mor /Fd mor revealed a 30‐fold preference for the NADH‐dependent reduction of nitroblue tetrazolium (NBT) and an absolute requirement for Fd mor in this reaction, compared with the NADH‐dependent reduction of cytochrome c . The quite low K m (5.3 ± 0.3 n m ) of FdR mor for Fd mor , measured with NBT as the electron acceptor, indicated high specificity. The addition of sequences providing His‐tags to the N‐ or C‐terminus of Fd mor did not significantly alter kinetic parameters, but led to competitive background activities of these fusion proteins. Production of P450 mor 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 P450 mor . The proposed substrates morpholine, piperidine or pyrrolidine failed to produce substrate‐binding spectra of P450 mor under any conditions. Pyridine, metyrapone and different azole compounds generated type II binding spectra and the K d values determined for these substances suggested that P450 mor might have a preference for more bulky and/or hydrophobic molecules. The purified recombinant proteins FdR mor , Fd mor and P450 mor were used to reconstitute the homologous P450‐containing mono‐oxygenase, which was shown to convert morpholine.