1887

Abstract

Cloning and sequencing of the operon region revealed the genes encoding the three components of a cytochrome P450 monooxygenase, which is required for the degradation of the N-heterocycle morpholine by sp. strain HE5. The cytochrome P450 (P450) and the FeS ferredoxin (Fd), encoded by and , respectively, have been characterized previously, whereas no evidence has hitherto been obtained for a specifically morpholine-induced reductase, which would be required to support the activity of the P450 system. Analysis of the operon has now revealed the gene , encoding the ferredoxin reductase of this morpholine monooxygenase. The genes , and were identical to the corresponding genes from sp. strain RP1. Almost identical genes in PCP-1, in addition to an inducible cytochrome P450, pointing to horizontal gene transfer, were now identified. No evidence for a circular or linear plasmid was found in sp. strain HE5. Analysis of the downstream sequences of revealed differences in this gene region between sp. strain HE5 and sp. strain RP1 on the one hand, and on the other hand, indicating insertions or deletions after recombination. Downstream of the genes, the gene ′, encoding a putative glutamine synthetase, was identified in all studied strains. The gene of sp. strain HE5 was heterologously expressed. The purified recombinant protein FdR was characterized as a monomeric 44 kDa protein, being a strictly NADH-dependent, FAD-containing reductase. The values of FdR for the substrate NADH (37·7±4·1 μM) and the artificial electron acceptors potassium ferricyanide (14·2±1·1 μM) and cytochrome (28·0±3·6 μM) were measured. FdR was shown to interact functionally with its natural redox partner, the FeS protein Fd, and with the FeS protein adrenodoxin, albeit with a much lower efficiency, but not with spinach ferredoxin. In contrast, adrenodoxin reductase, the natural redox partner of adrenodoxin, could not use Fd in activity assays. These results indicated that FdR can utilize different ferredoxins, but that Fd requires the specific NADH : ferredoxin oxidoreductase FdR from the P450 system for efficient catalytic function.

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2005-08-01
2019-10-15
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