Analysis of the nearly identical morpholine monooxygenase-encoding mor genes from different Mycobacterium strains and characterization of the specific NADH : ferredoxin oxidoreductase of this cytochrome P450 system

Abstract

Cloning and sequencing of the morABC 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 Mycobacterium sp. strain HE5. The cytochrome P450 (P450 mor ) and the Fe 3 S 4 ferredoxin (Fd mor ), encoded by morA and morB, 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 mor system. Analysis of the mor operon has now revealed the gene morC, encoding the ferredoxin reductase of this morpholine monooxygenase. The genes morA, morB and morC were identical to the corresponding genes from Mycobacterium sp. strain RP1. Almost identical mor genes in Mycobacterium chlorophenolicum 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 Mycobacterium sp. strain HE5. Analysis of the downstream sequences of morC revealed differences in this gene region between Mycobacterium sp. strain HE5 and Mycobacterium sp. strain RP1 on the one hand, and M. chlorophenolicum on the other hand, indicating insertions or deletions after recombination. Downstream of the mor genes, the gene orf19, encoding a putative glutamine synthetase, was identified in all studied strains. The gene morC of Mycobacterium sp. strain HE5 was heterologously expressed. The purified recombinant protein FdR mor was characterized as a monomeric 44 kDa protein, being a strictly NADH-dependent, FAD-containing reductase. The K m values of FdR mor for the substrate NADH (37?7±4?1 mM) and the artificial electron acceptors potassium ferricyanide (14?2±1?1 mM) and cytochrome c (28?0±3?6 mM) were measured. FdR mor was shown to interact functionally with its natural redox partner, the Fe 3 S 4 protein Fd mor , and with the Fe 2 S 2 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 mor in activity assays. These results indicated that FdR mor can utilize different ferredoxins, but that Fd mor requires the specific NADH : ferredoxin oxidoreductase FdR mor from the P450 mor system for efficient catalytic function. INTRODUCTIONThe degradation of the secondary cyclic amines morpholine, piperidine and pyrrolidine has been reported for different mycobacteria (Cech et al., 1988;Knapp & Brown, 1988;Poupin et al., 1998Poupin et al., , 1999a. The detection of intermediates during morpholine degradation in Mycobacterium aurum MO1 and in an environmental Mycobacterium strain strongly indicates that the initial ring cleavage occurs at the C-N bond (Combourieu et al., 1998b(Combourieu et al., , 2000 Abbreviations: AdR, adrenodoxin reductase; Adx, adrenodoxin; CD, circular dichroism; Fd, ferredoxin; FdI, spinach ferredoxin I; FdR, ferredoxin reductase; NBT, nitro blue tetrazolium...

Functional Characterization of pGKT2, a 182-Kilobase Plasmid Containing the xplAB Genes, Which Are Involved in the Degradation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine by Gordonia sp. Strain KTR9

“…This is in contrast to the case in KTR9, where xplB and xplA occur downstream of glnA. The nucleotide sequence repeats present in the mor/pip loci (29,39,50) are absent in the xpl cluster.…”

“…PCR analysis of KTR9 DNA genomic template with primers that flank the gene fusion point confirmed that there is no gap between these genes (data not shown). Interestingly, the glutamine synthase portion of the glnA-xplB fusion and XplR share up to 70% and 49% sequence identity, respectively, with the glutamine synthases and GntR-type regulators encoded by genes associated with the mycobacterial cyp151 genes (29,39,50). The organization of the xpl gene cluster suggests that it may constitute an operon that is regulated by XplR.…”

“…Approximately 1.5 kb upstream of this cluster is xplR, encoding a GntR-type transcriptional regulator. Cyp151C shares up to 71% amino acid sequence identity with the mycobacterial pipA/morA-encoded cytochromes P450 that are involved in the utilization of the secondary amines piperidine, pyrrolidine, and morpholine (29,39,50). The glnA-xplB fusion is predicted to encode the first 85% of the residues of a glutamine synthetase fused to the entire XplB reductase.…”

“…More particularly, xplR, cyp151C, and the truncated glnA domain of pGKT2 are remarkably similar in sequence and order to their homologs in the mor and pip gene clusters responsible for the degradation of morpholine, piperidine, and related compounds in mycobacteria and rhodococci (29,39,50). Indeed, a recent comparison of the mor/pip loci in six strains suggests that this locus is subject to a relatively high frequency of recombination events.…”

Quantum chemical studies for oxidation of morpholine by Cytochrome P450

“…Later, Poupin et al [8] presented evidence for involvement of Cytochrome P450 (CYP) in the initial oxidation of morpholine utilizing an environmental Mycobacterium strain. For the first time proteins involved in morpholine degradation were isolated and characterized by Sielaff et al [15,16] who purified Cytochrome P450 mor and its putative redox partner, a Fe 3 S 4 ferredoxin (Fd mor ).…”