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

Bernhard Sielaff and Jan R. Andreesen

Institut für Mikrobiologie, Martin-Luther-Universität Halle, Kurt-Mothes-Str. 3, 06120 Halle, Germany

Correspondence
Jan R. Andreesen
j.andreesen{at}mikrobiologie.uni-halle.de


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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 (P450mor) and the Fe3S4 ferredoxin (Fdmor), 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 P450mor 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 orf1', 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 FdRmor was characterized as a monomeric 44 kDa protein, being a strictly NADH-dependent, FAD-containing reductase. The Km values of FdRmor for the substrate NADH (37·7±4·1 µM) and the artificial electron acceptors potassium ferricyanide (14·2±1·1 µM) and cytochrome c (28·0±3·6 µM) were measured. FdRmor was shown to interact functionally with its natural redox partner, the Fe3S4 protein Fdmor, and with the Fe2S2 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 Fdmor in activity assays. These results indicated that FdRmor can utilize different ferredoxins, but that Fdmor requires the specific NADH : ferredoxin oxidoreductase FdRmor from the P450mor system for efficient catalytic function.


Abbreviations: AdR, adrenodoxin reductase; Adx, adrenodoxin; CD, circular dichroism; Fd, ferredoxin; FdI, spinach ferredoxin I; FdR, ferredoxin reductase; NBT, nitro blue tetrazolium; P450, cytochrome P450 monooxygenase

The GenBank/EMBL/DDBJ accession numbers for the nucleotide sequences reported in this paper from Mycobacterium sp. strain HE5 and Mycobacterium chlorophenolicum PCP-1 are AY816211 and AY960119, respectively.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The 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., 1998, 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, 2000; Poupin et al., 1998). Studies have suggested that a cytochrome P450 catalyses the hydroxylation of the carbon atom of this bond (Combourieu et al., 1998a; Poupin et al., 1998, 1999b).

An environmental bacterium has been isolated in our laboratory and identified as a new Mycobacterium sp. strain HE5, which is able to utilize morpholine, piperidine and pyrrolidine as sole carbon, nitrogen, and energy source. A specifically induced expression of a cytochrome P450 is observed during the degradation of these N-heterocycles (Schräder et al., 2000), which supports the above-mentioned reports. As observed for other mycobacterial strains, no enzymic hydroxylation activity is detected in cell-free extracts of Mycobacterium sp. strain HE5. To tackle this problem, the cytochrome P450, designated P450mor, and its proposed redox partner, a Fe3S4 ferredoxin (Fdmor) have been purified separately to homogeneity (Sielaff et al., 2001). Thus, for the first time, proteins involved in morpholine degradation could be isolated.

P450 cytochromes are able to catalyse a wide range of reactions, mainly hydroxylations (Urlacher et al., 2004). The activation of molecular oxygen species at the haem cofactor of these enzymes requires electrons, which are derived from the oxidation of NAD(P)H by an oxidoreductase. The cytochrome P450 can be reduced either directly by an FAD- and FMN-containing reductase (class II system) or indirectly by electrons transferred from an FAD-containing reductase to the cytochrome P450 via a small iron-sulphur protein (class I system). Most bacterial P450 systems belong to the latter class (Munro & Lindsay, 1996). However, a specifically induced reductase cannot be detected in cell-free extracts of Mycobacterium sp. strain HE5 (Sielaff et al., 2001) or of other mycobacterial strains (Combourieu et al., 1998a; Poupin et al., 1998; Trigui et al., 2004). The determined internal peptide of P450mor is identical to the translated sequence of the gene pipA, encoding a P450 (CYP151) from Mycobacterium smegmatis mc2155. PipA is involved in piperidine and pyrrolidine metabolism, but the pip operon lacks a reductase-encoding gene (Poupin et al., 1999b). These results have led to the assumption that the reductase is a constitutively expressed protein (Sielaff et al., 2001), as was once supposed for other P450 systems from different Actinomycetales (O'Keefe & Harder, 1991). Transcription studies have demonstrated that in Streptomyces coelicolor, three ferredoxin reductases are sufficient to support the activity of 18 P450 cytochromes (Lei et al., 2004), which is in agreement with this hypothesis.

The determination of the amino acid sequences of P450mor and Fdmor provided the opportunity to determine the genetic basis of the P450mor monooxygenase. Of special interest was the possibility of identifying the ferredoxin reductase of the P450mor system. We report here the cloning of the operon encoding all structural genes of the P450mor monooxgenase. Sequence determination of this operon region revealed a gene encoding a ferredoxin reductase, which was expressed as an enzymically active recombinant protein. This is the first report of the characterization from a P450 system of a native NADH-dependent ferredoxin reductase that is specifically required for enzymic function with the Fe3S4 protein Fdmor.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Materials.
All chemicals, NADH and spinach ferredoxin (FdI) were purchased from Sigma-Aldrich and Fluka. For molecular biological work, all biochemicals and enzymes other than restriction endonucleases were provided by Roche Molecular Biochemicals. Restriction endonucleases were from Fermentas or New England Biolabs, based on availability. Oligonucleotides were provided by Metabion. The Lambda ZAPII system was obtained from Stratagene. Cloning vectors were from Fermentas. Expression vectors and Ni-NTA affinity column material was from Novagen. All other column materials were obtained from Pharmacia. Purified adrenodoxin reductase (AdR) and adrenodoxin (Adx) were a kind gift from Professor Rita Bernhardt and Dr Frank Hannemann (Universität des Saarlandes).

Bacterial strains.
Mycobacterium sp. strain HE5 (DSM 44238) was from our laboratory collection. Mycobacterium chlorophenolicum PCP-1 (DSM 43826T) was kindly provided by Timo Nieminen (University of Oulo, Finland). Escherichia coli XL-1 Blue MRF' and E. coli Rosetta (DE3) were purchased from Stratagene and Novagen, respectively.

Preparation of whole-cell DNA.
Mycobacterium sp. strain HE5 and M. chlorophenolicum PCP-1 were grown on 20 mM morpholine to OD600 ~1·0 and harvested as described previously (Schuffenhauer et al., 1999). Cells (400 µg) were resuspended in 400 µl TENS buffer (50 mM Tris/HCl, pH 8·0, 100 mM EDTA, 100 mM NaCl, 0·3 % SDS), transferred to a 2 ml tube containing 1·6 g of glass beads (0·25 µm diameter), and shaken in a bead beater (Retsch) at maximum power for 10 min. After removing the cell debris and glass beads by centrifugation at 20 000 g, the supernatant was collected and incubated with RNase A (200 µg ml–1) at 37 °C for 30 min. Subsequently, proteinase K was added (200 µg ml–1) and the solution incubated at 55 °C for 1 h. The following steps were standard procedures and were performed in sequence: extraction with phenol and phenol/chloroform (60 : 40); ethanol precipitation; and resuspension of precipitated DNA in 10 mM Tris/HCl, pH 8·0, containing 1 mM EDTA.

DNA techniques.
Molecular procedures were either standard techniques (Sambrook et al., 1989) or those recommended by the respective manufacturers. All PCRs and sequencing reactions were performed on a Mastercycler (Eppendorf). Nucleotide sequences were determined using the dRhodamine Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer Applied Biosystems) and analysed using an ABI PRISM 377 DNA Sequencer. Attempts to isolate a potential circular plasmid were performed according to Larsen (2000). Plugs for usage in PFGE were prepared as described by Hughes et al. (2001). PFGE was performed on a CHEF-DR II system (Bio-Rad) at 13 °C and 170 V, and the pulse time was raised linearly over 24 h from 20 to 80 s.

Amplification and cloning of DNA fragments.
A specific DNA fragment was amplified from whole-cell DNA using a degenerate primer (5'-CAC CAC CGS YTS CGS CGS YTS ATG AAC CC-3') designed based upon the 19 aa internal fragment of P450mor (Sielaff et al., 2001) and a primer (5'-GGC AGT GTG TTG GGT CCG GTG TTG C-3') derived from the C-terminal part (1154–1171) of pipA from M. smegmatis mc2155 (Poupin et al., 1999b). PCR was performed according to the following standard protocol: 94 °C for 4 min; (94 °C for 15 s, 55 °C for 20 s, 72 °C for 1 min) for 10 cycles; (94 °C for 15 s, 55 °C for 20 s, 72 °C for 1 min plus 5 s at each cycle) for 20 cycles. This standard protocol was used for all PCRs, except that annealing temperatures and extension times were changed if necessary. The product P450-F1 was ligated into the pGem-T vector (Promega) and transformed into E. coli XL-1 Blue MRF', and for positive clones the plasmid was sequenced using M13 forward and reverse primers. From the sequence of P450-F1, two specific primers, mor6 (5'-AAA CTC ATC GGC TCG CTC GTA CC-3') and mor7 (5'-ACT CGC TGT ATA GGT GGA CGG TG-3'), were derived and used to amplify, from whole-cell DNA, a 630 bp PCR product (annealing at 65 °C) which was directly sequenced and subsequently labelled using DIG High-Prime, yielding the probe P450m. This probe was used in Southern analysis of whole-cell DNA digests using a bank of restriction enzymes (PstI, XhoI, PvuI, SmaI and EcoRI) After separation by electrophoresis on a 0·8 % (w/v) agarose gel, DNA was transferred to a nylon membrane by vacuum blotting and probed with P450m. Chemiluminescence detection revealed a band of suitable size (4·5 kb) in the EcoRI digest. After repetition of digestion and separation, bands of appropriate size were recovered by gel extraction (Qiagen Gel Extraction Kit) and ligated into the EcoRI site of the Lambda ZAPII vector. Recombinant clones were packaged in vitro, and after infection of E. coli XL-1 Blue MRF', the resulting phage particles were screened by plaque hybridization with P450m. The phagemids of positive plaques were excised in vivo and the resulting clones were screened by colony hybridization and, as a control, by colony PCR. Because no positive clone could be detected, the recombinant clones were used as template in PCR with the primers mor6 and RPRN (modified M13 reverse primer: 5'-CAA TTT CAC ACA GGA AAC AGC TAT G-3'), which yielded the new fragment P450-F2 (annealing at 65 °C and extension for 1 min 30 s). P450-F2 was gel extracted and used as template for another PCR in order to obtain enough DNA for direct sequencing. Based upon the new sequence information of P450-F2, the primers mor8 (5'-GCG TAT CCG TAG ATC CCA CG-3') and mor9 (5'-GCG GTT ATA AGG CAG GTG TC-3') were designed for the new probe Fdfrm. PCR (annealing at 52 °C and extension for 30 s) with whole-cell DNA yielded a 318 bp product, which was labelled as described for P450m.

After restriction site analysis of the fragment P450-F2, digests of whole-cell DNA were performed using the enzymes BstXI, NruI, PvuII, SacI, PaeI and XmaIII. Digested DNA was separated by electrophoresis and transferred to a nylon membrane, and hybridization was performed once with P450m and, after stripping the membrane, a second time with Fdfrm. Bands of suitable size were detected in the NruI (2·0 kb), SfiI (2·2 kb) and PaeI (2·0 kb) digests. Recovered DNA fragments were ligated into NruI-digested pUC57 or into PaeI-digested pUC18, and transformed individually into E. coli XL-1 Blue MRF'. Colonies were transferred with a sterile stamp to two new agar plates, one of which was swabbed off with 3 ml 10 mM Tris/HCl, pH 8·0, and 1 µl of this suspension was used as template for PCR with the primer pairs mor6/mor7 or mor8/mor9. For positive pools, the corresponding plates were screened by colony lifts and subsequent Southern hybridization. One positive clone each was detected in the NruI library (2500 clones tested) and the PaeI library (1500 clones tested). The plasmids of these positive clones were isolated (Qiagen Plasmid Purification Kit) and designated pMN21 and pMP10, respectively.

PCR with whole-cell DNA of M. chlorophenolicum PCP-1 as template and the primers 5'-CGC TGA TCC GTC GTT CTC CAT-3' with mor7 (annealing at 48 °C), mor6 with mor9 (annealing at 52 °C), 5'-GTC GTA GGC GGC TCA CTG-3' with 5'-CCT CGT TGT TGT TTG GAC-3' (annealing at 48 °C), and 5'-CTA TGG ATC ACC TGC TCT G-3' with 5'-ATC GCT TGG AAA TAA ACG-3' (annealing at 45 °C and extension for 30 s) yielded the products MC-F1, MC-F2, MC-F3 and MC-F4, respectively. These amplified DNA fragments were directly sequenced.

Cloning of morC.
For both ends of morC, primers were designed that contained suitable restriction sites flanked by ‘spacer’ nucleotides at the 5' end to facilitate efficient digestion. A PagI site was incorporated in the N-terminal primer 5'-GAACTA TCATGA CCACCC CGCGG CACGTC-3' to allow for an in-frame ligation in the NcoI-treated vector pET28b(+) to express morC as a C-terminal His-Tag fusion protein. In the C-terminal primer 5'-CTAGAC AAGCTT TGCGGG CAGCTG GACGGC GG-3', a HindIII site was incorporated (restriction sites underlined). PCR was performed using whole-cell DNA as template according to the standard protocol (see above) with the annealing temperature set to 67 °C. The major 1·2 kb product was cut with PagI and HindIII, extracted from the gel and ligated in the NcoI/HindIII-digested vector pET28b(+). This plasmid was transformed into E. coli XL-1 Blue MRF' cells. The resulting recombinant cells were screened by PCR, and plasmids of positive clones were purified and sequenced to confirm that no PCR errors were incorporated. A plasmid containing the correct insert was designated pMRC28 and transformed into cells of E. coli Rosetta (DE3). Glycerol stocks were prepared by adding 200 µl 40 % (v/v) glycerol to 800 µl of a cell culture that had been previously grown to OD600 1·0, followed by storage at –80 °C.

Production and purification of recombinant FdRmor.
Luria–Bertani medium (4 ml) with 30 µg kanamycin ml–1 was inoculated with 5 µl of a glycerol stock of E. coli Rosetta (DE3) containing pMRC28 and cultured overnight at 30 °C. This culture was used to inoculate four Erlenmeyer flasks (2 l), each containing 500 ml Terrific Broth with 30 µg kanamycin ml–1. The flasks were incubated at 37 °C until OD600 0·8 was attained (~5 h). The cells were then induced with 1 mM IPTG and incubated at 25 °C for 18 to 20 h. Cells were harvested by centrifugation (7500 g, 20 min, 4 °C) and stored at –20 °C. After resuspension in 20 ml buffer A (50 mM NaH2PO4, pH 8·0, 300 mM NaCl, 20 %, v/v, glycerol) containing 10 mM imidazole, 0·1 mM PMSF and 5 µl Benzonase, cells were disrupted by two passages through a 20 K French pressure cell (SLM-Amicon) at 120 MPa, and the lysate was centrifuged at 18 000 g for 30 min (4 °C) to remove cell debris. The supernatant was loaded onto a 0·5 ml Ni-NTA His-Bind Resin flow-through column, previously equilibrated with 3 ml buffer A containing 10 mM imidazole. After washing with 5 ml buffer A containing 20 mM imidazole, FdRmor was eluted by stepwise addition of 0·25 ml buffer A containing 200 mM imidazole. Fractions containing FdRmor, monitored by the flavin-specific absorption at 452 nm, were pooled and concentrated in an ultrafiltration device (Vivascience). The second purification step was a gel filtration on Sephadex 75, run with buffer B (50 mM Tris/HCl, pH 7·5, 20 %, v/v, glycerol). Fractions were collected and concentrated, and stored in aliquots at –20 °C.

Purification of Fdmor.
Culture of Mycobacterium sp. strain HE5 cells and preparation of crude extracts were performed as described previously (Sielaff et al., 2001). The purification protocol for Fdmor was modified. After eluting Fdmor from Q-Sepharose fast flow in a linear gradient of 0–1 M KCl in buffer B, fractions containing Fd were identified by their brownish colour and collected according to their A280/A425 value. The collected fractions were concentrated in an ultrafiltration device and proteins were then separated on a Sephadex 75 gel filtration column using buffer B. Subsequently, Fdmor was applied to a MonoQ column. After elution in a linear gradient of 0–1·5 M KCl in buffer B, the protein was desalted using a PD10 column run with buffer B. Fdmor purified by this procedure was >95 % pure, as judged by SDS-PAGE analysis.

Molecular characterization methods.
SDS-PAGE was carried out as described previously (Sielaff et al., 2001). Analytical gel filtration analysis was performed on a FPLC system equipped with a Superdex 75 column (Pharmacia Biotech) run with buffer B. UV/visible spectra were recorded on an Uvikon 930 spectrophotometer (Kontron). The reduction of FdRmor with NADH was performed in a glove box (Coy) under nitrogen atmosphere at 4 °C. Buffers and solutions were made anaerobic prior to usage by several cycles of degassing and gassing with nitrogen using the sluice of the glove box. The quartz cuvette was sealed with a rubber cap. The extinction coefficient of the protein-bound flavin was determined spectrophotometrically by quantification of the FAD released from the holoprotein following SDS treatment (Aliverti et al., 1999). The identity of the enzyme-bound flavin was assessed fluorometrically. After thermal denaturation of 10 µM holoenzyme at 100 °C for 15 min, the released flavin was treated with 3 mU phosphodiesterase I (Aliverti et al., 1999). Emission spectra (480 nm to 600 nm) were recorded in a fluorescence cuvette of 1 cm path length on a FluoroMax2 (Jobin Yvon-Spex) at 20 °C, using an excitation wavelength of 450 nm and a slit width of 5 nm. Visible circular dichroism (CD) spectra (320 nm to 600 nm) were recorded at 20 °C using a JASCO J-810 spectropolarimeter with a quartz cell of 1 cm pathlength (scan speed 50 nm min–1). Spectra were recorded five times and averaged.

Activity assays.
The activity of FdRmor towards the artificial electron acceptors potassium ferricyanide, cytochrome c and nitro blue tetrazolium (NBT) was determined spectrophotometrically using an Uvikon 930 spectrophotometer (Kontron). Potassium ferricyanide reduction was monitored at 420 nm ({varepsilon}420=1020 M–1 cm–1), cytochrome c reduction at 550 nm ({varepsilon}550=21 100 M–1 cm–1) and NBT reduction at 535 nm ({varepsilon}535=18 300 M–1 cm–1). Reactions were performed with 10 nM FdRmor in 50 mM Tris/HCl, pH 8·5, at 30 °C, if not stated otherwise. For measurements of ferricyanide-reducing activities at different pH values, a buffer was used composed of 25 mM Tris and 25 mM glycine, which was adjusted to the appropriate pH with either NaOH or HCl. Activity assays of FdRmor with Fdmor were performed in 50 mM glycine-buffer, pH 8·5. AdR/Adx activity was measured according to Uhlmann et al. (1994). Steady-state kinetic parameters were determined by varying the concentrations of the substrates in the standard assay. Initial velocities (v) were fitted to a hyperbolic function to obtain the kinetic parameters Km and Vmax.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cloning and sequencing of the genes encoding the P450mor system
Degenerate primers were designed on the basis of an internal 19 aa fragment of P450mor and the N-terminal 30 aa fragment of Fdmor (Sielaff et al., 2001), as it could be expected that these proteins would be encoded by adjacent genes. Primers were chosen according to codon usage in mycobacteria in order to minimize inherent degeneracy. However, no specific fragment could be amplified by PCR with genomic DNA from Mycobacterium sp. strain HE5. A different approach was eventually successful: the 19 aa P450mor fragment is identical to the corresponding translated sequence of pipA, a gene encoding a P450 that is involved in piperidine and pyrrolidine metabolism in M. smegmatis mc2155 (Poupin et al., 1999b). The combination of a new primer designed from the 3' end of pipA and a degenerate primer derived from P450mor produced an 895 bp fragment (P450-F1), the internal sequence of which proved to encode the P450mor fragment. Based on this sequence, internal primers were designed to produce a probe that was then used in Southern hybridization experiments with different restriction-enzyme digests. This revealed that there is only a single copy of the gene encoding P450mor in the genome of Mycobacterium sp. strain HE5. A 4·5 kb EcoRI fragment was isolated and ligated into the Lambda ZAPII vector. After in vivo excision of the phagemids of plaques giving a positive reaction in Southern hybridization experiments, no positive clone could be detected. However, use of the ligated EcoRI fragment as template in PCR with an internal primer and a vector-encoded primer yielded a specific 1373 bp fragment (P450-F2) comprising 1272 bp from the 3' terminal portion of the cloned fragment (Fig. 1).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 1. Genetic organization of the mor operon region. Arrows indicate genes deduced from the nucleotide sequence, which was derived from PCR products P450-F1 and P450-F2 and from plasmids pMN21 and pMP10 (Mycobacterium sp. strain HE5). The PCR products MC-F1, MC-F2, MC-F3 and MC-F4 (M. chlorophenolicum PCP-1) are also shown. Only selected restriction sites of importance are indicated. Detailed information is given in the text.

 
Based on the new sequence of P450-F2, a second probe, Fdfrm, was amplified, the sequence of which was located downstream of probable suitable restriction sites. A restriction site map of the P450mor gene region (data not shown) was obtained by Southern analysis of several DNA digests with P450m, which was located upstream of these sites, or with Fdfrm. This now allowed the cloning of specific fragments based on their location and the expected extent of new sequence information. A 2·0 kb NruI fragment was cloned, which was about 1·4 kb shorter at its 5' terminal site than the EcoRI fragment. A positive clone was detected from this library and plasmid pMN21 was isolated. Sequencing of the internal fragment revealed the upstream region of the P450mor operon. Cloning of a 2·0 kb PaeI fragment led to the isolation of plasmid pMP10, which contained the downstream region of this operon. Summarizing, 4782 bp of the P450mor operon could finally be sequenced.

Analysis of the genes encoding the P450mor system
Sequencing of DNA fragments revealed a putative operon consisting of six ORFs, of which two were truncated (Fig. 1). MorA encoded a protein of 400 aa, which contained a sequence identical to that of the previously determined internal 19 aa peptide of P450mor (Sielaff et al., 2001). Thus, we concluded that morA encodes the cytochrome P450mor. There was a difference between the predicted molecular mass of 44 603 Da from morA and the molecular mass of 44 769 Da determined by mass spectrometry for P450mor (Sielaff et al., 2001). P450mor proved to be N-terminally blocked in Edman degradation, suggesting an N-terminal acylation, which could account for this difference. MorA, as well as the following genes morB, morC and orf1', was found to be identical to corresponding genes from Mycobacterium sp. strain RP1 (Trigui et al., 2004). This point will be dealt with later.

The two ORFs downstream of morA appeared to encode the potential redox partners for a catalytically functional P450 system. MorB encodes the ferredoxin Fdmor (62 aa), as confirmed by comparison of the translated sequence with the N-terminal 30 aa sequence determined for the previously purified protein Fdmor from Mycobacterium sp. strain HE5. In addition, the predicted molecular mass of 6793 Da was in good agreement with the 6795 Da determined for Fdmor (Sielaff et al., 2001). The following ORF, morC, encoded a 403 aa protein with a predicted molecular mass of 42 376 Da, which was, as mentioned above, identical to MorC from Mycobacterium sp. strain RP1, and which exhibited identities to several ferredoxin reductases, all identified from genome sequences: 39 % in different overlaps to FprC from Streptomyces avermitilis MA-4680 (Ikeda et al., 2003) and Rv0688 from Mycobacterium tuberculosis H37Rv (Cole et al., 1998), and 37 % to FprA from S. avermitilis MA-4680 (Ikeda et al., 2003). FprC and fprA were identified adjacent to genes encoding Fe3S4 ferredoxins and the P450s CYP105Q1 and CYP147B1, respectively (Lamb et al., 2003). The identification of MorC as being in fact a ferredoxin oxidoreductase was confirmed in the present study by heterologous expression of morC and analysis of the protein. All previously purified P450 coupled reductases have been reported to belong to the glutathione reductase family, all of which contain an FAD-binding consensus sequence (GxGxxG) in the N-terminal region (Dym & Eisenberg, 2001). In FdRmor, this motif is changed (GGSLAG), whereas a second consensus sequence (GxGxxGxE) was found to be conserved. Sequence analysis of FprC and FprA from S. avermitilis revealed that they also contain the changed motif. However, despite such local differences, the overall homology of FdRmor to putidaredoxin reductase (28 % identity) from the P450cam system from Pseudomonas putida (Sevrioukova et al., 2004) indicates that these proteins are nevertheless related to the glutathione reductase family.

orf1' downstream of morC was truncated, and the derived amino acid sequence (141 aa) was identical to the deduced 74 aa of the truncated orf1' from Mycobacterium sp. strain RP1, and also showed 85 % identity to the 130 aa protein encoded by the similarly truncated orf2' from M. smegmatis mc2155. Both sequences exhibit significant identities to the N-terminal sequences of putative glutamine synthetases (Poupin et al., 1999b; Trigui et al., 2004).

Identical P450 genes in different mycobacterial strains
Quite recently, the genes encoding a cytochrome P450 system involved in secondary amine utilization in Mycobacterium sp. strain RP1 became known (Trigui et al., 2004). The analysed PstI fragment, exhibiting the ORFs morA, morB, morC and orf1', is identical to the corresponding sequence of Mycobacterium sp. strain HE5. The existence of totally identical mor gene regions was surprising, since the homology of this gene region is higher than that of the corresponding 16S rDNA (98·0 % identity). M. chlorophenolicum PCP-1 is another relative of these strains, according to its 16S rDNA (98·4 % identity to Mycobacterium sp. strain HE5, 97·3 % to Mycobacterium sp. strain RP1) and is known to be capable of degrading polychlorinated phenols (Apajalahti & Salkinoja-Salonen, 1987; Häggblom et al., 1994). It has now been shown in our laboratory that M. chlorophenolicum PCP-1 is also able to use morpholine, piperidine and pyrrolidine as sole carbon, nitrogen and energy source, and that a cytochrome P450 is induced during growth on morpholine, but not on the putative intermediate diglycolic acid (Debbab, 2003). The specific DNA fragments MC-F1, MC-F2, MC-F3 and MC-F4 (Fig. 1) could be amplified by PCR using primers derived from the mor operon and whole-cell DNA isolated from M. chlorophenolicum PCP-1 as template. Sequencing 2727 bp of these fragments revealed the nearly identical genes morA, morB and morC (only one nucleotide was different in morB) in an identical order to that of the mor operon from Mycobacterium sp. strain HE5. But a pronounced difference was detected downstream of morC, beginning with a changed nucleotide in the stop codon of morC (Fig. 2). The intergenic region between morC and orf1' was 66 bp longer in Mycobacterium sp. strain HE5. The sequence following this stretch is again almost identical to that of M. chlorophenolicum PCP-1, although to a lesser extent (95·5 %) than the mor genes. From these results, it seemed clear that these mycobacterial strains might have exchanged DNA. However, no plasmid could be detected in Mycobacterium sp. strain HE5, either by standard procedures for the isolation of circular plasmids or by PFGE for the detection of linear plasmids.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 2. Alignment of the gene regions downstream of morC from Mycobacterium HE5 (HE5) and M. chlorophenolicum PCP-1 (PCP1). The start codon, stop codon and a putative ribosome-binding site (RBS) are framed. Identical nucleotides are shaded.

 
Analysis of the upstream region of morA
The 3·9 kb PstI fragment from Mycobacterium sp. strain RP1 (see restriction sites in Fig. 1) contained a 914 bp sequence upstream of morA that was previously identified as non-coding (Trigui et al., 2004). In this work, a larger extent of this region was sequenced. This enabled the identification of a stop codon upstream of morA at position 560 of the sequenced mor operon (Fig. 1), which terminated a truncated ORF, designated 'morR. The translated 186 aa showed identities of 80 % to a putative regulatory protein encoded by the gene pipR from M. smegmatis mc2155 (Poupin et al., 1999b), and of 39 % (in a 177 bp overlap) to SAV1742, a putative GntR-family regulator from S. avermitilis MA-4680 (Ikeda et al., 2003). PipR has been shown to be involved in the regulation of piperidine and pyrrolidine metabolism, which involves the cytochrome P450 CYP151 encoded by the gene pipA (87 % identity to morA) found downstream of pipR. Between pipR and pipA, an insertion element (IS1096) has been identified (Poupin et al., 1999b), which was not present between 'morR and morA in Mycobacterium sp. strain HE5. Instead, an ORF was identified in which the start codon overlapped with the stop codon of 'morR. This ORF, designated orfX, encoded a polypeptide of 260 aa, which showed identities of 31 % (in a 158 aa overlap) to the hypothetical proteins SAV1740 and SAV1124 from S. avermitilis MA-4680 (Omura et al., 2001) and to a low extent to chemotactic transducers from different bacteria. No function can be assigned to the hypothetical protein (260 aa) encoded by orfX, or to SAV1740 (265 aa) and SAV1124 (278 aa), as they lack, for example, the C-terminal portion of chemotactic transducers, which are composed in general of 600 to 700 aa. Interestingly, SAV1740 is found 39 bp downstream of SAV1742, which shows significant identities to the polypeptide encoded by 'morR. A possible ORF homologous to orfX could also be identified at the same position in the sequence from M. smegmatis mc2155, but two additional nucleotides are present in the latter at position 1111 (position 273 of orfX) that were not detected in the corresponding sequences of Mycobacterium sp. strain HE5 and Mycobacterium sp. strain RP1. If these two nucleotides were deleted in M. smegmatis mc2155, the predicted polypeptide (125 aa) would exhibit an identity of 65 % to that of orfX. However, in M. smegmatis mc2155, this possible ORF was disrupted after 375 bp by the IS element.

Production and purification of morC
MorA and morB have now been shown to encode the previously isolated proteins P450mor and Fdmor, thus establishing them as part of the morpholine-hydroxylating P450 system. A specific, morpholine-induced reductase could not be detected in Mycobacterium sp. strain HE5 by the methods employed (Sielaff et al., 2001), but we have now identified morC as encoding the ferredoxin reductase FdRmor of the P450mor system. Direct proof of this has been provided in a separate publication by the reconstitution of all three isolated proteins in an enzymically active morpholine monooxygenase (Sielaff & Andreesen, 2005). MorC was expressed as a C-terminal His-Tag fusion protein to study its characteristics as a ferredoxin reductase and to enable a comparison to previously purified ferredoxin reductases from other bacterial P450 systems.

An additional protein band was clearly visible in SDS-PAGE after growth at 37 °C of E. coli Rosetta (DE3) harbouring pMRC28 and induction with 1 mM IPTG. But nearly all of the protein was found to form inclusion bodies. Lowering the growth temperature after induction significantly increased the amount of soluble protein, which was found to be highest when cells were grown at 25 °C for 18 to 20 h. This protein, which from now on is called FdRmor, was isolated by chromatography on a Ni2+ affinity column and subsequent gel filtration on Sephadex G75. The purified protein was judged to be about 90 % homogeneous in SDS-PAGE (Fig. 3). Attempts to further purify FdRmor by anion-exchange chromatography on MonoQ resulted in the loss of the flavin cofactor and therefore of its activity. The cofactor could not be restored, either by the addition of FAD or of FMN. The amount of purified FdRmor was calculated to be about 30 nmol [~1·2 mg (l culture)–1], using the estimated extinction coefficient for FdRmor (see below).



View larger version (56K):
[in this window]
[in a new window]
 
Fig. 3. SDS-PAGE (12·5 %) of purified C-terminal His-tagged FdRmor (~2 µg). The molecular masses of the marker proteins are indicated in kDa.

 
Molecular properties of FdRmor
FdRmor showed an Mr of about 50 000 in denaturing PAGE (Fig. 2), which appeared to be in the same range as the calculated mass of 43 523 Da (42 376 calculated from the sequence of morC and 1147 from the linker sequence). The Mr of native FdRmor was determined by gel filtration to be 50 000, indicating that the protein was a monomer under these conditions. The pure FdRmor enzyme exhibited in its oxidized state spectral features typical of flavin-containing enzymes, with spectral maxima at 273, 378 and 452 nm. Shoulders were observed at 422 and 473 nm (data not shown). A value of 10·0 was calculated for the ratio of protein to flavin-specific absorption (A273/A452). Addition of excess sodium dithionite or NADH under anaerobic conditions led to full reduction of the flavin. No spectral signals attributable to flavin semiquinone species could be detected (data not shown). The non-covalently bound flavin in FdRmor was identified as FAD. The fluorescence of the released flavin increased about tenfold after addition of phosphodiesterase, as expected for the conversion from FAD to FMN. The extinction coefficient of FdRmor at 452 nm was calculated to be 11 070 M–1 cm–1 from the amount of FAD released after protein denaturation by SDS. A stoichiometry of 0·75 mol FAD (mol FdRmor)–1 was determined, indicating that not all of the purified protein contained the flavin cofactor. The visible CD spectrum of FdRmor (Fig. 4) is mainly related to the chiral signal from the FAD cofactor. Minima are located at 450 nm (close to the electronic absorption maximum at 452 nm) and 478 nm. The overall shape of the visible CD spectrum is similar to that reported for the AdR homologue FprA from M. tuberculosis (McLean et al., 2003).



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 4. Visible CD spectrum of FdRmor (28·6 µM) in 50 mM Tris/HCl, pH 7·5, 10 % (v/v) glycerol.

 
Catalytic properties of FdRmor
FdRmor was capable of oxidizing NADH and reducing the electron acceptors potassium ferricyanide and cytochrome c. The reduction of these acceptors by FdRmor was strictly dependent on NADH, and no activity was obtained using NADPH as substrate. The addition of FAD to the assay had no enhancing effect on the activity of FdRmor. The pH optimum for the NADH-dependent reduction of potassium ferricyanide by FdRmor was found to be 9·4. At pH 7·5, the activity of the enzyme declined to about 50 %. The optimal temperature for this reaction was found to be around 30 °C (data not shown). No NADH oxidase activity of FdRmor could be observed under these conditions. The steady-state kinetic parameters of FdRmor (Table 1) for the substrate NADH were determined with saturating concentrations of ferricyanide (1 mM), and those for the artificial electron acceptors ferricyanide and cytochrome c were determined with saturating concentrations of NADH (250 µM). The Km values measured for these substrates were all found to be in the same range. The lowest Km was obtained for ferricyanide, whereas those determined for cytochrome c and NADH were around two- and threefold higher, respectively. The efficiency (kcat/Km) of NADH-dependent ferricyanide reduction by FdRmor was about 25-fold higher, compared to that of cytochrome c reduction. This was mainly due to an approximately 14-fold higher kcat for the reduction of ferricyanide.


View this table:
[in this window]
[in a new window]
 
Table 1. Steady-state kinetic parameters for NADH-dependent ferricyanide- and cytochrome c-reducing activities of FdRmor

Measurements were performed in triplicate in 50 mM Tris/HCl, pH 8·5, with 10 nM FdRmor. Kinetic parameters were obtained by varying substrate concentrations in the standard assay. Standard errors for the obtained parameters are included.

 
Analysis of the mor operon in this study revealed that the P450mor monooxygenase is a class I system, composed of three components: the NADH-oxidizing ferredoxin reductase FdRmor, the ferredoxin Fdmor as electron-transfer protein and the cytochrome P450mor, which acts as monooxygenase. Thus, FdRmor should be able to interact catalytically with its proposed natural redox partner Fdmor. The reduction of Fdmor by FdRmor was directly monitored by the decrease of the ferredoxin peak at 412 nm in the spectrum of Fdmor after the addition of NADH and a catalytic amount of FdRmor (Fig. 5). Subsequently, the ability of Fdmor to mediate the FdRmor-catalysed reduction of different electron acceptors was studied. The addition of Fdmor had no effect on the FdRmor-dependent reduction of ferricyanide, whereas Fdmor enhanced the reaction of FdRmor towards cytochrome c up to fivefold. In addition, it has been reported previously that the presence of recombinant Fdmor enables the reduction of NBT by FdRmor, which cannot be catalysed by FdRmor on its own (Sielaff & Andreesen, 2005). Altogether, these results proved that FdRmor reduces Fdmor, which acts as an electron shuttle to different artificial electron acceptors. Thus, the systematic name for FdRmor should be NADH : ferredoxin oxidoreductase.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 5. Absorption spectra of oxidized and reduced Fdmor. Solid line, 50 µM Fdmor; dotted line, Fdmor reduced by the addition of 1 nM FdRmor and 0·25 mM NADH; dashed line, Fdmor reduced by the addition of a few grains of sodium dithionite. Spectra were recorded ~20 s after addition of reductants to Fdmor in 50 mM Tris/HCl, pH 7·5.

 
The natural redox partners of previously characterized ferredoxin reductases have always been Fe2S2 ferredoxins. In contrast, it has been clearly demonstrated that Fdmor contains a Fe3S4 cluster (Sielaff & Andreesen, 2005). To elucidate whether or not FdRmor and Fdmor specifically require each other for efficient catalysis, the cross-reactivity of these proteins with AdR and Adx was examined. AdR and Adx serve as the electron transport system of the mitochondrial P450 cytochromes and are the mammalian counterparts of putidaredoxin reductase and putidaredoxin (Schiffler & Bernhardt, 2003). The AdR homologue FprA from M. tuberculosis has been reported to interact functionally with Adx and spinach FdI (Fischer et al., 2002). Therefore, the ability of FdRmor to utilize FdI in the reduction of NBT or cytochrome c was examined, but no reactivity between these proteins was observed. In contrast, Adx could replace Fdmor functionally in the NBT standard assay, although concentrations in the micromolar range were necessary (indicating a Km value of FdRmor for Adx of about 2 µM), compared to nanomolar concentrations in the case of Fdmor. The reduction of cytochrome c by FdRmor was not enhanced by the addition of Adx. More interestingly, AdR was not able to use Fdmor, either in the reduction of cytochrome c or in the reduction of NBT. These results suggest that FdRmor can interact with different ferredoxins, but that the reduction of the Fe3S4 ferredoxin Fdmor requires the specific NADH : ferredoxin oxidoreductase FdRmor from the P450mor system.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cloning and sequencing of the mor operon from Mycobacterium sp. strain HE5 revealed six ORFs, of which three were found to encode the components of the P450mor system: morA, encoding the cytochrome P450mor; morB, encoding the Fe3S4 ferredoxin Fdmor; and morC, encoding the NADH : ferredoxin reductase FdRmor. These genes were found to be identical to the corresponding genes from Mycobacterium sp. strain RP1. Only the gene morA from Mycobacterium sp. strain RP1 has recently been expressed, and the protein can convert the heterocycles piperidine, pyrrolidine and morpholine in a heterologous system with the alternative ferredoxin NADP+ oxidoreductase and ferredoxin from spinach (Trigui et al., 2004). MorA exhibits high identities to PipA, a cytochrome P450 which has been shown to be involved in piperidine and pyrrolidine metabolism of M. smegmatis mc2155 (Poupin et al., 1999b).

In this study, almost identical morA, morB and morC genes were also identified in M. chlorophenolicum PCP-1. Differences were detected downstream of these genes, where a shorter intergenic region is present in M. chlorophenolicum PCP-1. The following gene, orf1', is also highly conserved, but the lower identity compared to the mor genes correlates much better to the lower identity of the 16S rDNA. This indicates that only the mor genes have integrated recently into this gene region. In contrast, no differences between Mycobacterium sp. strain HE5 and Mycobacterium sp. strain RP1 were observed downstream of morC, suggesting that the mor genes have been exchanged together with downstream sequences including orf1'. Subsequently, this gene region might have undergone deletions or insertions, thus indicating a lesser importance for the putative glutamine synthetase encoded by orf1' for the degradation of morpholine. Transcription studies should indicate if orf1' is functionally related to the mor operon, for example, by scavenging the nitrogen. The identification of identical mor genes in different mycobacterial strains suggests that this P450 system is more widely distributed within this genus. In fact, a number of different mycobacteria, all able to degrade morpholine, piperidine and pyrrolidine, have been shown to specifically express a cytochrome P450 during growth on these heterocycles (Poupin et al., 1999a). It seems very likely that these enzyme systems are also identical to the P450mor system, or at least that they exhibit high identities to it. Interestingly, the distribution of this P450 system does not seem to follow the degree of relationship between mycobacterial strains. Mycobacterium gilvum, which has been identified as the closest relative of Mycobacterium sp. strain HE5, is not able to grow on any of the heterocycles metabolized by the P450mor system (Schräder et al., 2000). Similar results have been obtained for five distinct haloalkane-utilizing Rhodococcus strains, which all share the completely conserved gene dhaA encoding a haloalkane dehalogenase. The highly conserved gene region is detected on the chromosome as well as on plasmids in all these strains (Poelarends et al., 2000). It has been suggested that an ancestral plasmid was transferred and subsequently integrated into the chromosome. A plasmid could also meet the requirements for horizontal gene transfer in morpholine-degrading mycobacterial strains, but no evidence for any sort of plasmid was found in Mycobacterium sp. strain HE5.

The main discrepancy between the P450 systems in Mycobacterium sp. strain HE5, Mycobacterium sp. strain RP1, M. chlorophenolicum PCP-1 and M. smegmatis mc2155 on the genomic level is the lack of a ferredoxin reductase-encoding gene in the last strain. In the pip operon of M. smegmatis mc2155, the gene orf1, encoding the ferredoxin, is immediately followed by the gene orf2', encoding a putative glutamine synthetase (Poupin et al., 1999b). Sequencing the genome of M. tuberculosis has revealed 22 genes encoding P450 cytochromes (Cole et al., 1998), and the genome of M. smegmatis mc2155 has been shown to exhibit 40 P450 genes (Jackson et al., 2003). This is the highest number found in a bacterium so far, but large sets of CYP genes have also been identified in the genomes of other actinobacteria. Many of these CYP genes have an isolated position in the genome, while a lower number are close to genes encoding ferredoxins. Only a few CYP genes are organized in operons that include genes encoding both reductase and ferredoxin. For instance, in the genome of S. avermitilis, 33 CYP genes have been identified, but only two are linked to ferredoxin and ferredoxin reductase genes (Lamb et al., 2003). Interestingly, both these reductases show significant homologies to FdRmor. Gene expression studies with S. coelicolor reveal that only three reductases and six ferredoxins seem to be sufficient to support the activity of the 18 P450 cytochromes of this organism, which are all expressed during the life cycle (Lei et al., 2004). Thus, it seems likely that in M. smegmatis mc2155, the missing reductase is functionally replaced by another ferredoxin reductase. The organization of the P450mor system, recruiting a specific ferredoxin reductase, is consistent with that of the classical bacterial P450cam system (Koga et al., 1985). Other biodegradative P450s, such as P450terp (Peterson et al., 1992), P450cin (Hawkes et al., 2002) and P450RRI (Nagy et al., 1995), are also organized into operons with their electron transfer proteins. This might imply that xenobiotic-metabolizing P450s generally utilize specific redox partners to ensure efficient functionality.

The amino acid sequence of FdRmor showed identities to different ferredoxin reductases from different Streptomyces and Mycobacterium strains. So far, all of these have only been derived from nucleotide sequences and have not been characterized at the protein level. MorC has now been expressed as a C-terminal His-Tag fusion protein and the recombinant enzyme FdRmor characterized as an NADH-dependent, FAD-containing ferredoxin reductase and shown to interact functionally with the Fe3S4 ferredoxin Fdmor. FdRmor shows some instability, which might explain the fact that no specifically induced ferredoxin reductase could previously be identified in crude extracts of Mycobacterium sp. strain HE5, which led us to propose that the reductase was constitutively formed (Sielaff et al., 2001). Instability, as well as a low level of expression, might be the reason for the paucity of reports on purified reductases of bacterial P450 systems. One of the best-known examples is the putidaredoxin reductase from Pseudomonas putida, which uses putidaredoxin as electron transfer protein to reduce P450cam (Koga et al., 1985). This redox system is similar to the mammalian one, in which AdR and Adx reduce mitochondrial P450 cytochromes (Schiffler & Bernhardt, 2003). FprA from M. tuberculosis was identified as an AdR homologue, and the heterologously expressed flavoprotein was able to reduce Adx, the Fe2S2 protein FdI from spinach and a 7Fe ferredoxin from M. smegmatis (Fischer et al., 2002). While AdR is clearly an NADPH-dependent ferredoxin reductase, FprA also oxidizes NADH, although with a lower efficiency compared to NADPH. This distinguishes these proteins from FdRmor, which is strictly NADH-dependent and cannot use NADPH as reductant. A soybean flour-induced NADH-dependent ferredoxin reductase has been purified from Streptomyces griseus and shown to couple electron transfer to cytochrome P450soy in the presence of a 7Fe ferredoxin from S. griseus (Ramachandra et al., 1991). This 7Fe ferredoxin and those used in the studies of FprA are not the natural redox partners of these reductases. So far, only the specific redox partners of AdR and putidaredoxin reductase have been purified and characterized. Both proteins, Adx and putidaredoxin, contain an Fe2S2 cluster, distinguishing them from Fdmor, which has been clearly identified as a Fe3S4 ferredoxin (Sielaff & Andreesen, 2005). In contrast to FprA, FdRmor is not able to interact functionally with FdI. FdRmor is able to utilize Adx in the NBT reduction, but the low catalytic efficiency of this reaction indicates a high specificity of FdRmor for its natural redox partner, Fdmor. This is supported by the measured low Km value (5·6 nM) of FdRmor for Fdmor in the reduction of NBT (Sielaff & Andreesen, 2005). Interestingly, the reduction of cytochrome c by FdRmor is not enhanced by the addition of Adx, although this electron acceptor is widely used to investigate AdR–Adx interactions (Grinberg et al., 2000). Furthermore, Fdmor cannot replace Adx enzymically in AdR/Adx activity assays with cytochrome c. Similarly, putidaredoxin and Adx cannot substitute each other in activity assays of their respective reductases, although these ferredoxins share 37 % homology in their sequences (Geren et al., 1986). In summary, we conclude from these data that for higher enzymic efficiency, Fdmor requires the specific NADH : ferredoxin reductase FdRmor, thus reflecting the genomic organization of this P450 system, in which all genes are found adjacent in the same operon.


   ACKNOWLEDGEMENTS
 
We gratefully acknowledge the gift of purified AdR and Adx from Professor Dr R. Bernhardt and Dr F. Hannemann (Institut für Biochemie, Universität des Saarlandes). We also thank T. Nieminen (University of Uolu, Finland) for providing Mycobacterium chlorophenolicum PCP-1, and Dr J. Köditz (Institut für Biotechnologie, Martin-Luther-Universität Halle) for assistance in CD spectroscopy. This work was supported by grants from the Land Sachsen-Anhalt and the Deutsche Forschungsgemeinschaft (Graduiertenkolleg: ‘Adaptive physiologisch-biochemische Reaktionen auf ökologisch relevante Wirkstoffe’).


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Aliverti, A., Curti, B. & Vanoni, M. A. (1999). Identifying and quantitating FAD and FMN in simple and iron-sulfur-containing flavoproteins. In Methods in Molecular Biology, pp. 9–23. Edited by S. K. Chapman & G. A. Reid. Totowa, NJ: Humana Press Inc.

Apajalahti, J. H. & Salkinoja-Salonen, M. S. (1987). Dechlorination and para-hydroxylation of polychlorinated phenols by Rhodococcus chlorophenolicus. J Bacteriol 169, 675–681.[Medline]

Cech, J. S., Hartman, P., Slosarek, M. & Chudoba, J. (1988). Isolation and identification of a morpholine-degrading bacterium. Appl Environ Microbiol 54, 619–621.[Medline]

Cole, S. T., Brosch, R., Parkhill, J. & 22 other authors (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537–544.[CrossRef][Medline]

Combourieu, B., Poupin, P., Besse, P., Sancelme, M., Veschambre, H., Truffaut, N. & Delort, A. M. (1998a). Thiomorpholine and morpholine oxidation by a cytochrome P450 in Mycobacterium aurum MO1. Evidence of the intermediates by in situ 1H NMR. Biodegradation 9, 433–442.[CrossRef][Medline]

Combourieu, B., Besse, P., Sancelme, M., Veschambre, H., Delort, A. M., Poupin, P. & Truffaut, N. (1998b). Morpholine degradation pathway of Mycobacterium aurum MO1: direct evidence of intermediates by in situ 1H nuclear magnetic resonance. Appl Environ Microbiol 64, 153–158.[Abstract/Free Full Text]

Combourieu, B., Besse, P., Sancelme, M., Godin, J. P., Monteil, A., Veschambre, H. & Delort, A. M. (2000). Common degradative pathways of morpholine, thiomorpholine, and piperidine by Mycobacterium aurum MO1: evidence from 1H-nuclear magnetic resonance and ionspray mass spectrometry performed directly on the incubation medium. Appl Environ Microbiol 66, 3187–3193.[Abstract/Free Full Text]

Debbab, M. (2003). Weitere Untersuchungen zum Stoffwechsel von Mycobacterium sp. Stamm HE5. Diploma thesis, Martin-Luther-Universität Halle-Wittenberg.

Dym, O. & Eisenberg, D. (2001). Sequence-structure analysis of FAD-containing proteins. Protein Sci 10, 1712–1728.[Abstract/Free Full Text]

Fischer, F., Raimondi, D., Aliverti, A. & Zanetti, G. (2002). Mycobacterium tuberculosis FprA, a novel bacterial NADPH-ferredoxin reductase. Eur J Biochem 269, 3005–3013.[Abstract/Free Full Text]

Geren, L., Tuls, J., O'Brien, P., Millet, F. & Peterson, J. A. (1986). The involvement of carboxylate groups of putidaredoxin in the reaction with putidaredoxin reductase. J Biol Chem 261, 15491–15495.[Abstract/Free Full Text]

Grinberg, A. V., Hannemann, F., Schiffler, B., Müller, J., Heinemann, U. & Bernhardt, R. (2000). Adrenodoxin: structure, stability, and electron transfer properties. Proteins 40, 590–612.[CrossRef][Medline]

Häggblom, M. M., Nohynek, L. J., Palleroni, N. J., Kronqvist, K., Nurmiaho-Lassila, E. L., Salkinoja-Salonen, M. S., Klatte, S. & Kroppenstedt, R. M. (1994). Transfer of polychlorophenol-degrading Rhodococcus chlorophenolicus (Apajalahti et al., 1986) to the genus Mycobacterium as Mycobacterium chlorophenolicum comb. nov. Int J Syst Bacteriol 44, 485–493.[Abstract]

Hawkes, D. B., Adams, G. W., Burlingame, A. L., Ortiz de Montellano, P. R. & De Voss, J. J. (2002). Cytochrome P450(cin) (CYP176A), isolation, expression, and characterization. J Biol Chem 277, 27725–27732.[Abstract/Free Full Text]

Hughes, V. M., Stevenson, K. & Sharp, J. M. (2001). Improved preparation of high molecular weight DNA for pulsed-field gel electrophoresis from mycobacteria. J Microbiol Methods 44, 209–215.[CrossRef][Medline]

Ikeda, H., Ishikawa, J., Hanamoto, A., Shinose, M., Kikuchi, H., Shiba, T., Sakaki, Y., Hattori, M. & Omura, S. (2003). Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis. Nat Biotechnol 21, 526–531.[CrossRef][Medline]

Jackson, C. J., Lamb, D. C., Marczylo, T. H., Parker, J. E., Manning, N. L., Kelly, D. E. & Kelly, S. L. (2003). Conservation and cloning of CYP51: a sterol 14 alpha-demethylase from Mycobacterium smegmatis. Biochem Biophys Res Commun 301, 558–563.[CrossRef][Medline]

Knapp, J. S. & Brown, V. R. (1988). Morpholine biodegradation. Int Biodeterior 24, 299–306.[CrossRef]

Koga, H., Rauchfuss, B. & Gunsalus, I. C. (1985). P450cam gene cloning and expression in Pseudomonas putida and Escherichia coli. Biochem Biophys Res Commun 130, 412–417.[CrossRef][Medline]

Lamb, D. C., Ikeda, H., Nelson, D. R., Ishikawa, J., Skaug, T., Jackson, C., Omura, S., Waterman, M. R. & Kelly, S. L. (2003). Cytochrome P450 complement (CYPome) of the avermectin-producer Streptomyces avermitilis and comparison to that of Streptomyces coelicolor A3(2). Biochem Biophys Res Commun 307, 610–619.[CrossRef][Medline]

Larsen, M. H. (2000). Some common methods in mycobacterial genetics. In Molecular Genetics of Mycobacteria, pp. 313–317. Edited by G. F. J. Hatfull & W. R. Jacobs, Jr. Washington, DC: American Society for Microbiology.

Lei, L., Waterman, M. R., Fulco, A. J., Kelly, S. L. & Lamb, D. C. (2004). Availability of specific reductases controls the temporal activity of the cytochrome P450 complement of Streptomyces coelicolor A3(2). Proc Natl Acad Sci U S A 101, 494–499.[Abstract/Free Full Text]

McLean, K. J., Scrutton, N. S. & Munro, A. W. (2003). Kinetic, spectroscopic and thermodynamic characterization of the Mycobacterium tuberculosis adrenodoxin reductase homologue FprA. Biochem J 372, 317–327.[CrossRef][Medline]

Munro, A. W. & Lindsay, J. G. (1996). Bacterial cytochromes P-450. Mol Microbiol 20, 1115–1125.[Medline]

Nagy, I., Schoofs, G., Compernolle, F., Proost, P., Vanderleyden, J. & de Mot, R. (1995). Degradation of the thiocarbamate herbicide EPTC (S-ethyl dipropylcarbamothioate) and biosafening by Rhodococcus sp. strain NI86/21 involve an inducible cytochrome P-450 system and aldehyde dehydrogenase. J Bacteriol 177, 676–687.[Abstract/Free Full Text]

O'Keefe, D. P. & Harder, P. A. (1991). Occurrence and biological function of cytochrome P450 monooxygenases in the actinomycetes. Mol Microbiol 5, 2099–2105.[Medline]

Omura, S., Ikeda, H., Ishikawa, J. & 11 other authors (2001). Genome sequence of an industrial microorganism Streptomyces avermitilis: deducing the ability of producing secondary metabolites. Proc Natl Acad Sci U S A 98, 12215–12220.[Abstract/Free Full Text]

Peterson, J. A., Lu, J. Y., Geisselsoder, J., Graham-Lorence, S., Carmona, C., Witney, F. & Lorence, M. C. (1992). Cytochrome P-450terp. Isolation and purification of the protein and cloning and sequencing of its operon. J Biol Chem 267, 14193–14203.[Abstract/Free Full Text]

Poelarends, G. J., Zandstra, M., Bosma, T., Kulakov, L. A., Larkin, M. J., Marchesi, J. R., Weightman, A. J. & Jannsen, D. B. (2000). Haloalkane-utilizing Rhodococcus strains isolated from geographically distinct locations possess a highly conserved gene cluster encoding haloalkane catabolism. J Bacteriol 182, 2725–2731.[Abstract/Free Full Text]

Poupin, P., Truffaut, N., Combourieu, B., Besse, P., Sancelme, M., Veschambre, H. & Delort, A. M. (1998). Degradation of morpholine by an environmental Mycobacterium strain involves a cytochrome P-450. Appl Environ Microbiol 64, 159–165.[Abstract/Free Full Text]

Poupin, P., Godon, J. J., Zumstein, E. & Truffaut, N. (1999a). Degradation of morpholine, piperidine, and pyrrolidine by mycobacteria: evidences for the involvement of a cytochrome P450. Can J Microbiol 45, 209–216.[CrossRef][Medline]

Poupin, P., Ducrocq, V., Hallier-Soulier, S. & Truffaut, N. (1999b). Cloning and characterization of the genes encoding a cytochrome P450 (PipA) involved in piperidine and pyrrolidine utilization and its regulatory protein (PipR) in Mycobacterium smegmatis mc2155. J Bacteriol 181, 3419–3426.[Abstract/Free Full Text]

Ramachandra, M., Seetharam, R., Emptage, M. H. & Sariaslani, F. S. (1991). Purification and characterization of a soybean flour-inducible ferredoxin reductase of Streptomyces griseus. J Bacteriol 173, 7106–7112.[Medline]

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Schiffler, B. & Bernhardt, R. (2003). Bacterial (CYP101) and mitochondrial P450 systems – how comparable are they? Biochem Biophys Res Commun 312, 223–228.[CrossRef][Medline]

Schräder, T., Schuffenhauer, G., Sielaff, B. & Andreesen, J. R. (2000). High morpholine degradation rates and formation of cytochrome P450 during growth on different cyclic amines by newly isolated Mycobacterium sp. strain HE5. Microbiology 146, 1091–1098.[Medline]

Schuffenhauer, G., Schräder, T. & Andreesen, J. R. (1999). Morpholine-induced formation of L-alanine dehydrogenase activity in Mycobacterium strain HE5. Arch Microbiol 171, 417–423.[CrossRef][Medline]

Sevrioukova, I. F., Li, H. & Poulos, T. L. (2004). Crystal structure of putidaredoxin reductase from Pseudomonas putida, the final structural component of the cytochrome P450cam monooxygenase. J Mol Biol 336, 889–902.[CrossRef][Medline]

Sielaff, B. & Andreesen, J. R. (2005). Kinetic and binding studies with purified recombinant proteins ferredoxin reductase, ferredoxin and cytochrome P450 comprising the morpholine mono-oxygenase from Mycobacterium sp. strain HE5. FEBS J 272, 1148–1159.

Sielaff, B., Andreesen, J. R. & Schräder, T. (2001). A cytochrome P450 and a ferredoxin isolated from Mycobacterium sp. strain HE5 after growth on morpholine. Appl Microbiol Biotechnol 56, 458–464.[CrossRef][Medline]

Trigui, M., Pulvin, S., Truffaut, N., Thomas, D. & Poupin, P. (2004). Molecular cloning, nucleotide sequencing and expression of genes encoding a cytochrome P450 system involved in secondary amine utilization in Mycobacterium sp. strain RP1. Res Microbiol 155, 1–9.[CrossRef][Medline]

Uhlmann, H., Kraft, R. & Bernhardt, R. (1994). C-terminal region of adrenodoxin affects its structural integrity and determines differences in its electron transfer function to cytochrome P-450. J Biol Chem 269, 22557–22564.[Abstract/Free Full Text]

Urlacher, V. B., Lutz-Wahl, S. & Schmid, R. D. (2004). Microbial P450 enzymes in biotechnology. Appl Microbiol Biotechnol 64, 317–325.[CrossRef][Medline]

Received 16 March 2005; revised 18 May 2005; accepted 19 May 2005.



This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Sielaff, B.
Articles by Andreesen, J. R.
Articles citing this Article
PubMed
PubMed Citation
Articles by Sielaff, B.
Articles by Andreesen, J. R.
Agricola
Articles by Sielaff, B.
Articles by Andreesen, J. R.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS
Copyright © 2005 Society for General Microbiology.