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
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ABSTRACT |
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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.
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INTRODUCTION |
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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.
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METHODS |
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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 ml1) at 37 °C for 30 min. Subsequently, proteinase K was added (200 µg ml1) 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 (11541171) 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.
LuriaBertani medium (4 ml) with 30 µg kanamycin ml1 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 ml1. 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 01 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 01·5 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 min1). 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 (420=1020 M1 cm1), cytochrome c reduction at 550 nm (
550=21 100 M1 cm1) and NBT reduction at 535 nm (
535=18 300 M1 cm1). 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.
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RESULTS |
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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.
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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).
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DISCUSSION |
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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 AdRAdx 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.
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ACKNOWLEDGEMENTS |
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Received 16 March 2005;
revised 18 May 2005;
accepted 19 May 2005.
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