Institute of Microbiology, University of Stuttgart, Allmandring 31, 70550 Stuttgart, Germany1
DuPont de Nemours Company, Wilmington, Delaware, USA2
Author for correspondence: Gesche Heiss. Tel: +49 711 685 5491. Fax: +49 711 685 5725. e-mail: gesche.heiss{at}po.uni-stuttgart.de
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ABSTRACT |
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Keywords: picric acid, two hydride transferases, NADPH-dependent F420 reductase
Abbreviations: H--PA, hydride -complex of picric acid; 2H--PA, dihydride
-complex of picric acid; 2,4-DNP, 2,4-dinitrophenol; H--2,4-DNP, hydride
-complex of 2,4-dinitrophenol; HTES, hydride transferring enzyme system
b The GenBank accession number for the sequence reported in this paper is AF435009.
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INTRODUCTION |
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Picric acid and 2,4-DNP are degraded by an initial reductive attack on the aromatic ring (Lenke et al., 1992 ; Lenke & Knackmuss, 1992
). The orange-red hydride
-complex of picric acid (H--PA, or hydride Meisenheimer complex) is the first metabolite produced during mineralization of picric acid by Rhodococcus (opacus) erythropolis HL PM-1 (Lenke et al., 1992
; Lenke & Knackmuss, 1992
, 1996
; Rieger et al., 1999
). However, because of the difficulty in lysing the cells, the enzymes involved have, until now, remained unknown.
Nocardioides simplex FJ2-1A was shown to contain a hydride transferase (HTES, or hydride transferring enzyme system) involved in the hydride transfer to the aromatic ring of picric acid, giving rise to H--PA (Ebert et al., 1999 ). The HTES also catalyses a second hydride transfer, converting H--PA to the dihydride
-complex of picric acid (2H--PA; Ebert et al., 2002
). Both hydride transfer reactions require the NADPH-dependent F420 reductase (previously referred to as component A) as an electron shuttle from NADPH to F420.
Recently, a gene cluster of about 12·5 kb, involved in picric acid degradation, was identified in R. (opacus) erythropolis HL PM-1 using differential display and sequenced (Russ et al., 2000 ; Walters et al., 2001
). We have named these genes npd for nitrophenol degradation. Our goal was to determine the function of npdC (previously ORF3), npdG (previously ORF7), npdH (previously undetected) and npdI (previously ORF8) by overexpressing the genes in Escherichia coli. Herewith, we have proven that the genes encode enzymes involved in picric acid catabolism.
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METHODS |
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Fatty acid and mycolic acid analyses.
Fatty acid analysis and analysis of mycolic acids were performed by the DSMZ GmbH (Braunschweig, Germany).
Sequence comparisons and database searches.
Pairwise sequence comparisons and database searches were carried out using BLASTN, BLASTP, BLASTX and NCBI ORFfinder software (Altschul et al., 1990 ; Tatusova & Madden, 1999
). Translations were achieved using the Translation Machine from the EMBL Outstation European Bioinformatics Institute. Conserved motif searches were performed using the programs CD-search, Pfam, Proscan and Block (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi; http:/www.sanger.ac.uk; http://npsa-pbil.ibcp.fr/cgi-bin/pattern_prosite.pl; http://blocks.fhcrc.org/blocks-bin/blocks_search).
Cloning and expression of npdC, npdG, npdH and npdI.
npdI was amplified from R. (opacus) erythropolis HL PM-1 chromosomal DNA with specific primers. Primer P63 was designed with a BamH1 site (underlined) (5'-GGATCCATGATCAAAGGCAT-3') and primer P68 was designed with a HindIII site (underlined) (5'-GCCTGCGTGCAAGCTTTCATGCG-3'). Following digestion with BamHI and HindIII, the resulting PCR product was ligated into the expression vector pQE-30 to yield plasmid pDMW4. npdC was amplified with primers P33 (5'-ATGAAGGTCGGAATCAGGAT-3') and P34 (5'-GGGCAGGTTGTGTGCGGTGG-3'). The resulting PCR product was cloned into the pBAD/topo thio fusion expression system (Invitrogen) to yield pDMW7. npdG was amplified with primers P56 (5'-ATGAAGAGCAGCAAGATCGC-3') and P57 (5'-CGCAGCTCGTGGATCATGAA-3'). The resulting PCR product was cloned into pTrcHis2-topo (Invitrogen) to yield pDMW10.
For expression, the npdC and npdI genes were amplified from pDMW7 and pDMW4, respectively. Primers were designed to incorporate NdeI and HindIII sites (underlined), and the His-tag sequence at the 3' end of npdC (5'-TTTTCATATGAAGGTCGGAATCAGGATCC-3' and 5'-TTTAAGCTTTCAATGGTGATGGTGATGATG-3') and the 5' end of npdI (5'-CGTCATATGAGAGGATCGCATCACCATCAC-3' and 5'-CCCAAGCTTGGGTCATGCGAGCTCCGGCAGGAC-3').
npdH was cloned into pBW22 by amplification, the primers being designed to contain NdeI and BamHI sites (underlined) (5'-TTTTCATATGATCCACGAGCTGCG-3' and 5'-TTTTGGATCCGGAGCGGGCGTATGAC-3'). Subsequently, npdH was subcloned from pBW22 (including the His-tag) into pET11a*; pBW22 containing npdH was cut with NdeI and HindIII. The HindIII site was filled in with DNA polymerase I (large Klenow fragment; NEB) to create a blunt end and ligated into pEt11a* (NdeI/SmaI).
The PCR reaction mixtures contained 1 ng template DNA, 0·5 U Vent polymerase (New England Biolabs), 2 mM MgSO4, 4% DMSO, 0·2 mM each dNTP and 50 pmol each primer. Twenty-five amplification cycles were performed as follows: 95 °C for 30 s, 55 °C for 40 s, 72 °C for 1 min 40 s. Primers were purchased from MWG Biotech. PCR fragments were eluted from agarose using an Easypure DNA purification kit (Biozym Diagnostics), restricted with the appropriate restriction enzymes and ligated into the vector (Table 1).
Preparation of cell extracts and SDS-PAGE
. Cells suspended in 50 mM KH2PO4/K2HPO4 (pH 7·8) plus imidazole (5 mM) were lysed with a French press (Aminco) at 80 MPa. Cell debris was removed by centrifugation at 100000 g for 45 min at 4 °C. The protein concentration was determined by the method of Bradford (1976) using a dye reagent concentrate (Bio-Rad protein assay). Bovine serum albumin served as the standard. SDS-PAGE was performed by the method of Laemmli (1970)
on a Mini-PROTEAN II electrophoresis cell (Bio-Rad).
Purification of His-tag fusion proteins.
His-tag fusion proteins were purified by Ni-NTA metal affinity chromatography (Qiagen), as described by the manufacturer. Fractions were tested for enzyme activity. Purified fractions were desalted by utilizing pD10 Desalting Columns (Amersham Pharmacia Biotech) and subsequently concentrated with a Vivaspin 2 ml concentrator (Sartorius).
Determination of the concentration of F420.
F420-2, purified from Methanobacterium thermoautotrophicum using a QAE column, was obtained from Lacy Daniels (Peck, 1989 ). The lyophilized powder was dissolved in 50 mM Tris, pH 7·5, and the absorbance was determined at 420 nm. The concentration was calculated using an extinction coefficient of 41400 M-1 cm-1 (Purwantini & Daniels, 1996
).
Enzyme assays and kinetic measurements.
Enzyme assays and kinetic assays were performed with a Varian Cary 50 Biospectrophotometer controlled by Cary WinUV Biopackage software. All enzyme reactions were done in a total volume of 1 ml. The enzyme activity of NpdG in standard tests was measured in citrate-phosphate buffer (50 mM, pH 5·5) containing 10100 µM NADPH, 10 µM F420 and 0·25 µg enzyme. The decrease in absorbance was monitored at 400 nm. The Km constant and Vmax were determined for NpdG only. Reaction rates were calculated by taking an extinction coefficient of 25700 M-1 cm-1 (Eirich et al., 1978 ). One unit of enzyme activity was taken as the amount of enzyme which converts 1 µmol F420 min-1. For determination of the Km constant, the concentration of F420 was varied from 2 to 35 µM. The Km and Vmax values for F420 were calculated by using Sigma Plot for Windows, version 5.00 (SPSS).
Enzyme activities of NpdC, NpdH and NpdI were detected by repeated recording of UV-visible spectra (280600 nm). Spectra were recorded every minute. Reactions with NpdC and NpdI were performed in 50 mM KH2PO4/K2HPO4, pH 7·8, containing 125 µM NADPH, 11 µM F420, 100 µM substrate (picric acid, H--PA or 2,4-DNP), 5 µg NpdG and 30 µg NpdI or NpdC. The first recording was performed in buffer containing F420 and NADPH only. After addition of NpdG, the second spectrum was recorded. The reaction was then commenced by the addition of NpdI or NpdC. Testing for enzyme activities in the absence of F420 or NADPH served as negative controls. Reactions with NpdH were performed in 50 mM KH2PO4/K2HPO4, pH 7·8, containing 100 µM 2H--PA and 30 µg NpdH.
HPLC analyses.
Metabolites were detected by means of HPLC (Chromeleon Chromatography Data Systems 4.30, equipped with a Dionex UV/Vis detector, UVD 170S/340S, a Dionex pump, P 580, and a Dionex autosampler, Gina50). Samples were resolved on Gromsil 100 Octyl-4 columns (125x4 mm or 250x4 mm, particle size 5 µm; Grom) using 30 or 40% (v/v) methanol, respectively, plus 5 mM PicA (tetrabutylammonium hydrogensulfate), pH 7·8, as the mobile phase. The precolumn was a Gromsil 100 Octyl-4 column (20x4 mm, particle size 5 µm; Grom). Enzyme reactions were terminated by freezing samples in liquid nitrogen prior to HPLC analysis.
Mass spectra.
Liquid chromatography (LC) mass spectra were obtained by negative-mode electrospray ionization (ESI) on a high performance liquid chromatograph (Dionex P580A HPG) with a Dionex diode array detector, DAD 340, coupled to a TSQ 7000 mass spectrometer. Metabolites were resolved on an LC column [Luna RP18(2); Phenomenex; 3 µm, 150 mmx2·1 mm] cooled to 15 °C by a Dionex column oven, STH 585. The initial mobile phase comprised 4% (v/v) acetonitrile/0·0005% (v/v) ammonia which was maintained at a flow rate of 250 µl min-1 for 3 min. The percentage acetonitrile was then linearly increased to 50% (v/v) over a time period of 8 min and sustained for 1 min. Finally, the gradient was linearly decreased to 4% (v/v) within 1 min and maintained for 6 min.
Chemicals.
H--PA was synthesized as described previously with modifications (Rieger et al., 1999 ). Picric acid (650 mg; lyophilized) was dissolved in 7 ml absolute acetonitrile. Subsequently, tetramethylammonium octahydridotriborate (345 mg, dissolved in 7 ml absolute acetonitrile) was added to the picric acid/acetonitrile solution under argon as a protective atmosphere at -20 °C. The precipitating dark powdery crystals were filtered through a sintered glass filter, followed by washing with ice-cold, absolute acetonitrile and finally dried in a vacuum (Christ Alpha I-5). 2H--PA was prepared according to Severin & Schmitz (1962)
.
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RESULTS |
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Expression of npdC, npdG, npdH and npdI
All genes were expressed as His-tag fusion proteins (Table 1). npdG was cloned into pTrcHis2-TOPO and the resulting recombinant plasmid was designated pDMW10. In induced cultures of E. coli TOP10(pDMW10), the enzyme constituted approximately 20% of the total cellular protein as estimated by SDS-PAGE. In non-induced cultures, no protein band of the expected size was detected. Hence, NpdG was purified from E. coli TOP10(pDMW10).
npdC and npdI were originally cloned into pBAD/Thio-TOPO and pQE-30, respectively. Recombinant cells carrying npdC or npdI in pBAD/Thio-TOPO or pQE-30 contained a substantial amount of inclusion bodies. Hence, npdC and npdI were amplified from the respective vectors and ligated with pJoe2702 to create pNTG6 and pNTG3, respectively. SDS-PAGE showed no enhanced protein bands of the expected sizes in cell extracts prepared from JM109(pNTG3) or JM109(pNTG6), although the respective enzyme activities could be measured. Hence, the strains were used for subsequent protein production and purification.
npdH was first cloned into pBW22. The resulting recombinant cells showed very low levels of activity. npdH was then subcloned into pET11a* to form pNTG11. Cell extracts of BL21 (DE3)(pNTG11) indicated that NpdH comprised 30% of the total cellular protein. Consequently, BL21 (DE3)(pNTG11) was used for protein purification.
Characterization of NpdC, NpdG, NpdH and NpdI
NpdG showed a 44% sequence similarity to an F420-dependent NADP reductase of Methanobacterium thermoautotrophicum in the database (Russ et al., 2000 ; Smith et al., 1997
; Walters et al., 2001
). Further, the first 27 aa of the translation product, NpdG, exhibited a 66% sequence identity to the amino-terminal region of the NADPH-dependent F420 reductase of N. simplex FJ2-1A (Ebert et al., 1999
). NpdC exhibited a sequence similarity of 42% to an N5,N10-methylene-tetrahydromethanopterin reductase of Methanococcus jannaschii (Bult et al., 1996
; Russ et al., 2000
) and NpdI possessed a sequence similarity of 46% to an F420-dependent N5,N10-methylene-tetrahydromethanopterin reductase from Archaeoglobus fulgidus (Klenk et al., 1997
; Russ et al., 2000
). In addition, the first 27 aa of NpdI exhibited a 77% sequence identity to the amino-terminal region of the hydride transferase of N. simplex FJ2-1A (Ebert et al., 1999
).
Detailed sequence analyses of the picric acid degradation gene cluster revealed a short ORF (npdH) of 318 bp between npdG and npdI (Fig. 1). Database searches with NpdH showed no significant similarity to any known sequence.
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From SDS polyacrylamide gels, the molecular masses of NpdG, NpdC, NpdI and NpdH were calculated to be 28·8, 43·4, 41·7 and 13·8 kDa, respectively. The sizes of NpdG and NpdH were consistent with the predicted molecular masses of 27 and 12·9 kDa, respectively (taking fusion of the vector-encoded His-tag, myc and V5 epitopes into account). The predicted molecular masses for NpdC and NpdI were considerably smaller (35·9 and 32·9 kDa, respectively) than those calculated from SDS-PAGE. We have no explanation for this presently, except that the conditions used for SDS-PAGE (salts and SDS) may cause abnormal migration of the proteins.
NpdC, NpdG and NpdH were largely purified to complete homogeneity. NpdI could only be purified to 90%. NpdC, NpdG and NpdI were unstable in the presence of imidazole, a constituent of the elution buffer used for purification. Hence, imidazole was removed from purified protein solutions by pD10 desalting columns. All four enzymes were stable at -20 °C for several weeks in 50 mM KH2PO4/K2HPO4 buffer (pH 7·8) plus 20% glycerol.
Reduction of F420 by NpdG
Purified enzyme was incubated with NADPH and F420 in citrate-phosphate buffer (50 mM, pH 5·5). A reduction in absorbance was observed at 400 nm, indicating the reduction of F420 and the formation of F420H2 (Purwantini et al., 1992 ). The Km value was determined to be 9·68±2·89 µM. The enzyme possessed a Vmax of 17·27±2·46 U mg-1. The standard deviations were calculated from at least three repetitions. No activity was observed when F420 was replaced by FAD (flavin adenine dinucleotide) or FMN (flavin mononucleotide).
NpdI catalyses a hydride transfer to picric acid or 2,4-DNP
To demonstrate that NpdI catalyses the conversion of picric acid to H--PA, NpdI was incubated with picric acid, NADPH, F420 and NpdG. No enzyme activity was detected in the absence of F420. Enzyme activities were detected by repeated spectral scans. An increase in absorbance at 420 and 490 nm (max of H--PA at pH 7·8) was the same as described for H--PA formation (Behrend & Heesche-Wagner 1999
; Lenke et al., 1992
). Furthermore, the spectral changes corresponded to a colour change from yellow to orange. The spectrum measured during HPLC analysis (
max 422, 490 nm) was indistinguishable from the chemically synthesized H--PA and from that described previously. The retention volumes were 4·0 ml [40% (v/v) methanol plus 5 mM PicA, pH 7·8; 250 mm column] or 4·9 ml [30% (v/v) methanol plus 5 mM PicA; 125 mm column]. Coupled HPLC-ESI-MS proved the identity of the chemically synthesized H--PA. At a retention volume of 4·3 ml, the mass spectrum of the H--PA revealed a single peak at an ion mass of m/z 230, which corresponded to the molecular ion [M·]-. A decrease in the concentration of picric acid coincided with a stoichiometric increase in the concentration of H--PA, indicating that picric acid was completely converted to H--PA.
Incubation of NpdI with 2,4-DNP, NADPH, F420 and NpdG showed that NpdI converted 2,4-DNP to the hydride -complex of 2,4-DNP (H--2,4-DNP). Spectrophotometrically, an increase in absorbance was detected at 420 nm, which was typical for H--2,4-DNP (Behrend & Heesche-Wagner, 1999
). Furthermore, the spectrum measured during HPLC analysis (
max 258, 306 and 440 nm) corresponded to the UV/Vis spectrum of the H--2,4-DNP described by Behrend & Heesche-Wagner (1999)
. The retention volume of the H--2,4-DNP was 1·3 ml [40% (v/v) methanol plus 5 mM PicA, pH 7·8; 250 mm column] or 1·7 ml [30 (v/v) methanol plus 5 mM PicA; 125 mm column].
NpdC catalyses a hydride transfer to H--PA
Spectroscopic scans were performed with NpdC plus H--PA, NADPH, F420 and NpdG. As for NpdI, no enzyme activity of NpdC was detected when omitting F420 from the enzyme assay. Enzyme activity was detected as spectral changes exhibiting a decrease in absorbance at 420 and 490 nm (Fig. 2). This coincided with a colour change from orange to yellow. HPLC analysis showed that the new product possessed a retention volume of 2·6 ml [40% (v/v) methanol plus 5 mM PicA, pH 7·8; 250 mm column] or 2·4 ml [30% (v/v) methanol plus 5 mM PicA; 125 mm column]. The spectrum derived from HPLC analysis showed absorbance maxima at
max of 229 and 383 nm. These corresponded to 2H--PA as described previously (Ebert et al., 2002
) and to the chemically synthesized 2H--PA. Coupled HPLC-ESI-MS of the latter confirmed the structure: 2H--PA was detected at a retention volume of 0·9 ml. Two signals at m/z 232 and 185 were due to the molecular ion [M·]- and the molecular ion after elimination of HNO2, [M-HNO2]-.
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DISCUSSION |
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Comparable to the hydride transferase of strain FJ2-1A, NpdI converted 2,4-DNP to H--2,4-DNP. Behrend & Heesche-Wagner (1999) initially described H--2,4-DNP as a metabolite of picric acid degradation by Nocardioides sp. CB 22-2. Furthermore, H--2,4-DNP would be expected to be the product of nitrite release from 2H--PA, as proposed for N. simplex FJ2-1A (Ebert et al., 2002
). Indeed, cell-free extracts of R. (opacus) erythropolis HL PM-1 convert 2H--PA to H--2,4-DNP, suggesting it to be a true metabolite of picric acid degradation in the strain.
NpdG was shown to be essential for the hydride transfer reactions to take place by regenerating F420H2. An interesting aspect is that NpdG appears to only use F420 as a substrate. It cannot reduce flavins such as FAD or FMN. Enzymes playing a role in general metabolism, with F420 being involved, are known in mycobacteria, streptomycetes and Archaea (Berk & Thauer, 1997 ; Eker et al., 1989
; Purwantini & Daniels, 1996
). For this reason, even though specific for F420, NpdG may not necessarily be restricted to picric acid or 2,4-DNP degradation strains. Research is under way to test this hypothesis.
Although we cannot assign an explicit function to npdH, the gene product may well play an essential role in preparing 2H--PA for nitrite release. Since the molecular masses of the 2H--PA and product X were indistinguishable, while the retention volumes and the absorbance spectra differed, this suggests that product X may be a tautomeric form of protonated 2H--PA. This might be required to facilitate subsequent nitrite release. Vorbeck et al. (1998) made a similar observation in the course of TNT degradation, where the hydride complex of TNT (H--TNT) undergoes a second hydride attack, followed by protonation and subsequent tautomerization of the resulting dihydride complex (2H--TNT). Various other groups studying bacterial TNT degradation have detected several protonated forms of 2H--TNT (French et al., 1998
; Pak et al., 2000
). That the UV/Vis spectrum of product X (pH 7·8) was indistinguishable from that of 2H--PA (pH 12; at
280 nm) could be due to the same longer wavelength chromophore in both structures. Work is in progress to characterize product X, to show it to be part of a productive catabolic route of picric acid degradation and to assign an explicit function to NpdH.
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ACKNOWLEDGEMENTS |
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Received 9 August 2001;
revised 24 October 2001;
accepted 1 November 2001.