JOURNAL OF BACTERIOLOGY, Dec. 1997, p. 7796–7802 0021-9193/97/$04.0010 Copyright © 1997, American Society for Microbiology
Vol. 179, No. 24
Purification, Properties, and Sequence of Glycerol Trinitrate Reductase from Agrobacterium radiobacter JASON R. SNAPE,1† NEAL A. WALKLEY,1 ANDREW P. MORBY,1 STEPHEN NICKLIN,2 1 AND GRAHAM F. WHITE * School of Molecular and Medical Biosciences, University of Wales Cardiff, Cardiff CF1 3US,1 and Explosives Systems Centre, D.E.R.A., Fort Halstead, Sevenoaks TN14 7BP,2 United Kingdom Received 3 July 1997/Accepted 7 October 1997
Glycerol trinitrate (GTN) reductase, which enables Agrobacterium radiobacter to utilize GTN and related explosives as sources of nitrogen for growth, was purified and characterized, and its gene was cloned and sequenced. The enzyme was a 39-kDa monomeric protein which catalyzed the NADH-dependent reductive scission of GTN (Km 5 23 mM) to glycerol dinitrates (mainly the 1,3-isomer) with a pH optimum of 6.5, a temperature optimum of 35°C, and no dependence on metal ions for activity. It was also active on pentaerythritol tetranitrate (PETN), on isosorbide dinitrate, and, very weakly, on ethyleneglycol dinitrate, but it was inactive on isopropyl nitrate, hexahydro-1,3,5-trinitro-1,3,5-triazine, 2,4,6-trinitrotoluene, ammonium ions, nitrate, or nitrite. The amino acid sequence deduced from the DNA sequence was homologous (42 to 51% identity and 61 to 69% similarity) to those of PETN reductase from Enterobacter cloacae, N-ethylmaleimide reductase from Escherichia coli, morphinone reductase from Pseudomonas putida, and old yellow enzyme from Saccharomyces cerevisiae, placing the GTN reductase in the a/b barrel flavoprotein group of proteins. GTN reductase and PETN reductase were very similar in many respects except in their distinct preferences for NADH and NADPH cofactors, respectively. al. (2) found a similar activity in cell extracts of a strain of Enterobacter cloacae, designated PB2, except that this enzyme, which has since been sequenced and characterized in more detail (13), was dependent on NADPH. In the present study, we report the purification, gene isolation, and characterization of the NADH-dependent GTN reductase from A. radiobacter. The structural gene encoding this enzyme has been designated nerA (for nitrate ester reductase).
Nitrate esters such as glycerol trinitrate (GTN) and pentaerythritol tetranitrate (PETN) are widely used both as explosives and as vasodilators in the treatment of angina. During their long history of exploitation in these applications (24, 34), there has been ample opportunity during production, storage, and use for significant contamination of land sites and water courses to occur, so that site remediation of explosive residues is now an urgent issue worldwide. Moreover, the current acceleration in demilitarization of ordnance and rocket formulations will produce further waste material, raising new environmental concerns. The environmental issues, together with the rarity of naturally occurring analogs (14), make the discovery of microorganisms which can degrade such compounds and thus influence their environmental fate of particular interest. Earlier interest in the elimination of nitrate ester explosives in wastewater treatment plants (35, 36) and in their metabolism in fungal organisms (6, 7, 29, 30) led to the first report (21) of denitration of GTN in pure bacterial cultures of Bacillus thuringiensis plus B. cereus and Enterobacter agglomerans, apparently occurring via a hydrolytic pathway, although formation of nitrate was not demonstrated. In contrast, White et al. showed unequivocally that assimilation of nitrogen from GTN in pure cultures of a Pseudomonas sp. (37) and Agrobacterium radiobacter (38) occurred via nitrite (not nitrate) with the concomitant formation of mainly glycerol 1,3-dinitrate (1,3-GDN) and small amounts of the corresponding 1,2-isomer. Cells were able to denitrate both of the dinitrates to mononitrates but not beyond, and they also converted PETN to its tri- and dinitrates. The enzyme responsible was identified in crude cell extracts as an NADH-dependent GTN reductase. Subsequently, Binks et
MATERIALS AND METHODS Materials and reagents. MW-GF-200 molecular weight markers (12 to 200 kDa), SigmaMarker molecular weight standards, Tricine, and oxidized and reduced glutathione were obtained from Sigma Chemical Co., Poole, United Kingdom. Blue dextran was from Bio-Rad Laboratories Ltd., Hemel Hempstead, United Kingdom. The Superose 12 HR 10/30 gel exclusion chromatography column was from Pharmacia Biotech, St Albans, United Kingdom. NADP1, NADPH, and flavin adenine dinucleotide (FAD) were from Boehringer Mannheim, Lewes, United Kingdom, and the polyvinylidene difluoride membrane was from Millipore Limited, Watford, United Kingdom. With the exception of nitrate esters, all the other reagents were the purest available commercially. Luria-Bertani medium, Luria-Bertani agar, NZYCM agar, and NZYCM top agar were obtained in capsule form from Anachem 20, Luton, United Kingdom, and made up as specified by the manufacturer. Nitrate esters. Research quantities of most nitrate ester explosives were provided by Fort Halstead and stored at 4°C. PETN, hexahydro-1,3,5-trinitro-1,3,5triazine (RDX), and 2,4,6-trinitrotoluene (TNT) were supplied as pure solids. GTN was supplied as an ethanolic solution (10%, wt/vol). The preparation of working solutions of GTN and the determination of concentrations from known extinction coefficients (8) have been described elsewhere (38). Ethylene glycol dinitrate (EGDN) was also supplied as an ethanolic solution (10%, wt/vol). Isosorbide dinitrate (ISDN) (as a 40% [wt/wt] solid preparation in lactose) and isopropyl nitrate (IPN) were obtained from Sigma and Aldrich (Gillingham, United Kingdom), respectively. Bacterial strains and plasmids. The isolation, maintenance, and culture conditions for growing A. radiobacter on broth or on defined minimal medium containing 1% (vol/vol) glycerol and 0.14 mM GTN as C and N sources, respectively, have been described elsewhere (38). Nitrate esters were added as ethanolic solutions to culture flasks to provide nitrogen for growth; the small amounts of ethanol thus introduced to culture media were considered to be insignificant in comparison with the much larger quantities of glycerol already present. Bacterial growth at 25°C was measured by monitoring the absorbance at 450 nm (A450) for basal salt media and the A540 for nutrient broth. For the preparation of fully induced cell extracts, cells were grown on basal salts-glycerol medium
* Corresponding author. Mailing address: School of Molecular and Medical Biosciences, University of Wales Cardiff, P.O. Box 911, Cardiff CF1 3US, United Kingdom. Phone: 44 1222 874188. Fax: 44 1222 874116. E-mail:
[email protected]. † Present address: Brixham Environmental Laboratory, ZENECA Ltd, Freshwater Quarry, Brixham, Devon TQ5 8BA, United Kingdom. 7796
VOL. 24, 1997
GLYCEROL TRINITRATE REDUCTASE
7797
TABLE 1. Summary of purification of GTN reductase Treatment
Vol (ml)
Total amt of protein (mg)
Total enzyme activity (units)
Sp act (U mg21)
Yield (%)
Purification
Stage 1 (crude extract) Stage 2 (affinity chromatography) Stage 3 (ion-exchange chromatography)
60 24 33.6
57.6 4.2 0.34
6.34 5.16 5.11
0.11 1.23 15.02
100 81.4 80.6
1 11.2 136.5
containing 1 mM GTN. Bacteria previously maintained on 0.14 mM GTN were adapted to 1 mM GTN by serial passage through several intermediate concentrations. The following strains were used in the gene cloning experiments: Escherichia coli JM109 [e142 (McrA2 recA1 endA1 gyrA96 thi-1 hsdR17 (rK2 mK1) supE44 relA1 D(lac-proAB) (F9, traD36 proAB lacIqZDM15)] (40), E. coli XL1-blue MRF9 [D(mcrA)182 D(mcrCB-hsdSMR-mrr)172 endA1 supE44 thi-1 recA gyrA96 relA1 la l2 (F9, proAB lacIqZDM15 Tn10 (Tetr))] (16), and E. coli SOLR [e14(mcrA) D(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac (F9, proAB lacIqZDM15 Tn10 (Tetr))] (15). The cloning vectors used were pCR Blunt, pGEM-T vector, Lambda ZapII, and pBS. Assay of GTN reductase activity. Standard assay conditions to determine GTN reductase activity consisted of 0.5 ml of 0.2 mM GTN, 0.1 ml of 1 mM NADH, and 0.4 ml of cell extract, or multiples thereof. All substrate, cofactor, extract, and enzyme solutions were prepared in 50 mM phosphate buffer (pH 6.5) unless otherwise stated. GTN reductase activity was monitored by detecting the liberation of nitrite from GTN by a microtiter plate assay described previously (38). One unit of activity was defined as the amount of enzyme required to release 1 mmol of nitrite per min from GTN under the standard assay conditions. For determination of kinetic constants, the reaction conditions were the same as those of the standard assay except that the concentration of NADH was fixed at 0.1 mM and the concentration of GTN varied between 0 and 0.2 mM. The rate of NADH oxidation was determined for each concentration of GTN from changes in the A340. Total nitrite release was also determined at the end of the incubation period to demonstrate stoichiometry with the limiting substrate (GTN or NADH). Quantitative determination of GTN and its metabolites. Nitrate esters were analyzed by high-pressure liquid chromatography (HPLC) (Dionex DX300 series) as described previously (38), with a 10-mm bead size Lichrosorb C18 column eluted with a water-methanol gradient from 5 to 50% (vol/vol) methanol in water, over a 30-min period. Nitrate esters were detected in the column effluent and quantified by measurement of the A217 (LDC/Milton Roy detector). The system was calibrated with standard solutions of GTN, whose concentrations were determined from UV absorbance measurements with known extinction coefficients (8). The GTN calibration curve was adapted for use with the other esters by scaling the absorbance axis according to the relative extinction coefficients. Protein assay. Protein concentrations in solution were measured by the Bradford method (3) with bovine serum albumin standards. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was based on the method of Laemmli (18) with gels containing 10% (wt/vol) polyacrylamide, 1% SDS, and 0.375 M Tris-HCl (pH 8.8). The stacking gels contained 4% acrylamide in 0.126 M Tris-HCl (pH 6.8). Electrophoresis buffer contained 0.192 M glycine, 0.025 M Tris base, and 0.1% (wt/vol) SDS. After electrophoresis for 1 h at 100 V, the gels were fixed, stained for 1 h in Coomassie brilliant blue solution (0.025% [wt/vol] in 50% [vol/vol] aqueous methanol containing 7.5% [vol/vol] glacial acetic acid), and destained in 5% (vol/vol) aqueous methanol containing 7.5% (vol/vol) glacial acetic acid. Purification of GTN reductase. (i) Stage 1. Preparation of the cell extract. GTN-grown cells of A. radiobacter from a 3-liter culture in the late exponential phase of growth were harvested by centrifugation, washed, and resuspended in 60 ml of 50 mM phosphate buffer (pH 6.5). The ice-chilled cells were ruptured by passage three times through a French pressure cell (American Instrument Co., Bethesda, Md.) operated at 126 MPa. Residual whole cells and cell debris were removed by centrifugation (7,800 3 g for 1 h at 4°C). (ii) Stage 2. Affinity chromatography. Freshly prepared cell extract was applied to a 5-ml HiTrap Blue column preequilibrated in 50 mM phosphate buffer (pH 6.5). The column was eluted with 20 ml of the same buffer to remove unbound proteins and then with buffer containing 0.5 M NaCl and 0.5 mM NADH to elute bound proteins. Fractions (2 ml) were collected and assayed for GTN reductase activity and protein. The two highest-activity fractions were pooled, made up to 5 ml with 50 mM phosphate buffer (pH 6.5), and desalted in two 2.5 ml batches on a PD10 column. (iii) Stage 3. Ion-exchange chromatography. Stage 2 desalted protein was applied to a Mono Q-Sepharose 5/5 column, equilibrated with 50 mM phosphate buffer (pH 6.5) in a Pharmacia fast protein liquid chromatography (FPLC) system, at a flow rate of 1 ml min21. After the column was washed with buffer for 15 min to remove unbound material, a buffered salt gradient (0 to 0.4 M NaCl for
35 min) was used to elute the bound proteins. Fractions (1 ml) were assayed for GTN reductase, and the active fractions 19 and 20, eluting at 50 mM NaCl, which also contained a single protein peak (A280), were pooled and desalted on a PD-10 column. The purification protocol is summarized in Table 1. Determination of the relative molecular mass of the protein. Samples of enzyme (10 mg) were loaded onto a Superose-12 HR 10/30 FPLC gel exclusion column preequilibrated with 100 mM NaCl in 50 mM phosphate buffer (pH 6.5) in a Pharmacia FPLC system operated at 0.5 ml min21. Elution of the molecular weight markers was measured by monitoring the A280, but for GTN reductase, which has few aromatic amino acid residues (see below), fractions (0.5 ml) of column effluent were analyzed for GTN reductase activity by the nitrite assay. The molecular mass standards were cytochrome c (12.4 kDa), carbonic anhydrase (29 kDa), bovine serum albumin (66 kDa), alcohol dehydrogenase (150 kDa), and amylase (200 kDa). The subunit size of the GTN reductase was determined by SDS-PAGE in gels containing 12.5% acrylamide with 5% stacking gels; the molecular mass standards (Amersham International Plc, Little Chalfont, United Kingdom) were myosin (220 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (66 kDa), ovalbumin (46 kDa), carbonic anhydrase (29 kDa), trypsin inhibitor (21.5 kDa), and lysozyme (14.3 kDa) and were prepared, used, fixed, stained, and destained as described above. Determination of the N-terminal sequence of NerA. GTN reductase was separated from any minor contaminants by SDS-PAGE with a 25 mM Tris-HCl–192 mM Tricine electrolyte running buffer (pH 9.2). Enzyme solution (15 ml, containing 1.41 mg), preboiled after being mixed with an equal volume of sample denaturation solution, was loaded into each of six wells. Tricine replaced glycine in the electrophoretic buffer to avoid interference with subsequent amino acid analyses. GTN reductase resolved by SDS-PAGE was transferred to a polyvinylidene difluoride membrane by electroblotting with a semidry blotter (Sartorius Ltd., Epsom, United Kingdom) in the presence of anode buffer 1 (0.3 M Tris, 20% [vol/vol] methanol), anode buffer 2 (0.025 M Tris, 20% [vol/vol] methanol), and cathode buffer 3 (0.025 M Tris, 20% [vol/vol] methanol, 6-amino-n-hexanoic acid). Electroblotting was carried out at 145 mA for 45 min. The blotted membrane was rinsed in distilled water for 5 min and stained with a freshly prepared 0.1% (wt/vol) Coomassie blue R-250 solution in 50% (vol/vol) methanol for 5 min. The blot was then destained for 2 min in methanol-acetic acid (5:1, vol/vol) before being rinsed thoroughly in distilled water. The membrane was air dried and stored at 220°C between two layers of Whatman 3M paper. Samples were sequenced, using automated Edman degradation, by B. Parten (University of Florida, Gainesville, Fla.) with an Applied Biosystems model 470A protein sequenator. DNA isolation. Genomic DNA was prepared from A. radiobacter with QiaTip 100 (Qiagen, Crawley, United Kingdom) as specified by the manufacturer. Plasmid DNA was prepared with Wizard Miniprep kits from Promega UK Ltd., Southampton, United Kingdom, and PCR products and restriction enzyme digestion fragments were excised from 1% (wt/vol) agarose gels and purified with the QIAquick gel extraction kit (Qiagen). Oligonucleotide primers. Primers for DNA amplification, supplied by Gibco BRL, Life Technologies, Paisley, United Kingdom, were nerA-D (GCI AAY CGI ATY GTI ATG GC), nerA-E (ATI SWI CCI CCR TAY TCR TC), and nerA-H (ACR TCI GGR TTI GCD ATR AA), where R 5 A or G, W 5 A or T, S 5 C or G, D 5 A or G or T, Y 5 C or T, and I 5 A or C or G or T. PCR amplification. A. radiobacter genomic DNA (100 ng) was used as the template for PCR amplification. The specific primer nerA-D, designed against the amino terminus of NerA, and one or other of the degenerate primers nerA-E or nerA-H were used to amplify the PCR products ner-DE and ner-DH, respectively, in a Techne Progene thermal cycler by denaturation at 94°C for 1 min, annealing at 47°C for 1 min, and extension at 72°C for 2 min for a total of 30 cycles in the presence of 1 mM MgCl2, 10 mM tetramethylammonium chloride, and 300 ng of each primer. After being visualized on 1% agarose gels, the PCR products were cloned directly into the pGEM-T vector. To confirm that the correct PCR products were being amplified, the DNA sequence was determined, translated, and checked against the known amino-terminal sequence established previously. Screening a genomic DNA library. An A. radiobacter genomic DNA library was constructed in the DNA cloning vector lambda ZapII (32), and approximately 12,000 unamplified clones were probed with [a-32P]CTP-labelled PCR products.
7798
SNAPE ET AL.
J. BACTERIOL.
FIG. 1. SDS-PAGE analysis of purified GTN reductase. Lanes: 1, molecular mass markers (see the text for details); 2, pure enzyme after Stage 3.
Hybridization was carried out at 65°C in buffer containing 63 SSC (13 SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 53 Denhardt’s solution, 0.5% SDS, and 50 mg of sonicated and denatured salmon sperm DNA per ml. The filters were washed under stringent conditions (0.53 SSC, 0.1% SDS) at 65°C for 30 min prior to autoradiography. DNA sequencing. DNA was sequenced with ABI PRISM dye terminator cycle sequencing kits from Applied Biosystems (Warrington, United Kingdom). Nucleotide sequence accession number. The nucleotide sequence data reported in this paper have been deposited in the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession no. Y13942.
enzyme preparation before assaying for GTN reductase activity. Catalytic properties. GTN reductase converted GTN rapidly to 1,3-GDN, with only small amounts of the 1,2-GDN isomer being formed (9:1). Nitrite liberation was exactly stoichiometric with the total production of dinitrates and with the disappearance of GTN (Fig. 2). Neither disappearance of the dinitrates nor production of the mononitrate isomers was observed. When samples of 1,3-GDN and 1,2-GDN isolated from spent culture medium as described previously (38) were incubated with pure enzyme under the standard conditions of the GTN reductase assay, there was no liberation of nitrite. The steady-state kinetics of the GTN reductase reaction, based on monitoring NADH oxidation at 340 nm, conformed to Michaelis-Menten kinetics and yielded an apparent Km for GTN of 22.7 mM at 0.1 mM NADH. From the measured Vmax and taking the relative molecular mass to be 39,637 Da (see below), with one active site per molecule, the kcat for GTN reductase was 487 min21. Nitrite analyses at the completion of each reaction period confirmed that the total amount of nitrite produced was equal to the total amount of limiting reactant initially present (NADH or GTN, depending on the assay mixture), confirming the 1:1 involvement of these substrates in the reaction catalyzed. In assay mixtures having GTN in excess, reaction curves for NADH disappearance were almost entirely linear and showed signs of divergence from zero-order kinetics only at very low absorbances corresponding to NADH concentrations of ,1 mM. The substrate specificity was assessed in relation to a range of nitrate esters and related compounds (Table 2). GTN reductase showed good activity toward PETN but much less activity toward ISDN and EGDN. IPN, RDX, and TNT were not substrates under the conditions used. Incubations of the purified GTN reductase in the presence of either NAD1 or NADH (0.1 mM) with ammonia, nitrate, or nitrite (each at 0.1
RESULTS Purification of GTN reductase. The previously demonstrated requirement of GTN reductase in crude extracts for NADcontaining cofactors (38) was exploited in the purification of the enzyme on Blue Sepharose, which relies on the dye binding at nucleotide binding sites for its selectivity. Enzyme in crude extracts adsorbed to the HiTrap Cibacron Blue column, and elution with buffer containing 0.5 M NaCl and 0.5 mM NADH yielded a 12-fold purification with approximately 80% recovery of enzyme activity (Table 1). This step offers the added advantage of 2.5-fold concentration of the enzyme. The enzyme was further purified from the pooled dialyzed samples by FPLC ion-exchange chromatography. After sample application and initial column washing to remove significant amounts of unbound protein, the GTN reductase activity was eluted at about 50 mM NaCl in a salt gradient as a single, sharp, symmetrical peak coincident with an identical protein peak. Pooled, desalted active fractions contained a single band on SDS-PAGE gels (Fig. 1). The GTN reductase purification protocol was repeated eight times. The final specific activity was 15.02 6 0.22 units mg21 (mean and standard deviation) and the final percent yield was 82.67% 6 0.63% (mean and standard deviation). The overall purification factors were about 140-fold. Partially purified and pure preparations of GTN reductase could be stored at either room temperature or 4°C without any significant loss in activity for 1 month or more. GTN reductase activity was lost by incubation with trypsin or by boiling the
FIG. 2. Stoichiometric conversion of GTN to its dinitrates and inorganic nitrite by GTN reductase purified from A. radiobacter. The standard enzyme assay with 0.1 mM GTN and 0.1 mM NADH was scaled up 10-fold to provide sufficient material for the standard nitrite assay and for HPLC analysis. Volumes (1 ml) were removed from the incubation mixture at 1-min intervals and boiled rapidly to inactivate GTN reductase. Subsamples (0.1 and 0.9 ml) were assayed for nitrite by the standard nitrite assay and for GTN and its metabolites by HPLC, respectively. Symbols: E, GTN; h, 1,3-GDN; ‚, 1,2-GDN; ƒ, nitrite.
VOL. 24, 1997
GLYCEROL TRINITRATE REDUCTASE
TABLE 2. Activity of GTN reductase toward various organonitro compoundsa Substrate
Sp act (mmol of nitrite mg21 min21)
GTN....................................................................................... 15.02 PETN..................................................................................... 3.75 ISDN...................................................................................... 0.45 EGDN ................................................................................... 0.02 IPN......................................................................................... 0 RDX ...................................................................................... 0 TNT ....................................................................................... 0 a Compounds were tested at 0.1 mM under the standard assay conditions. The relatively insoluble PETN and TNT were introduced into the assay mixture dissolved at 1 mM (i.e., 103 concentrated) in 50 mM phosphate (pH 6.5) containing 10% (vol/vol) acetone. Activity measurements were based on the amount of nitrite liberated.
mM) gave rise to no production (or in the last case, no disappearance) of nitrite. A number of inorganic metal ions were tested for their effects on the activity of GTN reductase with GTN in the standard enzyme assay. None of the metal ions tested (Zn21, Fe21, Cu21, Ni21, Mg21, and Mn21 as the sulfate salts and Co21 as its chloride), at 0.1 mM, gave rise to any change in GTN reductase activity. The requirement for NADH as the source of reducing power for GTN reductase activity has been established. Of the other cofactors tested, only NADPH gave rise to GTN reductase activity. The specific activity of GTN reductase obtained by the standard enzyme assay with NADPH replacing NADH was 1.3 mmol of NO22 min21 mg21, i.e., approximately 11-fold lower. No GTN reductase activity was observed in incubations in which NAD1, NADP1, FAD, FADH, or oxidized or reduced glutathione replaced NADH. Incubations with either oxidized or reduced glutathione in the presence of NADH did not result in any change in the observed specific activity for GTN reductase. GTN reductase activity increased as the assay incubation temperature was raised in the range 10 to 30°C but decreased at higher temperatures and had disappeared by 50°C. When samples of GTN reductase, preincubated at 10, 15, 20, 30, or 35°C for 10 min, were subsequently assayed at 25°C (the standard assay incubation temperature), the GTN reductase activities observed were restored to approximately 15 mmol of NO22 min21 mg21. However, GTN reductase preincubated at 45 or 50°C for 10 min retained 20 and 0% of the original activity, respectively, when assayed subsequently at 25°C. Thus, the temperature profile was a composite of Arrhenius activation of catalytic activity up to about 35°C and thermal denaturation at higher temperatures. Optimal GTN reductase activity was observed at pH 6.5. The enzyme activity was much more sensitive to decreases in pH below this value than to increases above it. Thus, decreasing the pH by 0.5 unit to 6.0 resulted in a loss of 60% of the optimum activity (i.e., 12% per 0.1 pH unit) whereas raising the pH by 1.5 units caused a decrease of about 37% (2.4% per 0.1 pH unit). Molecular size. The molecular mass of the native GTNreductase determined by gel permeation chromatography was 30.7 kDa, and the subunit molecular mass determined by SDSPAGE was 36 to 40 kDa in replicate experiments (an example is given in Fig. 1), indicating a monomeric protein structure. N-terminal analysis and design of PCR primers. The Nterminal 35-amino-acid sequence of pure GTN reductase was (uncertain identities in parentheses) T(E/S)LFEPAQAGDIA
7799
LANRIVMAP(R/I/V)TRN(R/L)SPG(A/R/L)IPNN. The first 20 amino acids in the sequence were the most reliably determined, and these were compared to known protein sequences in the TREMBL database with the Basic Local Alignment Search Tool (BLAST [1]) from the Genetics Computer Group. Because the amino acid residue present at position 2 of the NerA sequence was uncertain, two sequences were searched, containing glutamic acid (E) and serine (S) at this position in sequences J2E and J2S, respectively. J2E showed homology to old yellow enzyme OYE3 (an NADPH-dependent flavin oxidase) isolated from Saccharomyces cerevisiae (25) (70% similarity and 55% identity) and to morphinone reductase isolated from Pseudomonas putida (11) (70 and 45%) and slightly less homology to a bile acid-inducible NADH:flavin oxidoreductase from a Eubacterium sp. (10, 19) (55 and 45%) and to old yellow enzyme KYE1 from Kluyveromyces lactis (22) (65 and 40%). For J2S, the identities were the same as for J2E, but the similarities for the old yellow enzymes were reduced by 5% in each case. The N-terminal sequence of NerA was reverse translated, and the sequence corresponding to the peptide ANRIVMA (residues 15 to 21 inclusive) was chosen to design a NerA-specific primer (nerA-D [see Materials and Methods]) for PCR amplification. This region was chosen for two main reasons: (i) the first 20 residues were considered to be the most accurate, and (ii) the end sequence MA reverse translates to ATG GCN, which, with the end base removed, yields a nondegenerate sequence ATGGC, thus making the primer more specific at its 39 end. Proteins homologous to NerA at their N termini contained two further conserved sequences (DEYGGSI and FIANPDL), and these were used to design further oligonucleotide primers designated nerA-E and nerA-H, respectively (see Materials and Methods). Cloning and sequence analysis of nerA. Primers nerA-D and nerA-E were used to amplify a PCR product of approximately 560 bp (as estimated by agarose gel electrophoresis), which was cloned into the pGEM-T vector (Promega). The identity of the DNA was confirmed by nucleotide sequence analysis. This insert was excised from the agarose gel, labelled with [a-32P] CTP, and used to screen an A. radiobacter genomic DNA library. A clone containing a 1.5-kb EcoRI-XhoI insert was isolated, and its nucleotide sequence was determined. The gene was found to contain an internal XhoI site, and therefore this initial screen isolated only part of the gene. The remaining 39 region of nerA was isolated from the same library with a PCR-generated probe derived from primers nerA-D and nerA-H. The full nucleotide sequence of nerA is shown in Fig. 3. The nerA coding region spans bp 1192 to 2307 (371 amino acids; molecular mass, 39,637 Da). The predicted amino acid sequence and those of similar proteins are shown in Fig. 4. In addition to the nerA coding region, another open reading frame (ORF1) is present in this sequence (bp 838 to 1068), and the predicted primary amino acid sequence shows similarity to the ArsR family of transcriptional regulators (26) (34% identity to a Synechocystis ArsR homolog [accession no. D90909] [17]). DISCUSSION The final GTN reductase purification protocol was quick and provided a consistently high yield of pure GTN reductase. Reproducible 140-fold purifications indicated that bacterial GTN reductase therefore represented approximately 0.7% of the total cell protein in crude extracts of A. radiobacter grown under the indicated conditions. The induced enzyme is thus a
7800
SNAPE ET AL.
J. BACTERIOL.
FIG. 3. DNA sequence of nerA and its flanking regions, and the deduced amino acid sequence of GTN reductase and the ORF1 product. Putative transcriptional promoters (capitals), ribosome binding sites (italics), transcription termination sites (` ), and regions of dyad symmetry (underlined) are shown.
very significant component in the cellular protein, and this situation is similar to that obtained for other inducible enzymes (5, 20, 31). In many respects (Mr, pH optimum, Km, and kcat for GTN reduction), GTN reductase closely resembled PETN reductase from E. cloacae PB2 (13), but on the other hand, the enzymes differed significantly in their preferences for NADH and NADPH, respectively. The NADH/NAD1 couple is involved almost exclusively in catabolic pathways leading to the generation of ATP, whereas NADPH/NADP1 is used mainly in biosynthetic pathways. The possession of the extra phosphate group as the sole distinguishing feature is critical in allowing enzymes to distinguish between the two pools of cofactors. It is therefore somewhat surprising to find that such similar enzymes fall into these distinct categories. The sequence of nerA (Fig. 3) contains an open reading frame beginning with the codons encoding the known N-terminal amino acid sequence of GTN reductase. The full deduced sequence corresponds to 371 amino acids, with an Mr of 39,637. This is consistent with the estimate of the subunit size of 36 to 40 kDa from SDS-PAGE. The deduced protein sequence of GTN reductase was compared with those in protein (SwissProt) and nucleic acid (GenBank and EMBL) sequence databases with the BLAST program. Proteins with similar structures were, in order of sequence identity, N-ethylmaleimide reductase from E. coli (67.7% similarity and 50.1% identity) (23), PETN reductase
from E. cloacae PB2 (68.8 and 48.9%) (13), morphinone reductase from P. putida M10 (68.0 and 48.3%) (11, 12), and old yellow enzyme OYE3 from S. cerevisiae (61.5 and 42.1%) (25). These proteins are all members of the old yellow enzyme subfamily of the a/b-barrel flavoprotein oxidoreductases, in that they possess the a/b-barrel domain but lack fusions either at the N or C terminus to accessory domains supporting the electron transfer processes which follow catalytic events involving flavin at the active sites of enzymes such as trimethylamine dehydrogenase (27). The sequence alignments are shown in Fig. 4. Old yellow enzyme OYE3 has been shown (9) to be an a/b-barrel structure in which the side chains of residues (numbered discounting the start methionine) Thr-37, Gln-114, Arg243, and Arg-348 and peptide bonds adjacent to Gly-324 and Gly-347 are hydrogen bonded to the flavin. Phe-374 and Tyr375 are also involved in forming a hydrophobic pocket. All of these residues, which are conserved in morphinone reductase and PETN reductase (13), are also conserved in GTN reductase (Fig. 4), except Arg-348, which undergoes the conservative change to lysine. Although the true function of old yellow enzyme is still unknown, its observed activity as an NADPH oxidase relies on its ability to bind NADPH by its “stacking” onto the flavin (9) through recognition of the nicotinamide mononucleotide portion. Similarly, in human glutathione reductase (and probably the corresponding E. coli enzyme too), NADPH is bound in a clearly delineated functional domain comprising the character-
VOL. 24, 1997
FIG. 4. Alignment of the deduced amino acid sequences of GTN reductase (NerA), N-ethylmaleimide reductase (Nem), PETN reductase (Onr), morphinone reductase (MorB), and old yellow enzyme (OYE3). The alignment was generated with the PILEUP program (Genetics Computer Group) with a gap weight of 5 and a gap length weight of 0.3. Boldface type, conserved residues involved in flavin-binding; bold-underlined type, Gly-X-Gly-X-X-Gly fold motif; bold-underlined-italic type, possible 29-phosphate binding site for NADPH. Numbers refer to the positions in each sequence, discounting the initiating methionine. The underlined sequence of 20 amino acids at the N-terminal end of NerA, determined by automated Edman degradation, was the sequence used initially to search the database for other proteins showing structural similarity in this region.
istic dinucleotide binding fold typical of NAD1-linked dehydrogenases. Here, too, the nicotinamide moiety stacks with the enzyme-bound flavin and makes additional contacts with the protein (28). Presumably, similar features operate in dinucleotide binding of the nitrate ester-reducing enzymes from E. cloacae and A. radiobacter, but the reasons for their different dinucleotide requirements remain obscure. NADH/NAD1 binding domains of many enzymes center around a bab fold containing a highly conserved Gly-X-Gly-X-X-Gly sequence, which introduces a tight turn between the first b strand and the a helix. The first glycine residue facilitates the tight turn, the second allows the dinucleotide to bind in the fold without steric hindrance from side chains at this position, and the third creates space for close interaction of the a and b components (39). This motif is fully conserved in GTN reductase (G-222, G-224, and G-227 [Fig. 4]) but not in the other homologous proteins. The first and third glycines are conserved in N-ethylmaleimide reductase, morphinone reductase, and old yellow
GLYCEROL TRINITRATE REDUCTASE
7801
enzyme, but only the third is present in PETN reductase. Dinucleotide binding sites (39) also include a hydrophobic core composed of six small residues at positions 24, 22, 19, 112, 120, and 122 relative to the first glycine. GTN reductase contains such residues but at shifted positions (24, 21, 18, 111, and 120). Similarly, a negatively charged residue expected to be present 23 to 25 amino acids downstream from the first glycine is present in GTN reductase as an Asp residue 21 amino acids downstream. The presence of the Gly-X-Gly-X-X-Gly sequence (and some other elements of the dinucleotide binding fold) in GTN reductase but not in PETN reductase may relate to the observed preferences for the NADH and NADPH cofactors, respectively. Site-directed mutagenesis of glutathione reductase (28) from E. coli has shown that positive charges on Arg-198 and Arg-204 make important contributions to the binding of NADPH via ionic interaction with the 29-phosphate group. PETN reductase contains arginines with this spacing (Arg-286 and Arg-292), although it lacks any Gly-X-Gly-X-XAla anticipated about 20 residues upstream for NADP binding proteins (28). Interestingly, in the GTN reductase sequence in this region, these Arg residues are absent. This is consistent with the observed replacement of arginine in NADPH binding proteins by glutamate in NADH binding enzymes (28). Thus, while GTN reductase and PETN reductase display some of the structural features hitherto associated with NADH and NADPH binding proteins, respectively, neither conforms fully. The otherwise close similarity of these enzymes thus presents a good opportunity to explore further the structural features dictating dinucleotide preference. In addition to the structural gene, the associated DNA sequence contains putative transcriptional control regions (promoters and terminators) and an open reading frame (ORF1) which precedes nerA (Fig. 3). The predicted primary amino acid sequence encoded by ORF1 shows similarity to a range of transcriptional regulatory proteins, and, given the proximity of ORF1 to nerA, it is possible that the gene product plays a role in the transcriptional control of nerA. This and most previous studies (2, 13, 38) have shown that the initial step in the bacterial denitration of GTN is reductive, yielding nitrite as the first-released nitrogenous product. In fungi, different mechanisms operate, possibly involving glutathione-S-transferase and hemoprotein-nitric oxide adducts (see reference 36 for a review), but nevertheless with a reductive theme. Recently, GTN was shown (4) to be completely mineralized without the accumulation of nitrate or nitrite under strictly anaerobic conditions by mixed cultures from an anaerobic digester, in keeping with the thermodynamic predictions of Smets et al. (33). In contrast, Meng et al. (21) reported that denitration of GTN occurred in dialyzed extracts of B. thuringiensis plus B. cereus and E. agglomerans in the absence of added cofactors. In light of the reductive pathways so far established, the implied hydrolytic scission in these strains becomes particularly intriguing. ACKNOWLEDGMENTS This work was supported by the Defence Evaluation and Research Agency and the Biotechnology and Biological Sciences Research Council, United Kingdom. REFERENCES 1. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic Local Alignment Search Tool. J. Mol. Biol. 215:403–410. 2. Binks, P. R., C. E. French, S. Nicklin, and N. C. Bruce. 1996. Degradation of pentaerythritol tetranitrate by Enterobacter cloacae PB2. Appl. Environ. Microbiol. 62:1214–1219. 3. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of
7802
4. 5. 6. 7.
8. 9. 10. 11. 12. 13. 14.
15. 16. 17.
18. 19. 20. 21.
SNAPE ET AL.
microgram quantities of proteins utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254. Christodoulatos, C., S. Bhaumik, and B. Brodman. 1997. Anaerobic biodegradation of nitroglycerin. Water Res. 31:1462–1470. Cloves, J. M., K. S. Dodgson, G. F. White, and J. W. Fitzgerald. 1980. Purification and properties of the P2 primary alkylsulphohydrolase of the detergent-degrading bacterium Pseudomonas C12B. Biochem. J. 185:23–31. Ducrocq, C., C. Servy, and M. Lenfant. 1989. Bioconversion of glyceryl trinitrate into mononitrates by Geotrichum candidum. FEMS Microbiol. Lett. 65:219–222. Ducrocq, C., C. Servy, and M. Lenfant. 1990. Formation of glyceryl 2mononitrate by regioselective bioconversion of glyceryl trinitrate: efficiency of the filamentous fungus Phanerochaete chrysosporium. Biotechnol. Appl. Biochem. 12:325–330. Dunstan, I., J. V. Griffiths, and S. A. Harvey. 1965. Nitric esters. II. Characterisation of the isomeric glycerol mononitrates. J. Chem. Soc. 120:1325– 1327. Fox, K. M., and P. A. Karplus. 1994. Old yellow enzyme at 2-Å resolution— overall structure, ligand-binding, and comparison with related flavoproteins. Structure 2:1089–1105. Franklund, C. V., S. F. Baron, and P. B. Hylemon. 1993. Characterization of the biaH gene encoding a bile acid-inducible NADH:flavin oxidoreductase from Eubacterium sp. strain VPI 12708. J. Bacteriol. 175:3002–3012. French, C. E., and N. C. Bruce. 1995. Bacterial morphinone reductase is related to Old-Yellow Enzyme. Biochem. J. 312:671–678. French, C. E., and N. C. Bruce. 1994. Purification and characterisation of morphinone reductase from Pseudomonas putida M10. Biochem. J. 301:97– 103. French, C. E., S. Nicklin, and N. C. Bruce. 1996. Sequence and properties of pentaerythritol tetranitrate reductase from Enterobacter cloacae PB2. J. Bacteriol. 178:6623–6627. Hall, D. R., P. S. Beevor, D. G. Campion, D. J. Chamberlain, A. Cork, R. D. White, A. Almestar, and T. J. Henneberry. 1992. Nitrate esters—novel sexpheromone components of the cotton leafperforator, Bucculatrix thurberiella Busck (Lepidoptera, Lyonetiidae). Tetrahedron Lett. 33:4811–4814. Hay, B., and J. M. Short. 1992. ExAssist™ helper phage and SOLR™ cells for Lambda ZAP® II excissions. Strategies 5:16–18. Jerpseth, B., A. Greener, J. M. Short, J. Viola, and P. L. Kretz. 1992. XL1-blue MRF9 E. coli cells: McrA2, McrCB2, McrF2, Mrr2, HsdR2 derivative of XL1-blue cells. Strategies 5:81–83. Kaneko, T., S. Sato, H. Kotani, A. Tanaka, E. Asamizu, Y. Nakamura, N. Miyajima, M. Hirosawa, M. Sugiura, S. Sasamoto, T. Kimura, T. Hosouchi, A. Matsuno, A. Muraki, N. Nakazaki, K. Naruo, S. Okumura, S. Shimpo, C. Takeuchi, T. Wada, A. Watanabe, M. Yamada, M. Yasuda, and S. Tabata. 1996. Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Res. 3:109–136. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. Mallonee, D. H., W. B. White, and P. B. Hylemon. 1990. Cloning and sequencing of a bile acid-inducible operon from Eubacterium sp. strain VPI 12708. J. Bacteriol. 172:7011–7019. Matts, P. J., G. F. White, and W. J. Payne. 1994. Purification and characterization of the short-chain alkylsulphatase of Coryneform B1a. Biochem. J. 304:937–943. Meng, M., W. Q. Sun, L. A. Geelhaar, G. Kumar, A. R. Patel, G. F. Payne,
J. BACTERIOL.
22.
23. 24. 25. 26. 27. 28. 29. 30. 31.
32. 33. 34. 35. 36. 37.
38. 39. 40.
M. K. Speedie, and J. R. Stacy. 1995. Denitration of glycerol trinitrate by resting cells and cell extracts of Bacillus thuringiensis/cereus and Enterobacter agglomerans. Appl. Environ. Microbiol. 61:2548–2553. Miranda, M., J. Ramirez, S. Guevara, L. Ongay-Larios, A. Pena, and R. Coria. 1995. Nucleotide sequence and chromosomal localization of the gene encoding the Old Yellow Enzyme from Kluyveromyces lactis. Yeast 11:459– 465. Miura, K., Y. Tomioka, H. Suzuki, M. Yonezawa, T. Hishinuma, and M. Mizugaki. 1997. Molecular cloning of the nemA gene encoding the N-ethylmaleimide reductase from Escherichia coli. Biol. Pharm. Bull. 20:110–112. Murrell, W. 1879. Nitro-glycerine as a remedy for angina pectoris. Lancet i:80–81, 151–152, 225–227. Niino, Y. S., S. Chakraborty, B. J. Brown, and V. Massey. 1995. A new Old Yellow Enzyme of Saccharomyces cerevisiae. J. Biol. Chem. 270:1983–1991. Sanfrancisco, M. J. D., C. L. Hope, J. B. Owolabi, L. S. Tisa, and B. P. Rosen. 1990. Identification of the metalloregulatory element of the plasmid-encoded arsenical resistance operon. Nucleic Acids Res. 18:619–624. Scrutton, N. S. 1994. a/b barrel evolution and the modular assembly of enzymes: emerging trends in the flavin oxidase/dehydrogenase family. Bioessays 16:115–122. Scrutton, N. S., A. Berry, and R. N. Perham. 1990. Redesign of the coenzyme specificity of a dehydrogenase by protein engineering. Nature 343:38–43. Servent, D., C. Ducrocq, Y. Henry, A. Guissani, and M. Lenfant. 1991. Nitroglycerin metabolism by Phanerochaete chrysosporium: evidence for nitric oxide and nitrite formation. Biochim. Biophys. Acta 1074:320–325. Servent, D., C. Ducrocq, Y. Henry, C. Servy, and M. Lenfant. 1992. Multiple enzymic pathways in the metabolism of glyceryl trinitrate in Phanerochaete chrysosporium. Biotechnol. Appl. Biochem. 15:257–266. Shaw, D. J., K. S. Dodgson, and G. F. White. 1980. Substrate specificity and other properties of the inducible S3 secondary alkylsulphohydrolase from the detergent degrading bacterium Pseudomonas C12B. Biochem. J. 187:181– 190. Short, J. M., J. M. Fernandez, J. A. Sorge, and W. D. Huse. 1988. LambdaZap—a bacteriophage lambda-expression vector with in vivo excision properties. Nucleic Acids Res. 16:7583–7600. Smets, B. F., R. T. Vinopal, D. Grasso, K. A. Strevett, and B.-J. Kim. 1995. Nitroglycerin biodegradation: theoretical thermodynamic considerations. J. Energ. Mater. 13:385–398. Urbanski, T. 1965. Chemistry and technology of explosives, p. 32–212. PWNPolish Scientific Publishers and Pergamon Press, Warsaw, Poland. Wendt, T. M., J. H. Cornell, and A. M. Kaplan. 1978. Microbial degradation of glycerol nitrates. Appl. Environ. Microbiol. 36:693–699. White, G. F., and J. R. Snape. 1993. Microbial cleavage of nitrate esters: defusing the environment. J. Gen. Microbiol. 139:1947–1957. White, G. F., J. R. Snape, and S. Nicklin. 1995. Bacterial biodegradation of nitrate ester explosives, p. 43.1–43.11. In Pyrotechnics—basic principles, technology, application. 26th International Annual Conference of ICT. DWS Werbeagentur und Verlag GmbH, Karlsruhe, Germany. White, G. F., J. R. Snape, and S. Nicklin. 1996. Biodegradation of glycerol trinitrate and pentaerythritol tetranitrate by Agrobacterium radiobacter. Appl. Environ. Microbiol. 62:637–642. Wierenga, R. K., M. C. H. De Meyer, and W. G. M. Hol. 1985. Interaction of pyrophosphate moieties with a-helixes in dinucleotide binding proteins. Biochemistry 24:1346–1357. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119.
