1 Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK
2 Department of Biomolecular Sciences, PO Box 88, UMIST, Manchester M60 1QD, UK
Correspondence
Howard Dalton
H.Dalton{at}warwick.ac.uk
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ABSTRACT |
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Present address: GlaxoSmithkline Plc, New Frontiers Science Park, Harlow, Essex CM19 5AW, UK.
Present address: Biomedical Research Centre, Sheffield Hallam University, Howard Street, Sheffield S1 1WB, UK.
Present address: Dept Clinical Pharmacology Q7642, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark.
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INTRODUCTION |
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Previous studies in our laboratory suggested that Mc. capsulatus (Bath) could oxidize formaldehyde to formate using a soluble formaldehyde dehydrogenase (FDH) that existed as a dimer of 57 kDa subunits, possessed NAD(P)+-dependent FDH activity and required a factor (apparently a low-molecular-mass protein) from heat-treated soluble extract (HTSE) (Stirling & Dalton, 1978). We conducted a subsequent investigation into the FDH of Mc. capsulatus (Bath) which suggested that the protein responsible for FDH activity was actually a homotetramer of 63 kDa subunits and that the factor in the HTSE required for activity with formaldehyde was a heat-stable 8·6 kDa protein, termed the modifin. The modifin preparation converted FDH from a general aldehyde dehydrogenase to a specific FDH and was thus a potential regulator of formaldehyde metabolism (Tate & Dalton, 1999
). Other reports have also indicated the presence of a membrane-associated FDH (Zahn et al., 2001
) and at least some of the enzyme activities that are necessary for oxidation of formaldehyde as a conjugate to tetrahydromethanopterin (H4MPT) (Vorholt et al., 1999
).
Here we report analysis of modifin-dependent FDH preparations similar to those which we prepared previously that show that the principal enzyme activities within these preparations are a quinoprotein MDH and a methylene tetrahydromethanopterin dehydrogenase (methylene H4MPT-DH), which is one of the enzymes of the H4MPT-dependent formaldehyde oxidation pathway (Vorholt et al., 1998).
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METHODS |
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Growth of bacteria.
Mc. capsulatus (Bath) was cultivated at 45 °C using methane (15 %, v/v, in air) as the growth substrate, in nitrate minimal salts medium (Dalton & Whittenbury, 1976) containing CuSO4.5H2O (0·5 or 1·5 mg l-1, as stated for each experiment) as the sole source of added copper. Cultures were grown in a 100 litre batch fermentation vessel or continuously, as described previously (Tate & Dalton, 1999
) except that the dilution rate was 0·05 h-1. Cells were harvested according to the published method (Tate & Dalton, 1999
) and stored at -80 °C.
Enzyme assays.
All enzyme assays were performed at 45 °C. Modifin/HTSE-dependent FDH assays were performed by spectrophotometric quantification at 340 nm of the production of NADH, as described by Tate & Dalton (1999). NAD+-dependent methylene H4MPT-DH activity was also monitored spectrophotometrically (Vorholt et al., 1998
). Dye-linked oxidation of methanol and formaldehyde was similarly measured at 600 nm by a modification of the method of Anthony & Zatman (1967)
, as follows. The enzyme sample was incubated in 50 mM Tris/HCl (pH 9·0) containing substrate (10 mM), NH4Cl (15 mM), KCN (5 mM) and dichloroindophenol (DCIP; 0·1 mM); the reaction was initiated by adding the mediator phenazine ethosulfate to 1·0 mM and the activity estimated from the decrease in absorbance at 600 nm due to reduction of DCIP, presuming an absorption coefficient of 1·91x104 M-1 cm-1.
Preparation of soluble extracts.
DNase A (to 20 µg ml-1) was added to the defrosted cell paste and the cells were broken by passing through a high-pressure cell disrupter (Constant Systems, Warwick, UK; 172 MPa, 4 °C). The broken cell suspension was centrifuged (150 000 g, 1·5 h, 4 °C) and the supernatant (the soluble extract) was removed. HTSE was prepared as described by Tate & Dalton (1999).
Partial purification of FDH (preparation FDH-1)
Step 1.
Ammonium sulfate was added to native soluble extract to 40 % saturation and the proteins that precipitated during a 30 min incubation on ice were removed by centrifugation (48 500 g, 15 min, 4 °C). The concentration of ammonium sulfate in the supernatant was adjusted to 60 % saturation and, after a further 30 min on ice, the precipitate, containing the FDH activity, was collected by centrifugation as above and resuspended in a minimal volume of buffer A (20 mM potassium phosphate buffer, pH 7·2, containing 1 mM benzamidine). The sample (23 ml) was then desalted into 5 column volumes of buffer A using a Pharmacia PD 10 desalt column equilibrated with the same buffer.
Step 2.
The sample was loaded onto a Pharmacia Mono Q anion exchange column (1·0 cmx10 cm) equilibrated with buffer A and then proteins were eluted with a linear gradient of 01 M NaCl in buffer A. Fractions containing NAD+-dependent FDH activity, which eluted in the void volume, were pooled and concentrated using an Amicon PM 30 ultrafiltration membrane.
Step 3.
The concentrated protein was loaded onto a Pharmacia Hi-Trap Blue affinity chromatography column (0·5 cmx1 cm) equilibrated with buffer A. Elution was effected using a linear gradient of 01 M NaCl in buffer A and fractions containing NAD+-dependent FDH activity, which again eluted in the void volume, were pooled and concentrated as above.
Step 4.
The protein was then subjected to size-exclusion chromatography using a Pharmacia Superdex 200 column (1·6 cmx70 cm) with a flow rate of 0·5 ml min-1 of buffer A. The active fractions, containing the partially purified FDH, were pooled and concentrated as above. This gave preparation FDH-1, which was drop-frozen in liquid nitrogen and stored at -80 °C.
General protein characterization.
Protein concentration was determined by using the Bradford assay reagent (Bio-Rad) according to the manufacturer's instructions. Bovine serum albumin was used as the standard. SDS- and native-PAGE were performed using the discontinuous buffer system of Laemmli (1970). Molecular masses of polypeptides analysed by SDS-PAGE were estimated by comparison with Dalton Mk VIIL standards (Sigma). Electroelution of protein samples from bands excised from polyacrylamide gels was performed in the gel-running buffer using a model 422 Electroeluter (Bio-Rad). Samples for N-terminal sequencing were subjected to SDS-PAGE and then electroblotted onto a PVDF membrane (Pharmacia-Amersham) and stained with Coomassie blue. Protein bands of interest were excised and the peptide sequence was determined using an Applied Biosystems 476A protein sequencer. Electrospray-mass spectrometry (ES-MS) was performed using a Quattro II QhQ tandem mass spectrometer (Micromass, Altrincham, UK) as detailed by Millar et al. (1998)
.
Equilibrium sedimentation.
Sedimentation equilibrium analysis of preparation FDH-1 was performed at 4 °C in a Beckman Optima XL-A analytical ultracentrifuge. Samples were diluted to 0·34, 0·17 and 0·04 mg ml-1 using 20 mM potassium phosphate buffer (pH 7·2). Each dilution (70 µl) was injected into a separate cell in the centrifuge rotor and the samples were centrifuged to equilibrium at 6000 and 9000 r.p.m. Scanning absorbance optics were used at wavelengths of 280 nm (for samples at 0·34 and 0·17 mg ml-1) and 223 nm (for samples at 0·04 mg ml-1). Sedimentation equilibrium was considered to have been achieved when a negligible difference between solute distributions was observed after a time interval of 3 h. After equilibrium at both rotor speeds, the rotor was accelerated to 45 000 r.p.m. to obtain a reading of non-redistributing absorbance, which was used as baseline reading in the analysis. The partial specific volume of the proteins and the density of the solvent were estimated from composition information using the program SEDNTERP (Laue et al., 1992). Solute distributions were analysed using non-linear least-squares fitting of exponential equations using the ORIGIN software package (MicroCal Software, Northampton, MA, USA). The data obtained were fitted to models that included association terms, in order to determine the stoichiometry of any intermolecular interactions.
Electron microscopy and single-particle analysis.
Samples were added to freshly glow-discharged (rendered hydrophilic) copper/carbon mesh grids for 30 s, the sides of which were then blotted with Whatman grade 50 filter paper, before negative staining in 4 % uranyl acetate (Rosenberg et al., 1997). Micrographs of the grids were taken with a Philips CM10 transmission electron microscope operating at 100 kV and scanned using a leaf microdensitometer (University of Leeds).
Particles from electron micrographs were selected using the SPIDER software package on an Indigo workstation (Silicon Graphics) and the densities normalized. Reference-free alignment was carried out and the results were statistically analysed by sorting particles into groups for averaging using hierarchical clustering (Holzenburg et al., 1994). Fourier ring correlation between two subaverages was used to assess the resolution. The point at which the Fourier ring correlation values dropped to 0·5, indicating higher resolution shells did not correlate significantly, was determined by fitting the data with a four-parameter Boltzman function (using ORIGIN). A threshold for each class that gave the best averages in terms of resolution was thus determined.
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RESULTS |
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The major protein components of preparation FDH-1 are identical to the components of FDH isolated previously
The major polypeptides of preparation FDH-1 were subjected to N-terminal sequencing and accurate mass analysis by means of ES-MS in order to investigate whether degradation of the proteins has occurred that might account for the low FDH activity. The N-terminal sequences of the 64 and 8 kDa species from preparation FDH-1 (Fig. 2a, b) were very similar to those found previously (Tate & Dalton, 1999
). The positions of the N termini in the two proteins were unchanged, which suggested that N-terminal degradation was not the cause of inactivation, although in the case of the 8 kDa moiety there was a discrepancy in the identification of the N-terminal residue. The molecular masses of the two major polypeptides in preparation FDH-1 were 63 615 and 8212·5 Da, which were identical to the masses of the major protein components of FDH and the modifin, respectively, determined previously (Tate & Dalton, 1999
). Consequently, the minor discrepancies in the N-terminal sequences could be put down to sequencing inaccuracies and degradation of the 64 and 8 kDa polypeptides could be discounted as a cause of the low NAD+-dependent FDH activity in preparation FDH-1. The mass spectrum of preparation FDH-1 also revealed a number of minor contaminants, including a polypeptide of 34 525 Da (data not shown).
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The 64 and 8 kDa polypeptides form a complex with the same 2
2 stoichiometry as known MDHs and similar molecular topology
Previously the native molecular mass of the major protein complex in FDH preparations was estimated at 250 kDa, based on gel filtration chromatography (Tate & Dalton, 1999). In order to obtain an accurate value for the native molecular mass that could be used to determine the stoichiometry of the complex, the molecular mass(es) of the protein complex(es) in preparation FDH-1 were determined in solution under native conditions by sedimentation equilibrium analysis. Analysis of residual plots after fitting of the experimental data from FDH-1 samples to specific models of association indicated that the data were best accounted for by a single species of molecular mass 140 600±1200 Da. Models which included self-association terms and/or non-interacting species did not describe the data well (data not shown). Assuming that this species is composed only of the 8 and 64 kDa polypeptides, the complex must have an
2
2 stoichiometry, with a molecular mass of 143 655 Da as calculated from the masses of the individual polypeptides determined by ES-MS. The slight discrepancy between sedimentation equilibrium results and the mass derived from the ES-MS data was attributed to inaccuracies in the estimation of solute partial specific volume and solvent density.
The topology of the major macromolecular constituents in the FDH preparations was investigated in electron micrographs of negatively stained particles of FDH by means of single-particle analysis. Two distinct species were identified by visual inspection of the micrographs. The first (species A) was small, thin and tubular; the second (species B) was broader and appeared to be a sandwich of the first. Particles of each species were separately selected by size into subpopulations before performing rotational and translational alignment. This process used an interactive procedure of cross-correlation and auto-correlation of images which were thereby (Frank, 1990) averaged to produce a refined image. Hierarchical cluster analysis of each aligned particle dataset (corresponding to species A and B, respectively) resulted in final averages from both datasets with two very different structures. The resolution of the species A image extends about 26 Å, and that of species B to 35 Å. Enlarged versions of the best averages of each species were compared with the 1·9 Å resolution crystal structure of the
2
2 tetramer of MDH from Methylophilus W3A1 (Xia et al., 1999
) (Fig. 3
). Species A was similar in size and shape to the MDH crystal structure, consistent with the proposal that the 64 and 8 kDa FDH components, like the subunits of MDH from Methylophilus W3A1, existed in an
2
2 configuration. Species B resembled a sandwich of two MDH heterotetramers and suggested that, under the conditions used for sample preparation, further aggregation of the
2
2 complex in preparation FDH-1 was possible.
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The FDH-1 preparations contain a methylene H4MPT-DH that may be involved in NAD+-dependent FDH activity
The observation that the 2
2 heterotetramer alone showed no NAD+-dependent FDH activity suggested that the enzyme(s) responsible for the NAD+-linked FDH activity must be among the minor contaminants in preparation FDH-1. The previous observation of enzyme activities associated with the H4MPT-dependent formaldehyde dissimilation pathway in cell-free extracts of Mc. capsulatus (Bath) (Vorholt et al., 1999
) suggested one possible explanation. In M. extorquens, in which this pathway is well characterized, the formation of methylene H4MPT is catalysed by a specific formaldehyde-activating enzyme (Vorholt et al., 2000
); however, formaldehyde and H4MPT also react spontaneously to form methylene H4MPT. Methylene H4MPT is the substrate for the NAD(P)+-dependent enzyme methylene H4MPT-DH (Chistoserdova et al., 1998
) and so, in the presence of H4MPT, methylene H4MPT-DH would oxidize formaldehyde in an NAD+-dependent manner. Moreover, since H4MPT is heat-stable (Romesser & Wolfe, 1982
) it, rather than the 8 kDa polypeptide, could be the active component of HTSE and the modifin that stimulates oxidation of formaldehyde. Consistent with this hypothesis, it was found that the NAD+-linked FDH activity of preparation FDH-1 increased from 167 nmol min-1 (mg protein)-1 to 23 000 nmol min-1 (mg protein)-1 when the HTSE in the assay was replaced by H4MPT (to 35 µM). Thus the FDH preparations contained an NAD+-dependent methylene H4MPT-DH and it was likely that H4MPT was at least one of the components of the modifin preparation that stimulated the NAD+-linked FDH activity.
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DISCUSSION |
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Our observation that the FDH preparation FDH-1, described here, contained a methylene H4MPT-DH showed that such an activity might be responsible for the NAD+-dependent FDH activity of preparation FDH-1 and offered a possible explanation for the FDH-stimulating activity of the HTSE and modifin preparations, by providing the heat-stable coenzyme H4MPT. These results confirm the previous observation of the activities of enzymes of the H4MPT pathway in Mc. capsulatus (Bath) (Vorholt et al., 1999) and demonstrate the importance of H4MPT as a C1-carrier during oxidation of formaldehyde by this methanotrophic bacterium.
The NAD+-dependent FDH activity observed by Tate & Dalton (1999), within a preparation that also contained principally the quinoprotein MDH, must likewise have been due to a minor enzyme in the preparation. It is possible that this was the same methylene H4MPT-DH observed during the present study; however, the complex kinetics, the effect of the modifin of inhibiting oxidation of aldehydes larger than formaldehyde and the effect of NAD+ in changing the spectral properties of the preparation cannot be explained by the model presented here. If H4MPT is indeed the active component of HTSE and the modifin preparations, the FDH activities in the presence of modifin and HTSE reported here and by Tate & Dalton (1999)
are overestimated by about 4·4-fold, because of the increase in A340 due to oxidation of methylene H4MPT to methenyl H4MPT (Vorholt et al., 1998
) that was not taken into account during calculation of these activities.
The available evidence suggests at least two routes via which formaldehyde can be oxidized in Mc. capsulatus (Bath): (1) via the H4MPT-dependent pathway; (2) by oxidation to formate by the membrane-associated FDH described by Zahn et al. (2001). It is also intriguing that the soluble FDH purified from Mc. capsulatus (Bath) by Stirling & Dalton (1978)
was clearly distinct from the enzymes identified in preparation FDH-1 because it used NAD+ as its electron acceptor but was dependent on a factor from HTSE which, since it was sensitive to trypsin, is unlikely to have been H4MPT. Thus, although the preparation that Tate & Dalton (1999)
classified as a soluble FDH is clearly principally composed of an MDH, the existence of a soluble NAD(P)+-dependent FDH in this organism cannot currently be excluded.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 13 August 2003;
revised 5 December 2003;
accepted 9 December 2003.
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