©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Flavinylation Reaction of Trimethylamine Dehydrogenase
ANALYSIS BY DIRECTED MUTAGENESIS AND ELECTROSPRAY MASS SPECTROMETRY (*)

Leonard C. Packman, Martin Mewies, and Nigel S. Scrutton (§)

From the (1) Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The flavinylation reaction products of wild-type and mutant forms of trimethylamine dehydrogenases purified from Methylophilus methylotrophus (bacterium WA) and Escherichia coli were studied by electrospray mass spectrometry (ESMS). The ESMS analyses demonstrated for the first time that wild-type enzyme expressed in M. methylotrophus is predominantly in the holoenzyme form, although a small proportion is present as the deflavo enzyme. ESMS demonstrated that the deflavo forms of the recombinant wild-type and mutant enzymes are not post-translationally modified and therefore prevented from assembling with flavin mononucleotide (FMN) because of previously unrecognized modifications. The data suggest that the higher proportion of deflavo enzyme observed for the recombinant wild-type enzyme is a consequence of the higher expression levels in E. coli. Mutagenesis of the putative flavinylation base (His-29 to Gln-29) did not prevent flavinylation, but the relative proportion of flavinylated product was substantially less than that seen for the recombinant wild-type enzyme. No flavinylation products were observed for a double mutant (His-29 to Cys-29; Cys-30 to His-30), in which the positions of the putative flavinylation base and cysteine nucleophile were exchanged. Taken together, the data indicate that the assembly of trimethylamine dehydrogenase with FMN occurs during the folding of the enzyme, and in the fully folded form, deflavo enzyme is unable to recognize FMN. Results of site-directed mutagenesis experiments in the FMN-binding site suggest that following mutation the affinity for FMN during the folding process is reduced. Consequently, in the folded mutant enzymes, less flavin is trapped in the active site, and reduced levels of flavinylated product are obtained.


INTRODUCTION

Trimethylamine dehydrogenase (EC 1.5.99.7) is an iron-sulfur flavoprotein and a member of a growing family of enzymes, which contains flavin-binding / barrel domains (Raine et al., 1994; Scrutton, 1994). The enzyme is found in several methylotrophic bacteria (Kasprzak and Steenkamp, 1983) where it catalyzes the oxidative demethylation of trimethylamine ((CH)N + HO = (CH)NH + HCHO + 2H + 2e). The enzyme is responsible for the ability of methylotrophic bacteria to subsist on trimethylamine as the sole source of carbon. The crystal structure of trimethylamine dehydrogenase has been solved at 2.4 Å resolution (Lim et al., 1986), revealing a homodimeric structure comprising three domains viz. a larger amino-terminal 8-fold / barrel domain (about 380 residues) and two smaller domains at the COOH terminus of the enzyme. The covalently attached flavin, the iron-sulfur center, and the substrate-binding residues are located in the / barrel domain. The other two domains contain 5-stranded parallel -sheets flanked by helices and other structural elements and are similar in fold to the dinucleotide-binding domains of glutathione reductase (Lim et al., 1988). ADP is bound to this part of the enzyme, and it occupies a position equivalent to the ADP moiety of FAD in the FAD-binding domain of glutathione reductase. This observation has led to the proposal that the COOH-terminal domains of trimethylamine dehydrogenase are the vestigial remains of dinucleotide-binding domains, which were modified by a process of divergent evolution (Lim et al., 1988; Scrutton, 1994).

Most covalently linked flavins are attached to proteins at the 8a methyl of the flavin isoalloxazine ring via a tyrosine, histidine, or cysteine side chain (Singer and McIntire, 1984). Trimethylamine and dimethylamine dehydrogenase are unique in that the enzyme-bound FMN() is covalently linked to the protein via a 6S-cysteinyl FMN bond (Steenkamp et al., 1978a, 1978b; Kenney et al., 1978). Interestingly, a structural solution of a flavoprotein related to the subunits of bacterial luciferase has revealed that the C6 of the flavin is in covalent linkage with the C3 atom of myristic acid (Moore et al., 1993). In trimethylamine dehydrogenase, the isoalloxazine ring of the flavin is highly non-planar, which may be a consequence of flavinylation (Lim et al., 1986), and this non-planarity may facilitate the reductive half-reaction of the enzyme by raising the flavin reduction potential. We have shown recently that biological activity is retained on removal of the 6S-cysteinyl FMN link by directed mutagenesis of Cys-30 to Ala-30 (Scrutton et al., 1994), but the effect of mutation on the reductive half-reaction, as yet, has not been investigated in detail. The cloning of the tmd gene (Boyd et al., 1992) has enabled trimethylamine dehydrogenase to be expressed in the heterologous host Escherichia coli (Scrutton et al., 1994). Flavinylation of the recombinant enzyme occurs in E. coli, albeit at reduced levels, demonstrating that flavinylation is autocatalytic, and a hypothetical mechanistic pathway for the reaction has been proposed (Scrutton et al., 1994).

The reductive half-reaction of trimethylamine dehydrogenase and the transfer of electrons between redox cofactors within the enzyme have been the subject of intensive research (Rohlfs and Hille, 1991; Rohlfs and Hille, 1995; Wilson et al., 1995), and a detailed picture of the enzyme chemistry is beginning to emerge. The mechanism by which the flavin cofactor is linked to each polypeptide in the enzyme dimer, however, has been less rigorously studied.

We report here a study by electrospray mass spectrometry of the products of the flavinylation reaction of wild-type and mutant trimethylamine dehydrogenases expressed in E. coli. Three mutant enzymes were studied: mutant C30A, in which the cysteine residue that forms the 6S-cysteinyl FMN link in the wild-type enzyme is replaced by alanine; mutant H29Q, in which the putative flavinylation base is replaced by glutamine; and a double mutant, H29C,C30H, which was designed to investigate the effects on flavinylation of switching the positions of these neighboring residues. Our results suggest that capture of the flavin in the active site occurs during the folding of the enzyme in vivo. We demonstrate that deflavo trimethylamine dehydrogenase produced in vivo is ``locked'' and cannot be reconstituted with FMN. The locked form is indistinguishable in terms of mass from the deflavo enzyme produced in vitro, which can assemble reversibly with FMN. We show that the putative flavinylation base, His-29, is not absolutely required for flavinylation but that its removal by directed mutagenesis may affect the recognition of FMN in the folding process. For the first time, we have also demonstrated that a small proportion of the trimethylamine dehydrogenase purified from the native host Methylophilus methylotrophus (bacterium WA) is present in the deflavo form.


EXPERIMENTAL PROCEDURES

Materials

Complex bacteriological media were from Difco Laboratories, and all media were prepared as described by Sambrook et al.(1989). Ethidium bromide was purchased from Bachem. Ultrapure agarose and CsCl were from Life Technologies, Inc. Trimethylamine, dicyclopentadienyliron (ferrocene), sodium hexafluorophosphate, 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine (ferrozine), riboflavin, and ascorbic acid were from Sigma. Acetonitrile (HPLC far-UV grade) was from Rathburn (Walkerburn, Scotland). Ferricenium hexafluorophosphate was synthesized as described by Lehman et al.(1990). Timentin was from Beecham Research Laboratories. All other chemicals were of analytical grade wherever possible. Glass-distilled water was used throughout except for HPLC work, where MilliQ-purified water was used (Millipore, Watford, UK).

Restriction enzymes EcoRI and BamHI were purchased from Pharmacia Biotech Inc. Calf intestinal alkaline phosphatase was obtained from Boehringer Mannheim. T4 DNA ligase and T4 polynucleotide kinase were from Amersham International. E. coli strain JM109 (r, m, rec A1, sup E, end A1, hsd R17, gyr A96, rel A1, thi, (lac-pro AB)/F` tra D36, pro AB, lac i, lac Z M15) was from Stratagene.

Mutagenesis, Plasmid Construction, and DNA Sequencing

Bacteria were cultured in 2 YT media supplemented, where appropriate, with timentin. Bacteriophage replicative DNA and plasmid DNA were prepared by CsCl density centrifugation (Sambrook et al., 1989). For the purposes of screening, plasmids were prepared on a miniscale using the alkaline lysis method (Sambrook et al., 1989). Restriction endonuclease digestion and ligation of DNA were carried out as recommended by the enzyme suppliers.

Site-directed mutagenesis was carried out on a derivative of M13 containing the coding strand of the tmd gene as described by Scrutton et al.(1994). The mutagenic oligonucleotides 5`-ATCGGATCCAGCACCGATACATTGCGGTACCTGGTA-3` (H29Q) and 5`-ATCGGATCCAGCACCGATATGACACGGTACCTGGTA-3` (H29C,C30H) were annealed to single-stranded template, and mutants were isolated using the phosphorothioate method as marketed by Amersham International. Putative mutants were screened directly by dideoxynucleotide DNA sequencing (Sanger et al., 1980; Biggin et al., 1983) using the T7 system purchased from Pharmacia. All mutant genes were fully resequenced to ensure spurious mutations had not arisen during the mutagenesis reactions. The construction of the C30A mutant has been described elsewhere (Scrutton et al., 1994). The H29Q and H29C,C30H mutant genes were subcloned as mutagenized EcoRI/BamHI cassettes, which replaced the corresponding wild-type cassette in the expression construct pSV2tmdveg (Scrutton et al., 1994).

Purification and Characterization of Wild-type and Mutant Enzymes

Recombinant enzymes were expressed from the appropriate pSV2tmdveg plasmid-based system in E. coli strain JM109. Enzyme preparations, analysis of iron, and ADP content in wild-type and mutant enzymes were performed as previously described (Scrutton et al., 1994). Wild-type trimethylamine dehydrogenase was purified from M. methylotrophus (WA) as described by Steenkamp and Mallinson (1976) and modified by Wilson et al.(1995). Protein concentrations were determined by performing amino acid analysis of hydrolysates using a Pharmacia Alpha plus II analyzer. The samples were hydrolyzed with gas-phase 6 M HCl containing 0.2% 2-mercaptoethanol and 100 µM of phenol for 24 h in vacuo. Protein samples were submitted to SDS-polyacrylamide gel electrophoresis (Laemmli, 1970) in 12.5% Phast gels (Pharmacia) using the Phast system marketed by Pharmacia, and protein was visualized by staining with Coomassie Brilliant Blue R250. Each recombinant enzyme was purified three times, and flavin content was determined spectrophotometrically for each sample (see Results).

Wild-type and mutant trimethylamine dehydrogenases were assayed using the artificial electron acceptor ferricenium hexafluorophosphate (Lehman et al., 1990). Assays were performed in 100 mM potassium phosphate buffer, pH 7.5, at 30 °C in a total volume of 1 ml. The final concentration of ferricenium hexafluorophosphate in the assay cuvette was 200 µM, and stock solutions were prepared in 10 mM HCl. Various concentrations of trimethylamine were used during the determination of kinetic parameters. Rates of reaction were monitored as the decrease in absorbance at 300 nm and calculated using an extinction coefficient at 300 nm of 4300 M cm for ferricenium hexafluorophosphate (Lehman et al., 1990). Fits to rate equations were performed using Kaleidograph software (Abelbeck Software, CA).

Electrospray Mass Spectrometry

Analyses of wild-type and mutant trimethylamine dehydrogenases by ESMS were performed using a VG BioQ triple quadrupole instrument (VG Biotech, Altrincham, UK) but using only the first analyzer. Capillary potential was 4 kV, and the instrument resolution control setting was 12.5. Purified samples of wild-type and mutant trimethylamine dehydrogenases were prepared for mass spectrometry by reverse phase HPLC using a Brownlee C column (2.1 30 mm) equilibrated with 0.1% trifluoroacetic acid. Protein samples (approximately 250 µg) in 20 mM potassium phosphate buffer, pH 7.5, 20% ethylene glycol were diluted 2-3-fold with water and acidified with trifluoroacetic acid to a final concentration of 1%. The trimethylamine dehydrogenase remained soluble. Following the injection of protein samples, trimethylamine dehydrogenase was eluted by employing a rapid gradient (0-70% in 10 min, 0.2 ml/min) of acetonitrile/0.1% trifluoroacetic acid. Protein samples prepared in this way were analyzed as soon as possible (within 48 h) by mass spectrometry. Samples from the HPLC fraction (containing 0.1% trifluoroacetic acid, approximately 40% acetonitrile) were injected directly into the mass spectrometer. The mobile phase of the instrument was 50% aqueous acetonitrile, flowing at 4 µl/min. Data were collected over a scan range of 1050-1250 Da/e (encompassing charge states 66+ to 77+) and 15 s/scan for 30-90 scans, depending on the signal strength. Calibration was by horse heart myoglobin. Data processing was by MassLynx software (VG Biotech), and each sample was transformed to the mass scale. Replicate measurements were taken from the transformed data.


RESULTS

Electrospray Mass Spectrometry of Native and Recombinant Wild-type Enzyme

Homogeneous preparations of trimethylamine dehydrogenase purified from M. methylotrophus (bacterium WA) were analyzed by ESMS. For the native enzyme, a major peak of mass 81,956 ± 5 Da and a minor peak of 81,500 ± 6 Da were obtained. The major peak corresponds to a mass of 81,951 Da expected for subunit coupled with FMN linked via the 6S-cysteinyl FMN bond. Neither the 4Fe-4S center nor ADP, which are associated with each subunit of trimethylamine dehydrogenase purified from M. methylotrophus, were associated with the sample in ESMS analysis. The minor peak obtained for the M. methylotrophus enzyme corresponds to a mass expected (81,498 Da) for deflavo trimethylamine dehydrogenase. This finding illustrates the power of electrospray mass spectrometry since it was previously thought trimethylamine dehydrogenase purified from M. methylotrophus contained a full complement of flavin. It is difficult to quantify precisely the amount of deflavo trimethylamine dehydrogenase in the sample by ESMS as it is not necessarily a quantitative technique, but since the apo- and holoenzymes are closely related, it is likely that their respective ionization efficiencies are similar. It is therefore reasonable to expect the relative abundances of the apo- and holo- forms in ESMS to reflect approximately their relative proportions in solution. Measuring the areas beneath the peaks of the transformed data (Fig. 1), ESMS suggests that about 5% of the purified enzyme lacks covalently bound flavin. This small amount is not easily detected by spectrophotometry and amino acid analysis, as the combined errors of the two measurements would approach 10%. It should be noted that, in principle, some loss of flavin could occur by a fragmentation mechanism in the nozzle-skimmer region of the mass spectrometer. However, the cone voltage used in these experiments (65 V) was the minimum necessary to produce a good signal and much lower than that commonly used (100V) in cone-voltage fragmentation studies. Certainly these conditions are mild enough not to break a disulfide linkage between mercaptoethanol and a surface cysteine of aldolase (Packman and Berry, 1995). It is therefore extremely unlikely that the stronger thioether bridge linking flavin to trimethylamine dehydrogenase would be susceptible to cleavage under these conditions.


Figure 1: Electrospray mass spectra, deconvoluted to the mass scale, of native wild-type (WT) and recombinant wild-type (rWT) trimethylamine dehydrogenase.



Recombinant trimethylamine dehydrogenase expressed from the cloned tmd gene in E. coli was also amenable to analysis by ESMS. In this case, the amount of deflavo trimethylamine dehydrogenase in the sample compared with fully flavinylated enzyme was increased (Fig. 1). Again, as seen for the native wild-type protein, the analysis of recombinant enzyme by ESMS indicated that this sample did not ionize and desorb with an intact 4Fe-4S center or ADP, although these latter cofactors are associated with the recombinant enzyme when purified from E. coli (Scrutton et al., 1994). The larger proportion of deflavo enzyme seen for the recombinant wild-type protein (approximately 25%) is in line with previous UV-visible and EPR spectral measurements, which suggested that recombinant wild-type trimethylamine dehydrogenase is about 30% flavinylated (Scrutton et al., 1994). The ESMS data have shown that the remainder of the sample (81,505 ± 2.1 Da) is bona fide deflavo trimethylamine dehydrogenase and that full flavinylation of the enzyme is not prevented by any detectable post-translation processing/modification of the enzyme sample. It therefore appears that the flavinylation process is functional in E. coli but that a larger proportion of the enzyme is isolated in the deflavo form. It is probable that the higher levels of expression in E. coli compared with the native enzyme expression drains the FMN pool more effectively, leading to a higher proportion of deflavo enzyme. Attempts to reconstitute the deflavo form of wild-type enzyme by adding FMN in the presence of 1 M potassium bromide were unsuccessful (Scrutton et al., 1994). The deflavo form is therefore ``locked'' with respect to reconstitution with FMN.

Analysis of Mutant Enzymes Altered in the FMN Binding Site

In previous work, we demonstrated that the C30A mutant of trimethylamine dehydrogenase when expressed in E. coli was assembled with a full complement of ADP and 4Fe-4S center (Scrutton et al., 1994). For various preparations of the C30A mutant, spectral analysis of FMN released from perchloric acid-precipitated enzyme indicated that only about 30% of the enzyme was assembled non-covalently with FMN, and the remainder of the sample was devoid of FMN. When analyzed by ESMS, the C30A mutant was identified as a single peak of mass 81,468.3 ± 2 Da, corresponding to subunit protein minus all cofactors (). As well as confirming that the flavin is non-covalently linked in this mutant protein, the data also indicate that, like the recombinant wild-type enzyme, the non-flavinylated protein is authentic deflavo enzyme (expected mass, 81,466 Da) and is not prevented from assembling with FMN because of any chemical modification/processing of the enzyme. The proportion of holoenzyme (25%) corresponds well with the proportion of flavinylated recombinant wild-type (30%) obtained from expression in E. coli. Given that the expression levels of the C30A enzyme and recombinant wild-type are similar in E. coli, this finding supports the view that free FMN is limiting. The C30A deflavo is also locked with respect to reconstitution with FMN (Scrutton et al., 1994).

ESMS analysis of the H29Q mutant and the double H29C,C30H mutant trimethylamine dehydrogenases suggested that these mutant proteins are predominantly present in the deflavo form or, alternatively, contain non-covalently bound FMN, which is lost in the ESMS analysis (). In each case, subunit masses corresponding to enzyme devoid of all cofactors were obtained. The detection of very small amounts of flavinylated enzyme against a high background of deflavo enzyme is difficult owing to the accumulation of salt adducts on the high mass side of the apoenzyme peak. Fig. 1 does show a likely holoenzyme peak beginning to resolve from the main peak of the H29Q mutant, but, taken alone, this would not be reliable evidence for the presence of holoenzyme in this case. Spectroscopic methods (see below) were therefore also employed to investigate further the presence/absence of FMN in the active sites of these proteins.

Spectral and Kinetic Characterization of the H29Q and H29C,C30H Mutant Enzymes

Steady-state activity determinations of the H29Q mutant enzyme indicated that the enzyme was able oxidatively to demethylate trimethylamine in the presence of the artificial electron acceptor ferricenium hexafluorophosphate. The kinetic behavior was unusual in that the enzyme displayed marked substrate inhibition (K= 1 mM ± 0.14). The K is about an order of magnitude greater than the wild-type protein, and k is approximately equal to that of the wild-type enzyme when the proportion of active enzyme (i.e. enzyme associated with flavin; see below) is taken into account (). The possession of activity indicates that a proportion of the H29Q mutant must bind FMN, either covalently or non-covalently. The H29C,C30H mutant enzyme was found to be devoid of catalytic activity, which suggests that, in agreement with the ESMS data, the enzyme does not bind FMN.

The ESMS data suggested that the H29Q and the H29C,C30H (as purified) mutant enzymes do not contain significant amounts of covalently attached FMN. The possibility exists, however, that either or both of the mutant proteins might assemble non-covalently with FMN. To address this question, various spectral characterizations of the mutant proteins were undertaken. The spectra of the H29Q, H29C,C30H, wild-type, recombinant wild-type and C30A trimethylamine dehydrogenases (as purified) are displayed in Fig. 2 . The most striking difference between the wild-type enzyme isolated from bacterium WA and the recombinant wild-type enzyme is a reduction in the extinction at around 450 nm and a shift to shorter wavelength for the recombinant protein. This spectral difference is wholly attributable to the larger proportion of deflavo enzyme present in the recombinant wild-type preparation, since the 4Fe-4S center and ADP cofactors are stoichiometrically associated with both the wild-type and recombinant wild-type proteins (Scrutton et al., 1994). The H29Q mutant shows a marked reduction in extinction at around 450 nm, which is suggestive of there being little FMN in the enzyme sample. The spectrum of the H29C,C30H mutant (as purified) closely resembles that of the C30A enzyme, in which the non-covalently attached FMN has been removed by exhaustive dialysis against 1 M potassium bromide (Scrutton et al., 1994). The implication is that the H29C,C30H mutant is purified in the deflavo form and, as a consequence, is devoid of any catalytic activity. The spectral changes observed for the H29Q and H29C,C30H mutant enzymes are dependent only on changes in the flavinylation state of these mutant enzymes. Iron and ADP titrations for all the enzyme preparations described here indicated that each sample was stoichiometrically associated with the 4Fe-4S cluster and ADP.


Figure 2: Absorption spectra of native and recombinant forms of trimethylamine dehydrogenase. Millimolar extinction values shown refer to a single active site. a, native wild-type trimethylamine dehydrogenase; b, C30A recombinant wild-type trimethylamine dehydrogenase; c, recombinant wild-type trimethylamine dehydrogenase; d, H29Q recombinant trimethylamine dehydrogenase; e, H29C,C30H recombinant trimethylamine dehydrogenase. Spectra were recorded for enzyme samples dissolved in 0.1 M potassium phosphate buffer, pH 7.5. Spectra for the wild-type enzymes and the C30A mutant are taken from Scrutton et al. (1994).



To corroborate the conclusions drawn from the ESMS and spectrophotometric data, the wild-type, recombinant wild-type, H29Q, and H29C,C30H enzymes were precipitated with 0.5 M perchloric acid to release any non-covalently linked FMN and ADP from the enzyme and also to disrupt the structure of the 4Fe-4S cluster. Precipitated protein was harvested by centrifugation, and the pellet was redissolved in 6 M guanidine hydrochloride contained in 0.1 M potassium phosphate buffer, pH 7.5. Spectral measurements of clarified supernatants indicated that none of the enzymes treated in this way released FMN into the supernatant. For the resolubilized protein pellets, and as previously reported for the wild-type proteins (Scrutton et al., 1994), FMN was found to be attached covalently to the native and recombinant wild-type enzymes (Fig. 3). The difference in extinction between the native wild-type and recombinant wild-type enzymes reflects the greater proportion of deflavo enzyme in the latter sample. A similar analysis revealed that no FMN was found linked to the H29C,C30H mutant, but a small amount of flavin (<5% (mol:mol) of that seen for native trimethylamine dehydrogenase) was linked to the H29Q enzyme (Fig. 3). The data, therefore, illustrate unequivocally that the H29Q trimethylamine dehydrogenase retains the ability to form the 6S-cysteinyl FMN link, but the mole ratio of flavin to protein is reduced by >6-fold. These results therefore support the tentative identification of holoenzyme in the H29Q mutant by ESMS analysis.


Figure 3: Absorption spectra of perchloric acid-precipitated native and recombinant trimethylamine dehydrogenase resolubilized in 6 M guanidine hydrochloride and 0.1 M potassium phosphate buffer, pH 7.5. a, native wild-type trimethylamine dehydrogenase; b, recombinant wild-type trimethylamine dehydrogenase; c, H29Q trimethylamine dehydrogenase; d, 10 expansion of spectra c. Spectra for the wild-type enzymes are taken from Scrutton et al. (1994).




DISCUSSION

In previous work, we demonstrated that expression of trimethylamine dehydrogenase from the cloned gene in a heterologous host (E. coli) yielded a mixed population of flavinylated enzyme and ostensibly deflavo enzyme (Scrutton et al., 1994). We also demonstrated that it was not possible to reconstitute the deflavo recombinant wild-type protein by adding excess FMN even in the presence of a mild chaotropic agent, e.g. KBr, a method that has been used to reconstitute many other flavoproteins (Massey and Curti, 1966). Like the recombinant wild-type enzyme, a C30A mutant, which lacks the 6S-cysteinyl FMN link, is also purified from E. coli as a mixed population of holo- and deflavo enzyme, and we were also unable to reconstitute the deflavo portion by adding FMN in the presence of KBr (Scrutton et al., 1994). We were, however, able to remove the non-covalently linked FMN in the holo- portion of the C30A mutant by dialysis against 1 M KBr and subsequently reconstitute the same portion of enzyme with FMN (Scrutton et al., 1994). It therefore appears that two classes of deflavo enzyme can be produced viz. that synthesized in vivo, which is refractory to reconstitution, and that generated in vitro by treatment of the holoenzyme with 1 M KBr, which can be reconstituted. In work presented here, we have addressed the question whether the deflavo enzyme synthesized in vivo is chemically altered in some way so as to prevent its reconstitution with FMN.

The results of the ESMS experiments clearly indicate that when the mixed holo/deflavo preparation of the C30A enzyme is analyzed, only one peak corresponding to the subunit mass is obtained. The data demonstrate that the in vivo synthesized deflavo enzyme is not post-translationally processed or modified and is identical (in terms of mass and within the resolution of the technique) to the in vitro generated deflavo enzyme. The reason for the difference in reconstitution behavior between the two forms of deflavo enzyme must therefore reside in some local changes in structure within the FMN-binding site, perhaps through misfolding in vivo of the deflavo enzyme. Any structural change must be small since the in vivo synthesized deflavo enzyme assembles with the 4Fe-4S center (which is only 6 Å from the FMN-binding site) and ADP, and in all other respects it is indistinguishable from the holoenzyme.

Similarly, the recombinant wild-type enzyme (as purified) also produces a bona fide deflavo enzyme peak in addition to the flavinylated enzyme peak in ESMS. The inability to reconstitute deflavo recombinant wild-type enzyme purified from E. coli probably also resides in local misfolding of the FMN-binding site.

The relatively high levels of deflavo recombinant wild-type and C30A enzyme purified from E. coli is likely to be a consequence of the high levels of expression in this host system. Many other flavoproteins overexpressed in E. coli are isolated as a mixture of deflavo and holoenzyme, and the conversion of deflavo to holoenzyme is easily accomplished by adding excess flavin. Since the deflavo form of trimethylamine dehydrogenase is locked, in that it cannot be reconstituted with FMN even in the presence of 1 M KBr, it is likely that flavinylation of the wild-type protein occurs as part of the folding process. The ESMS data has, for the first time, demonstrated that enzyme isolated from M. methylotrophus (WA) is also a combination of deflavo and holoenzyme. In this case, however, the proportion of deflavo enzyme is very low, which no doubt accounts for it not being identified in previous work. Importantly, the perchloric acid precipitation experiment on the wild type enzyme failed to release any flavin into solution; the apoenzyme detected in this preparation by ESMS must therefore represent genuine deflavo enzyme and not enzyme capable of binding flavin non-covalently. Because the level of expression in M. methylotrophus is modest (3% total cell protein as opposed to about 15% in E. coli), it is likely that the rate of synthesis and supply of FMN is sufficient to keep up with enzyme synthesis, and as a result only a very small proportion of enzyme becomes locked in the deflavo form.

Both the ESMS data and spectral analyses indicate that the H29C,C30H double mutant is devoid of flavin. The mutant was originally designed to test whether there was any flexibility in the positioning of the cysteine nucleophile (Cys-30) and putative flavinylation base (His-29). Perhaps the most surprising result is that, in addition to there being no flavinylated enzyme, flavin was found to be totally absent from the active site of the enzyme. One must conclude that the local structure in the FMN-binding site has been sufficiently disrupted to prevent FMN associating with the polypeptide during the folding process, and as a consequence flavin is not trapped in the fully folded enzyme.

A much reduced level of flavinylated enzyme was found for the H29Q mutant following the replacement of the putative flavinylation base, His-29, with a glutamine residue. The degree of flavinylation is significantly less (<6-fold) than that seen for the recombinant wild-type, and this cannot be attributed to higher levels of expression since all the recombinant proteins are expressed to the same level in E. coli. Like the other enzymes reported in this paper, various preparations of the H29Q mutant were made, and in each case the level of flavinylation was found to be only about 5% that seen for the native wild-type protein. A priori, the much reduced flavinylation levels of the H29Q mutant might be attributed to the effects of removing the putative flavinylation base, which is predicted to enhance the nucleophilicity of Cys-30 (Scrutton et al., 1994). However, an attractive and more likely alternative, especially in the light of the results obtained for the H29C,C30H double mutant, is that the formation of the FMN-binding site is affected during folding, and consequently less trapping of flavin in the active site occurs. Indeed, the finding that the total flavin occupancy in the H29Q mutant (as purified) (<5% mol:mol) is much less than either the recombinant wild type enzyme (30%) or the C30A mutant (30%) implies a weaker association of FMN with the partially folded polypeptide. As such, the H29Q mutant would be mid-way between the recombinant wild-type or C30A enzymes and the H29C,C30H double mutant in terms of binding affinity for FMN during folding. Following the trapping of FMN in the active site of the H29Q mutant, the mutation is not sufficient to arrest the flavinylation reaction. This is to be expected, since the presence of a flavinylation base would serve only to accelerate the reaction by enhancing the nucleophilicity of Cys-30, and after trapping of the flavin, the rate at which flavinylation proceeds would not affect the end product.

One ideally needs to develop a flavinylation assay to ascertain whether His-29 accelerates flavinylation following the trapping of FMN in the active site. A time-dependent flavinylation assay, which can be performed at different values of pH for the wild-type and H29Q mutant, would determine whether the rate of flavinylation was reduced as a consequence of removing the putative base. In future work, we will attempt to prepare reconstitutable (i.e.in vitro generated) deflavo wild-type and H29Q trimethylamine dehydrogenases. Flavinylation of these samples in the presence of 1 M KBr as a function of pH might then resolve the issue.

  
Table: Molecular masses determined by electrospray mass spectrometry for wild-type and mutant trimethylamine dehydrogenases

Standard deviation and number of determinations are given in parentheses. Numbers in italics represent expected mass. ND, not determined.


  
Table: Kinetic parameters of wild-type and mutant trimethylamine dehydrogenase

Turnover numbers (k) are based on flavin content determined spectrophotometrically (see ``Results'') rather than total protein concentration. ND, no detectable activity.



FOOTNOTES

*
This work was funded by the Leverhulme Trust and the Royal Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 44-223-333654; Fax: 44-223-333345.

The abbreviations used are: FMN, flavin mononucleotide; ESMS, electrospray mass spectrometry; HPLC, high pressure liquid chromatography.


ACKNOWLEDGEMENTS

We thank Juliette Jacoby for skilled assistance with amino acid analysis.


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