From the
The flavinylation reaction products of wild-type and mutant
forms of trimethylamine dehydrogenases purified from Methylophilus
methylotrophus (bacterium W
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
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
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
W
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
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).
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
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.
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 W
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 (W
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 (
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.
Standard deviation and number of determinations are given in
parentheses. Numbers in italics represent expected mass. ND, not
determined.
Turnover numbers
(k
We thank Juliette Jacoby for skilled assistance with
amino acid analysis.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
A
) 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.
/
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 + H
O =
(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).
(
)
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).
A
) is present in the deflavo form.
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).
,
m
, rec A1, sup E,
end A1, hsd R17, gyr A96, rel A1,
thi,
(lac-pro AB)/F` tra D36, pro A
B
, 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.
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).
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.
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).
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.
A
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).
A
) 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.
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.
Table:
Molecular masses determined by electrospray mass
spectrometry for wild-type and mutant trimethylamine dehydrogenases
Table:
Kinetic parameters of wild-type and mutant
trimethylamine dehydrogenase
) are based on flavin content determined
spectrophotometrically (see ``Results'') rather than total
protein concentration. ND, no detectable activity.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.