Thauer1and Holger Dau2 1 Max-Planck-Institut fu¨r terrestrische Mikrobiologie and Laboratorium fu¨r Mikrobiologie, Fachbereich Biologie der Philipps-Universita¨t, Marburg, Germany;2Freie
Trang 1The role of zinc in the methylation of the coenzyme M thiol group
in methanol:coenzyme M methyltransferase from
Methanosarcina barkeri
New insights from X-ray absorption spectroscopy
Markus Kru¨er1, Michael Haumann2, Wolfram Meyer-Klaucke3, Rudolf K Thauer1and Holger Dau2
1
Max-Planck-Institut fu¨r terrestrische Mikrobiologie and Laboratorium fu¨r Mikrobiologie, Fachbereich Biologie der Philipps-Universita¨t, Marburg, Germany;2Freie Universita¨t Berlin, Fachbereich Physik, Berlin, Germany;
3
DESY, EMBL Outstation, Hamburg, Germany
Methanol:coenzyme M methyltransferase from
methano-genic archaea is a cobalamin-dependent enzyme composed
of three different subunits: MtaA, MtaB and MtaC.MtaA is
a zinc protein that catalyzes the methylation of coenzyme M
(HS-CoM) with methylcob(III)alamin.We report zinc
XAFS (X-ray absorption fine structure) results indicating
that, in the absence of coenzyme M, zinc is probably
coordinated by a single sulfur ligand and three oxygen or
nitrogen ligands.In the presence of coenzyme M, one
(N/O)-ligand was replaced by sulfur, most likely due to
ligation of the thiol group of coenzyme M.Mutations in
His237 or Cys239, which are proposed to be involved in ligating zinc, resulted in an over 90% loss in enzyme activity and in distinct changes in the zinc ligands.In the His237fi Ala and Cys239 fi Ala mutants, coenzyme M also seemed to bind efficiently by ligation to zinc indicating that some aspects of the zinc ligand environment are sur-prisingly uncritical for coenzyme M binding
Keywords:
1 zinc enzymes; methanogenic archaea; methyl transferases; thiol group alkylation; EXAFS
Methanosarcina barkeriand other Methanosarcina species
can grow on methanol as carbon source which is
dispro-portionated to CH4 and CO2 [1].The first step in this
metabolic pathway is the formation of methyl-coenzyme M
(CH3-S-CoM) from methanol and coenzyme M (HS-CoM)
[2]
CH3OHþ HS-CoM !MtaABCCH3-S-CoM þ H2O ð1Þ
DG¢ ¼ )27.5 kJÆmol)1
The reaction is catalyzed by methanol:coenzyme M
methyltransferase which is composed of the three subunits
MtaA (35.9 kDa), MtaB (50.7 kDa) and MtaC (27.9 kDa),
of which MtaC is a corrinoid protein.They catalyze the
following partial reactions [3–7]
CH3OHþ MtaC *)MtaB CH3-MtaCþ H2O ð1aÞ DG¢ ¼ )7 kJÆmol)1
CH3-MtaCþHS-CoM *)MtaACH3-S-CoMþMtaC ð1bÞ DG¢ ¼ )20.5 kJÆmol)1
MtaA is a zinc protein [3,7,8] that also catalyzes the methylation of coenzyme M with methylcob(III)alamin [9] Several isoenzymes of MtaA, designated MtbA and MtsA have been found [9–11]
The methylation of coenzyme M to methyl-coenzyme M
is a reaction in which a thiol group is alkylated.Enzymes catalyzing alkyl transfers to thiols have all been found to be zinc proteins [12].They include the E coli Ada protein [13], the cobalamin-dependent methionine synthase MetH [14], the cobalamin-independent methionine synthase MetE [15], betaine:homocysteine S-methyltransferase [16], S-methyl-methionine:homocysteine methyltransferase [17], epoxy-alkane:coenzyme M transferase [18] and protein farnesyl transferase [19].The postulated role of zinc in these enzymes
is that of a Lewis acid that activates the thiol group to be alkylated.Coordination of the thiol group to the active site zinc has been shown by extended X-ray absorption fine structure (EXAFS) spectroscopy [14,15], by UV spectros-copy [20] and in the case of protein farnesyl transferase by crystal structure analysis [19].It results in a decrease in the
pK value of the thiol group as shown by the release of a proton upon binding of the substrate to the zinc enzyme [21]
MtaA does not share sequence similarity to any of the other zinc enzymes catalyzing thiol group alkylation [8,22]
Correspondence to H.Dau, Freie Universita¨t Berlin, Fachbereich
Physik, Arnimallee 14, D-14195 Berlin, Germany.
Fax: + 49 30 838 56299, Tel.: + 49 30 83853581,
E-mail: holger.dau@physik.fu-berlin.de or
R.K.Thauer, Max-Planck-Institut fu¨r terrestrische Mikrobiologie,
Karl-von-Frisch-Strasse, D-35043 Marburg, Germany.
Fax: + 49 6421 178209, Tel.: + 49 6421 178200.
Abbreviations: HS-CoM, coenzyme M; CH 3 -S-CoM,
methyl-coen-zyme M; EXAFS, extended X-ray absorption fine structure; Mta,
methanol:coenzyme M methyltransferase; MtaA, protein subunit of
Mta; XANES, X-ray absorption near edge structure; XAS, X-ray
absorption spectroscopy.
(Received November 2001, revised 15 February 2002, accepted 28
February 2002)
Trang 2In the sequence, however, the motif HXCXnC is found
which in MetE has been shown to be the zinc binding motif
[15].It has been suggested that the thiol group of
coenzyme M is activated by MtaA via the same mechanism
as proposed for the other zinc enzymes [7].This suggestion
is mostly based on the finding that MtaA contains per mol
one mol of zinc and that upon binding of coenzyme M one
mol of H+is released [7].The zinc EXAFS results reported
here indeed now show an increase of one in the number of
sulfur ligands to zinc upon formation of the
MtaA-coenzyme M complex
After completion of this manuscript a paper was
published (30 October 2001) describing zinc EXAFS results
for the MtaA isoenzyme MtbA from M barkeri and
coming to the same conclusion with respect to the sulfur
ligation upon binding of coenzyme M [23].The isoenzyme
is involved in methyltransfer from methylamines to
coen-zyme M
ðCH3Þ2NHþ2 þ HS-CoM !MtbABCCH3-S-CoMþ CH3NHþ3
ð2Þ DG¢ ¼ )2 kJÆmol)1
MtbA and MtaA show only 40% sequence identity [22] and
cannot substitute for each other in the catalysis of reactions
1 and 2 [6,8,24,25]
M A T E R I A L S A N D M E T H O D S
M barkeristrain Fusaro (DSM 804) was obtained from the
Deutsche Stammsammlung fu¨r Mikroorganismen und
Zellkulturen (Braunschweig).Methylcob(III)alamin,
coen-zyme M and methylmethanethiosulfonate were purchased
form Sigma, 4-(2-pyridylazo)resorcinol from Fluka.The
QuickChange site-directed mutagenesis kit, Escherichia coli
XL1 Blue MFR and Pfu polymerase were from Strategene
T4 DNA Ligase was from Roche.Oligonucleotides were
obtained from MWG Biotech
Heterologous overproduction and purification
of the MtaA proteins
Heterologous overproduction and purification of the MtaA
proteins His6tagged at the N-terminus was performed as
described by Sauer & Thauer [7].The mtaA wild-type gene
was expressed heterologously in E coli M15 and the
mutated mtaA genes were expressed heterologously in
E coli XL1 Blue MFR.From 500 mL cell culture,
approximately 10 mg highly purified MtaA was obtained
For X-ray absorption spectroscopy (XAS) the enzyme was
equilibrated with 50 mMMops/KOH pH 7.0 by Centricon
ultrafiltration
Site-directed mutagenesis
Site-directed mutagenesis was performed using the
Quick-Change site-directed mutagenesis kit following the protocol
given by the manufacturer.Two different MtaA mutants
were made and confirmed by DNA sequencing: in the first
mutant, His237 was exchanged for Ala using the mutagenic
primers 5¢-CCGTGACTGTACTCgcCATCTGTGGTAA
GG-3¢ (sense) and 5¢-CCTTACCACAGATGgcGAGTAC
AGTCACGG-3¢ (antisense); in the second mutant, Cys239
was exchanged for Ala using the primers 5¢CGTGACTGT ACTCCACATCgcTGGTAAGGTTAACGC (sense) and 5¢GCGTTAACCTTACCAgcGATGTGGAGTACAGTC ACG (antisense).The mutated bases are given in lower-case letters.The mutated mtaA genes were obtained by ampli-fying the expression plasmid pUH28 [22] harbouring mtaA using the respective primers.The resulting linear strands were treated with DpnI, which digested the nonmutated template DNA.The linear strands were ligated using T4 DNA ligase following the manufacturer’s instructions and subsequently transformed into E coli XL1 Blue MFR which had been grown and induced as described above The resulting plasmids were designated as pMK2 for the His237fi Ala exchange and pMK5 for the Cys239 fi Ala exchange
Determination of specific activity Methylcob(III)alamin:coenzyme M activity was determined
at 37C and pH 7.0 by following the demethylation of methylcob(III)alamin (50 lM) photometrically at 520 nm (De ¼ 6.3 mM )1Æcm)1) [7].One unit ¼ 1 lmol methyl-cob(III)alamin demethylated per min under the assay conditions used in this paper.(Note that 1 unit ¼ 20 lmol methylcob(III)alamin demethylated per min under the assay conditions described by Gencic et al.[23].) Protein was determined using the Bradford method and bovine serum albumin as standard [26]
Determination of zinc content The zinc content was in principle determined as described by Zhou et al.[15].The zinc concentration was determined from the absorption change at 500 nm associated with a zinc complex formed with 4-(2-pyridylazo)resorcinol.With ZnCl2 as a standard an De ¼ 55 mM )1Æcm)1was deter-mined.To avoid zinc contamination, all plastic tubes and pipette tips were rinsed with 10 mM EDTA and distilled
H2O before use
XAS measurements and data analysis Zinc K-edge X-ray spectra were collected at beamline D2 of the EMBL Hamburg outstation (HASYLAB, DESY, Hamburg, Germany).The liquid samples were injected into 1-mm cuvettes with capton foil windows.Fluorescence-detected X-ray absorption spectra were measured at 20 K as described previously [27] (monochromator detuning to less than 60% of maximum intensity; scan range: 9400–
10 700 eV).An absolute energy calibration was performed
by monitoring the Bragg reflections of a crystal positioned
at the end of the beamline [28].For each element of the used 13-element solid-state detector the total count-rate was kept below 30 000; the output signal was corrected for detector saturation.The spot size of the X-ray beam on the sample was 4.75· 1.13 mm2; not more than four scans of 1-h duration were taken on the same spot of the sample Comparison of the first and fourth scan revealed no evidence for radiation damage to the samples.Ten to 12 scans were averaged for each EXAFS spectrum
Spectra were normalized and EXAFS oscillations were extracted as described in Schiller et al.[27].The energy scale
of all collected EXAFS spectra was converted to a k-scale
Trang 3using an E0of 9660 eV; k3-weighted spectra were used for
curve-fitting and calculation of Fourier transforms.For the
shown Fourier transforms, for k values ranging from 1.8 to
15.3 A˚)1, the data was multiplied by a fractional cosine
window (10% cosine fraction at low and high R-side); for
curve-fitting the energy range was 20–900 eV.For
simula-tion of k3-weighted spectra, complex backscattering
ampli-tudes were calculated using FEFF 7 [29]; the used value of
S2, the amplitude reduction factor, was 0.9 For the
least-squares curve-fitting of unfiltered k space the in-house
softwareSIMXwas used.By curve-fitting of various EXAFS
spectra we found consistently that DE0refined to a value of
9665 eV; this value has been fixed and used in all
simulations discussed in this work.For further details see
figure captions
The edge energy used to describe the position of the
absorption edge is the energy value corresponding to 0.65
units of normalized absorption.(The value of 0.65 was
chosen because it corresponds to roughly half of the
magnitude of the maximum absorption on top of the edge.)
Other approaches for determination of edge energies
resulted in similar edge-shifts
R E S U L T S
Typical ligands to zinc atoms in proteins are nitrogen and
oxygen atoms provided by the imidazole side chains of
histidines and carboxylic side chains, respectively, as well as
coordinated H2O species (H2O, OH–).Furthermore, the
sulfur atoms of the thiol groups of cysteine and methionine
are potential ligands.Distances between zinc and (N/O)
(meaning nitrogen or oxygen) of 2.03–2.12 A˚ and between
zinc and sulfur of 2.25–2.36 A˚ have been observed in proteins
[14,15,23,30,31] and in synthetic zinc compounds [32,33]
EXAFS of wild-type MtaA
Wild-type MtaA from M barkeri had a zinc content of
0.91 mol/mol and exhibited a specific activity of 0.3 U per
mg under our assay conditions.The enzyme was studied in
the absence and presence of coenzyme M (apparent
Km ¼ 40 lM) by XAS at the zinc K-edge EXAFS and
XANES spectra are shown in Figs 1 and 2, respectively
The Fourier transforms of the EXAFS spectrum of
wild-type MtaA (Fig.1A-I) reveals two prominent and closely
spaced peaks at reduced distances of about 1.6 A˚ (peak 1)
and 1.9 A˚ (peak 2).Peaks at these reduced distances are
indicative of zinc-ligand distances of 2.0 and 2.3 A˚,
suggesting the presence of oxygen or nitrogen ligands
around 2.0 A˚ and sulfur ligands at about 2.3 A˚.Addition of
coenzyme M resulted in a slight decrease of Peak 1 and
strong increase of Peak 2 (Fig.1A-II,III).Thus, already the
visual inspection of the EXAFS data suggests that the
number of sulfur atoms ligated to the zinc of MtaA
increases upon formation of the MtaA–coenzyme M
com-plex explainable by ligation of the thiol sulfur of
coen-zyme M to the active-site zinc
We simulated the experimental spectra in Fig.1A using
two shells of backscattering ligands, namely one shell of N
or O ligands and one shell of sulfur ligands to zinc.(In
general a discrimination between N and O ligands by
EXAFS analysis
2 is almost impossible because (a) the typical
bond-lengths are similar and (b) the EXAFS oscillations of
these two low-Z atoms are of similar shape.) Without using any constraints, a fit of the spectrum of wild-type MtaA without coenzyme M resulted in coordination numbers of 3.4 and 0.7 for the (N/O)-ligand and the sulfur ligands,
Fig 1 Fourier transforms (FT) of k 3 -weighted EXAFS spectra of wild-type MtaA and of two MtaA mutants (Cys239 fi Ala, His237 fi Ala)
in the absence and presence of coenzyme M Experimental spectra (thin lines) and simulations (thick lines) are shown.(A) The wild-type en-zyme in the absence (trace labeled by I) and presence of 0.5 mol/mol HS-CoM (II) or 2 mol/mol HS-CoM (III).(B) In the absence of HS-CoM, comparison of wild-type (I) and mutants (IV – Cys239 fi Ala, V – His237 fi Ala).(C) In the presence of 2 mol/mol HS-CoM, comparison of wild-type (III) and mutants (VI – Cys239 fi Ala, VII – His237 fi Ala).The insets show the corresponding EXAFS oscilla-tions in the k-space (same top-to-bottom sequence as used for the Fourier-transformed spectra).
Trang 4respectively (RF ¼ 19.5%, Table 1), immediately
suggest-ing the presence of three (N/O)-ligands and one sulfur
ligand.Fixing the number of zinc ligands to the nearest
integer values (i.e 3 and 1 for (N/O) and sulfur ligands,
respectively) yields a fit result of comparable quality
(RF ¼ 20.9%; see Table 1, parameters in parenthesis),
whereas a combination of 2· (N/O) plus 2 · S results in
unsatisfactory fits (RF ¼ 29.3%) and problematic
Debye-Waller factors (2r2 too high for (N/O)-shell, but
too low for sulfur shell).Constraining the sum of the two
coordination numbers to a value of 3 or 5 results in poorer
fits and significantly higher RFvalues (26.0% and 27.8%)
confirming that zinc is tetra-coordinate.We did not simulate
the weak features seen in the Fourier transform at (reduced)
distances greater than 3.2 A˚ (Fig.1A-I).Similar features
have been ascribed to single and multiple scattering effects
resulting from imidazole ligation to zinc [14,30].In
conclu-sion, the EXAFS data suggests that in the wild-type the zinc
is tetra-coodinated with one sulfur and three (N/O)-ligands
The EXAFS spectra are significantly altered (Fig.1A) by
addition of HS-CoM to wild-type MtaA.When the ratio of
MtaA to coenzyme M was 1 : 0.5, the spectrum is best simulated with 2.8 (N/O)-ligands and 1.3 sulfur ligands (Table 1, Fig.1A-II, thick line).These numbers compare reasonably well with the results expected for high-affinity binding of HS-CoM resulting in substitution of one (N/O)-ligand by 1 sulfur (N/O)-ligand in 50% of all MtaA proteins [i.e 2.5 (N/O) plus 1.5 sulfur at the MtaA–HS-CoM stoichi-ometry of 1 : 0.5] At a MtaA to HS-CoM ratio of 1: 2, unconstrained fit results suggest 1.7 (N/O)-ligands and 2.3 sulfurs (Fig.1A-III).These numbers are close to the ideal values for substitution of one (N/O)-ligand by a sulfur ligand in all zinc-containing MtaA proteins [i.e 2.0 (N/O) plus 2 (S)].We interpret these results as indicating the binding of the thiol sulfur of HS-CoM to the zinc atom of MtaA in a way that it replaces one of the nonsulfur ligands, possibly the oxygen of coordinated H2O or hydroxide
EXAFS of two MtaA mutants, Cys239fi Ala and His237fi Ala
His237, Cys239 and possibly Cys316 have been proposed to
be involved in zinc coordination in MtaA based on sequence comparisons of MtaA with MetE [15].By site-directed mutagenesis, we therefore exchanged His237 and Cys239 to alanine.The His237fi Ala mutated enzyme and the Cys239fi Ala mutated enzyme had zinc contents of 0.25 mol/mol and 0.4 mol/mol, respectively, and exhibited specific activities of 0.02 UÆmg)1and 0.01 UÆmg)1 Figure 1B shows the Fourier transforms of the experi-mental EXAFS spectra (thin lines) for wild-type MtaA (trace I), for Cys239fi Ala MtaA (trace IV), and for His237fi Ala MtaA (trace V), in the absence of coen-zyme M.The two mutant proteins exhibit EXAFS spectra that are clearly different from those of the wild-type, strongly suggesting that His237 and Cys239 are involved in zinc coordination
In comparison to the wild-type, in the spectrum of Cys239fi Ala (Fig.1B-IV) Peak 2 is shifted to shorter distances and a new peak appears at a reduced distance of
2.4 A˚ (corresponding to a zinc-ligand distance of
2.7 A˚).The latter feature, which indicates the presence
of relatively strong backscattering by atoms at 2.7 A˚, is not found in the wild-type.Only a poor simulation (RF ¼ 44%) of the EXAFS of the Cys239 fi Ala mutant
is obtained with similar parameters as used for the wild-type.A specific alternative approach yields satisfactory results [(N/O) at 2.0 A˚, (N/O) at 2.2 A˚, S at 2.7 A˚, Table 1, Fig.1B-IV)], whereas other simulation approaches using three shells of backscattering atoms did not result in high-quality fits.Interestingly, in Cys239fi Ala the zinc seems to be penta-coordinated.These results are highly suggestive that, in the wild-type, Cys239 is a zinc ligand.In the Cys239fi Ala mutant, the thiol group of Cys239 seems
to be replaced by two (N/O)-ligands, presumably the oxygens of two H2O molecules.Furthermore, there may
be a thiol group of an amino-acid residue which moves closer to the active-site zinc due the structural rearrange-ment resulting from the mutation
In the His237fi Ala mutant, peak 2 is strongly increased (in comparison to the wild-type, see Fig.1B-V) proving a significantly modified ligand environment of the active-site zinc.A simulation with two shells [(N/O), S] yielded coordination numbers of 2.4 and 1.8, respectively,
Fig 2 Normalized XANES spectra of wild-type MtaA and of two
MtaA mutants (Cys239 fi Ala, His237 fi Ala) in the absenc e and
presence of added coenzyme M (A) Wild-type MtaA (thick line),
type MtaA + 0.5 mol/mol HS-CoM (line of medium thickness),
wild-type MtaA + 2 mol/mol HS-CoM (thin line).(B) Wild-wild-type MtaA
(solid line), Cys239 fi Ala (dotted line), His237 fi Ala (dashed line).
(C) His237 fi Ala (thick dashes), His237 fi Ala + 2 mol/mol
HS-CoM (thin dashes).(D) Cys239 fi Ala (thick dots), Cys239 fi
Ala + 2 mol/mol HS-CoM (thin dots).(E) ZnCl 2 in aqueous solution
(dash-dotted line).
Trang 5pointing towards the presence of two (N/O)-ligands and two
sulfur ligands in the mutant (Table 1).Possibly, one of the
(N/O)-ligands in the wild-type, probably His237, is replaced
by a sulfur ligand in the His237fi Ala mutant.It is
tempting to speculate that this sulfur ligand is the same that
is observed in the Cys239fi Ala mutant at a zinc-sulfur
distance of 2.7 A˚
In Cys239fi Ala and His237 fi Ala, the pronounced
changes in the EXAFS spectra resulting from HS-CoM
addition are indicative of an increase in the number of
sulfur ligands (visual inspection of Fig.1C).Thus, in both
mutants the HS-CoM binding seems to involve ligation of
the thiol group of HS-CoM to the active-site zinc.For the
Cys239fi Ala mutant plus HS-CoM the simulation
results suggest the presence of tetra-coordinated zinc with
two (N/O) and two sulfur ligands (Fig.1C-VI, Table 1)
showing that the HS-CoM addition may lead to an
increase in the number of sulfur ligands of 2.We
tentatively propose that either more than 1 HS-CoM
molecule binds to a single zinc site or, alternatively, one
HS-CoM sulfur as well as the sulfur detectable at 2.7 A˚
in the absence of HS-CoM become direct ligands in its
presence.In His237fi Ala, in the presence of HS-CoM
the spectrum is well simulated using two shells of
backscatterers [(N/O), S] with coordination numbers of
1.4 and 2.5 (Fig 1C-VII, Table 1); fixing the coordination
numbers to integer values results in a satisfactory
simula-tion for 1· (N/O) and 3 · S.The clear increase in the
coordination number for sulfur upon HS-CoM addition
indicates that also in His237fi Ala the HS-CoM binding
results in sulfur ligation to the active-site zinc
XANES spectra of wild-type and mutants
As shown in the following, the interpretation of the EXAFS
of the mutant enzymes is confirmed by a comparative
analysis of the zinc K-edge spectra (XANES spectra, see
Fig.2) of the wild-type and the mutants (His237fi Ala,
Cys239fi Ala) in the presence and absence of added HS-CoM
For the wild-type, the EXAFS analysis unambiguously indicates that HS-CoM addition results in an increase in the number of sulfur atoms in the first coordination sphere of zinc.This increase in the number of sulfur ligands is accompanied by the following changes in the XANES (Fig.2A, Table 1): (a) the edge position shifts down from 9663.5 eV to 9663.1 eV; (b) the magnitude of an absorption peak at the top of the edge ( 9671 eV) becomes reduced (Fig.2A) Seemingly, additional sulfur ligands result in down-shift of the edge-energy as well as in reduced absorption at the top of the edge.In inorganic models, peptide models and zinc proteins, Penner-Hahn and coworkers observed the same relations between XANES spectra and the number of sulfur ligands [14,23,30]
In His237fi Ala (Fig.2B, dashed line), in comparison
to the wild-type enzyme (Fig.2b, solid line) the K-edge is shifted to lower energies (Table 1) and the absorption on top of the edge is reduced.In the Cys239fi Ala mutant (Fig.2B, dotted line), the K-edge energy is increased (Table 1) and the absorption on top of the edge is significantly increased.We conclude that not only the EXAFS but also the XANES spectra are suggestive of a reduced number of sulfur ligands in the Cys239fi Ala and
an increased number of sulfur ligands in His237fi Ala (in comparison to the wild-type enzyme)
As a model for the zinc in the absence of any sulfur ligand
we use ZnCl2dissolved in H2O (Fig.2E, dash-dotted line) The Cys239fi Ala (Fig.2D, thick dots) and the ZnCl2
spectra (Fig.2E) are obviously very similar (edge energy of 9663.7 eV, single edge peak at 9669 eV) confirming the absence of any sulfur ligands in the Cys239fi Ala mutant For both mutants (Fig.2C,D), comparison of edge positions and edge-peak magnitudes strongly suggests that coenzyme M addition (Fig.2C,D; thin lines) results in additional sulfur ligands in the first coordination sphere of zinc.In the Cys239fi Ala mutant, coenzyme M addition
Table 1 ZincEXAFS simulation parameters and edge positions for wild-type (WT) MtaA and mutated MtaA (Cys239 fi Ala, His237 fi Ala) without and in the presence of 0.5 mol/mol coenzyme M (+ ½ HS-CoM) or 2 mol/mol coenzyme M (+ 2 HS-CoM) The values in parenthesis result from simulations using coordination numbers fixed to integer values.The edge position, E K-edge , was determined for the XANES spectra shown in Fig.2; EXAFS data and simulations are shown in Fig.1.Simulation parameters: N, coordination number (coord.no.); 2r 2 , Debye-Waller parameter; R F , the filtered R-factor [34] representing the deviation between data and simulation (in percentage) for reduced distances ranging from
1 to 2 8 A˚.
Sample E K-edge (eV) Shell Coord.no Distance, R (A˚) 2r 2 (A˚ 2 ) R F (%)
WT MtaA 9663.5 (N/O) 3.4 (3.0) 2.02 (2.02) 0.009 (0.007) 19.5 (20.9)
S 0.7 (1.0) 2.32 (2.31) 0.003 (0.006)
WT MtaA + ½ HS-CoM 9663.3 (N/O) 2.8 (2.5) 2.02 (2.03) 0.010 (0.009) 24.6 (25.6)
S 1.3 (1.5) 2.32 (2.32) 0.005 (0.006)
WT MtaA + 2 HS-CoM 9663.1 (N/O) 1.7 (2.0) 2.06 (2.06) 0.005 (0.005) 11.4 (13.3)
S 2.3 (2.0) 2.32 (2.32) 0.005 (0.004) Cys239 fi Ala 9663.6 (N/O) 2.9 (3.0) 2.04 (2.04) 0.004 (0.004) 21.4 (21.3)
O 2.2 (2.0) 2.17 (2.17) 0.003 (0.003)
S 0.9 (1.0) 2.72 (2.72) 0.006 (0.007) Cys239 fi Ala + 2 HS-CoM 9663.1 (N/O) 2.1 (2.0) 2.07 (2.07) 0.007 (0.007) 17.5 (18.0)
S 1.9 (2.0) 2.32 (2.32) 0.005 (0.005) His237 fi Ala 9663.0 (N/O) 2.4 (2.0) 2.03 (2.03) 0.006 (0.005) 16.5 (17.1)
S 1.8 (2.0) 2.29 (2.29) 0.010 (0.009) His237 fi Ala + 2 HS-CoM 9662.6 (N/O) 1.4 (1.0) 2.07 (2.06) 0.003 (0.003) 13.8 (14.1)
S 2.5 (3.0) 2.32 (2.32) 0.005 (0.006)
Trang 6results in particularly pronounced changes.The absorption
at 9671 eV and the edge position reach the values found for
the wild-type in the presence of coenzyme M (Fig.2C, thin
dotted line).These findings point towards two sulfur ligands
in the Cys239fi Ala mutant supplemented with
coen-zyme M
In summary, the XANES analysis fully confirms the
EXAFS results on the number of sulfur ligands in the first
coordination sphere of the active-site zinc
D I S C U S S I O N
Zinc EXAFS spectra of wild-type MtaA from M barkeri
and of two mutant proteins, Cys239fi Ala and
His237fi Ala, in the absence and presence of
coen-zyme M, indicate how zinc interacts with its substrate
coenzyme M and how zinc is most probably coordinated in
the active site of this methyltransferase
Upon binding of coenzyme M to MtaA the number of
sulfur ligands to zinc increased by at least one whereas the
total number of ligands remained four; one (N/O) ligand
was replaced by a sulfur ligand.This, and the previous
finding that upon coenzyme M binding to MtaA one mol of
protons was released [7], is consistent with an activation of
coenzyme M via coordination of its thiol group to the active
site zinc.The EXAFS data reported for the isoenzyme
MtbA show that the isoenzyme uses the same activation
mechanism [23]
The EXAFS data do not prove this activation
mecha-nism definitely as they only show that upon coenzyme M
binding to MtaA the number of sulfur ligands increased
from 1 to 2.However, this increase could also be due to the
binding of a cysteine residue to the active site zinc upon
enzyme–substrate complex formation.Final proof could
come from EXAFS studies using seleno coenzyme M as
substrate [31]
The simulation of the EXAFS spectrum of wild-type
MtaA revealed that zinc is likely coordinated by 1 sulfur
ligand and 3 (N/O) ligands, its total coordination number is
4.In both mutants, zinc is quite differently coordinated,
namely by 2 (N/O) and 2 S ligands in His237fi Ala, and
by 5 (N/O) ligands in the Cys239fi Ala mutant.These
results, together with the features of the XANES spectra
likely indicate that both residues, Cys239 and His237
provide direct ligands to zinc in wild-type MtaA.Cys239
was probably replaced by 2 H2O molecules and His237 by a
thiol group from a cysteine residue of which the enzyme
contains six
In the E coli Ada protein, which is a zinc dependent
methyltransferase, the active site zinc is coordinated to four
cysteine sulfur ligands [13].In the methyltransferase MetH
zinc is coordinated to 3 S and 1 (N/O) ligands and in MetE
to 2 S and 2 (N/O) ligands [14].In protein farnesyl
transferase the active site zinc is coordinated by 1 S and 3
(N/O) ligands, as revealed by the crystal structure [19].Zinc
thus can be quite differently coordinated and still activate
thiol groups for alkylation.Consistent with the
interpreta-tion is our finding that both MtaA mutants still bind
coenzyme M and exhibit some activity although in both
mutants the coordination of zinc differs from the situation
in the wild-type enzyme.Apparently, other ligands to zinc
(H2O thiol groups of cysteine residues) were able, at least in
part, to functionally replace Cys239 and His237
The EXAFS and XANES spectra recently reported for the MtaA isoenzyme MtbA from M barkeri revealed that
in the active site of MtbA zinc is coordinated by 2 S and
2 (N/O) ligands [23].These spectra are indeed clearly different to our spectra of MtaA showing that the active site zinc in MtaA is most probably coordinated by 1 S and 3 (N/O) ligands.MtaA and MtbA are only 40% sequence identical [22] but they share the putative HXCXnC zinc binding motif [15].The latter finding indicates that zinc coordination in MtaA and MtbA should be the same.The finding that the number of sulfur ligands to zinc can vary in different S-alkyl transferases from 1 to 4 (see above) and that both mutated MtaA proteins still showed some activity indicates, however, that some aspects of the ligand envi-ronment of zinc are surprisingly uncritical for thiol group activation.It could therefore well be that in MtaA the zinc
is ligated by 1 S and 3 (N/O) and in MtbA by 2 S and
2 (N/O)
In comparing the results for MtaA (this work) and MtbA [23] it has to be considered that the MtaA samples analysed
by X-ray absorption spectroscopy were His tagged (whereas the MtbA samples were not).Adventitiously picked up zinc
by the His tag might contribute to a higher number of (N/O) ligands and thus to a relatively lower number of S ligands The finding, however, that upon addition of coenzyme M to MtaA the number of S ligands increases from 1 to 2 whereas
in the case of MtbA the number increases from 2 to 3 suggests that it is unlikely that the His tagging is responsible for the differences between the MtaA- and MtbA-EXAFS results (although this possibility cannot be totally excluded) MtbA is inactivated when Cys241 or Cys316 of the putative zinc binding motif H239XC241XnC316 are mutated [23].In case of MtaA the mutation of Cys239 in the H237XC239XnC316 motif leads to an enzyme with only
a few percent activity.It is not known whether MtaA becomes inactive when Cys316 is mutated and how this mutation affects the Zn ligand environment.The apparent absence of any sulfur in the first coordination sphere of Zn
in the Cys239fi Ala mutant suggests that Cys316 is not a direct Zn ligand in MtaA.It would be interesting to investigate the XAFS of a Cys316 mutant to support (or disprove) the model of 1 S plus 3 (N/O) ligands in the resting state of MtaA
A C K N O W L E D G E M E N T S
We gratefully acknowledge financial support by the Max-Planck Society, by the Deutsche Forschungsgemeinschaft, and by the Fonds der Chemischen Industrie.
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