(Received for publication, June 1, 1995; and in revised form, October 5, 1995)
From the
The structure of S-adenosylmethionine synthetase (MAT,
ATP:L-methionine S-adenosyltransferase, EC 2.5.1.6.)
from Escherichia coli has been determined at 3.0 Å
resolution by multiple isomorphous replacement using a uranium
derivative and the selenomethionine form of the enzyme (SeMAT). The
SeMAT data (9 selenomethionine residues out of 383 amino acid residues)
have been found to have a sufficient phasing power to determine the
structure of the 42,000 molecular weight protein by combining them with
the other heavy atom derivative data (multiple isomorphous
replacement). The enzyme consists of four identical subunits; two
subunits form a spherical tight dimer, and pairs of these dimers form a
peanut-shaped tetrameric enzyme. Each pair dimer has two active sites
which are located between the subunits. Each subunit consists of three
domains that are related to each other by pseudo-3-fold symmetry. The
essential divalent (Mg/Co
) and
monovalent (K
) metal ions and one of the product,
P
ions, were found in the active site from three separate
structures.
In biological systems, there are a myriad of reactions in which
methyl groups are transferred from a few types of methyl donors to a
wide variety of methyl acceptors. Among biological methyl group donors, S-adenosylmethionine (AdoMet), ()discovered by
Cantoni in 1953(1) , is the most widely used, while
5-methyltetrahydrofolic acid, methylcobalamin, and betaine are involved
in far fewer methylation reactions. The activated methyl cycle
involving AdoMet is illustrated below (Fig. S1).
Figure S1: Scheme 1Activated methyl cycle.
AdoMet, synthesized from L-methionine and ATP, transfers a methyl group to a methyl acceptor molecule, yielding S-adenosylhomocysteine. S-Adenosylhomocysteine is subsequently hydrolyzed to adenosine and homocysteine, the latter of which can be metabolized to methionine.
Methylation reactions involving AdoMet are increasingly being recognized as significant control factors in the regulation of a variety of cellular functions. Methylation of nucleic acids is known to have regulatory effects on DNA replication and transcription and RNA translation. Protein methylation is involved in the regulation of a variety of metabolic processes such as bacterial chemotaxis(2, 3) , sperm mobility(4) , and release of neurotransmitters(5) . AdoMet is also the methyl donor to a vast number of small molecules (e.g. in the biosynthesis and/or metabolism of various catecholamine neurotransmitters). AdoMet is being used in clinical trials for treatment of depression(6) , liver injury(7) , and as a potential cancer chemopreventive agent(8) . In addition to its role as a methyl donor, AdoMet undergoes decarboxylation to generate the propylamine donor in the biosynthesis of the polyamines, spermine and spermidine, which are widely distributed in nature and appear to be involved in cellular proliferation(9) . Recently, inhibition of the metabolism of AdoMet has been found to be an important target for development of the chemotherapeutic agents for neoplastic and viral diseases(10, 11) . Regland and Gottfries (12) have proposed that slowed synthesis of AdoMet is a pathogenetic mechanism common to the dementia seen in Alzheimer's disease, Down's syndrome, and the acquired immunodeficiency syndrome.
The formation of AdoMet is catalyzed solely by AdoMet
synthetase (MAT, ATP:L-methionine S-adenosyltransferase, EC 2.5.1.6). The synthesis of AdoMet
occurs in an unusual two-step reaction in which the complete
tripolyphosphate chain is cleaved from ATP as AdoMet is formed, and the
tripolyphosphate is subsequently hydrolyzed to pyrophosphate
(PP) and orthophosphate (P
) before the
sulfonium product (AdoMet) is released, giving the overall reaction
shown below(13) .
Thus, the enzyme uniquely catalyzes reactions at both ends of
the tripolyphosphate chain in a defined temporal sequence. AdoMet
synthetase requires divalent metal ions for activity (e.g. Mg, Co
) and is greatly
activated by certain monovalent cations such as K
.
The amino acid sequences of the enzyme from Escherichia coli(14, 15) , Saccharomyces
cerevisiae(16, 17) , Arabidopsis
thaliana(18, 19) , rat liver(20) , rat
kidney (21) , human liver(22) , human
kidney(23) , and the leaf of Dianthus caryophyllus(24) have been reported deduced based on DNA sequences. In
all organisms studied so far, sequence homologies show that AdoMet
synthetase is an exceptionally well conserved enzyme through evolution.
No biological data regarding the amino acid residues involved in the
active site of MAT has been reported. The first crystallization of MAT
was reported by Gilliland et al.(25) ; however, no
subsequent work was reported. The determination of the
three-dimensional structure of the enzyme is essential for elucidation
of the unique catalytic mechanism and will also facilitate development
of effective inhibitors of this enzyme. In the present paper, we
describe the x-ray structures of three MAT complexes with the product
P ions and various metal ions.
Crystals of the native enzyme (MAT) and SeMAT from E. coli were grown in a buffer containing 100 mM potassium
phosphate (pH 7.0), 10 mM MgCl, 33% (v/v)
saturated ammonium sulfate with the protein concentration at 10 mg/ml.
The hexagonal bipyramidal shaped crystals were grown at 26 °C for 2
weeks. Crystals of the native enzyme containing Co
ions in the active site (MAT-Co) were grown from a similar
solution which contained 10 mM CoCl
instead of 10
mM MgCl
. The uranium derivative crystals were
prepared by the soaking method; native crystals were incubated in an
artificial mother liquor containing 50 mM Tris-malate buffer
(pH 7.0), 10 mM MgCl
, 40% saturated ammonium
sulfate, and 5 mM UO
(NO
)
for 5 h before they were mounted into the capillary.
The difference
electron density map calculated with coefficients (F - F
) and the phases obtained from
the SIR described above showed nine peaks clearly (>7
) for the
selenium atoms of the SeMet residues (Fig. 1). The positions of
the selenium atoms were refined by the locally developed program HEVY.
The relatively high B-values of selenium atoms refined by using the
centric reflections might be due to some systematic errors introduced
by the many crystals used to collect each data set, but all SeMet
residues in SeMAT structure are well-ordered in the final structure.
The calculated phasing power of the SeMAT data (1.76) was as strong as
that of the MAT-U derivative data (1.64). Therefore, the protein phases
were determined by the SeMAT isomorphous replacement data in
conjunction with solvent flattening, in order to test whether the SeMAT
data have enough phasing power as indicated by the calculation and
whether the positions of the selenium atoms are reliable. As shown in Fig. 2, the map calculated using the phases determined from the
SeMAT data had the similar electron density distribution seen in the
map calculated from MAT-U data. Thus, the MIR phases (4.0 Å
resolution) were obtained by combining the phase distribution functions
of uranium and selenium derivatives. The phases were refined and
extended from 4.0 Å to 3.0 Å resolution by solvent
flattening. It is noted that the SeMet forms of enzymes have recently
been used successfully for one of the MIR
derivatives(33, 34) .
Figure 1:
The difference electron density map
calculated with the coefficients of (F - F
) and the phase angles obtained by the single
isomorphous replacement (SIR) using the UO
derivative data.
Nine peaks (>7
) centered on the selenium atoms of the nine
selenomethionine residues are visible. The map was calculated with
10-4.0 Å resolution data, and the contour was drawn at
5.0
level.
Figure 2:
Electron density maps of the B5
-strand (
QGLMFGYATN
) containing
various sized residues. A, SIR map phased by the MAT-U
derivative; B, SIR map phased by the SeMAT; C, MIR
map phased by the MAT-U and SeMAT; D, final
(2F
- F
) map; E, omit map. The SIR and
MIR maps were calculated with the phases refined and extended by
solvent flattening. The omit map was calculated after 30 cycles of the
positional refinement by X-PLOR. The contours are drawn at 1.5
level for map A, B, C, and D and
3.0
for map E. The final model is superimposed in the
maps.
The map calculated with the
new phases was improved (Fig. 2). The entire main chain except
for Ala-His
,
Ile
-Asp
, and Gly
-Lys
was traced without ambiguity, and the side chain groups of 343
out of 383 residues were placed in the electron density peaks contoured
at 1.1
level. The initial model was built on an IRIS work station
using the program TOM/FRODO(35, 36) . The model was
refined with the positional protocol and then the simulated annealing
procedure of X-PLOR(37) . The model was rebuilt where
necessary, and previously undefined residues were built into the
electron density map.
During later stages of refinement, difference
maps calculated with coefficients of (F - F
), and calculated phases showed several
significant residual electron density peaks in the region of active
site (Fig. 3). The electron density peaks were assigned to two
P
, one K
, and two Mg
ions based on the size of the electron density, refined
temperature factors, and polar environment, and those ions were
introduced into the refinement. Assignment of the divalent ions
(Mg
) was confirmed with the structure analysis of
MAT-Co which is described below. The additional residual electron
density peak found near the center of tight dimer and on the 2-fold
axis was significant and assigned to a K
ion based on
the size of the electron density peak and polar environment. Refinement
of isotropic temperature factors for individual atoms was carried out
by the individual B-factor refinement procedure of X-PLOR using bond
and angle restraints. The thermal parameters of the two P
and metal ions were relatively large in comparison with those of
the amino acid residues constituting the active site, suggesting that
the ion sites are partially occupied. After four cycles of model
building and refinement, all residues from 1 to 101 and 108 to 383 were
built into the electron density map. The residues 102 to 107, which are
outside the active site, were not visible in the electron density maps
and were assumed to be heavily disordered. Residues 102 to 107 were
arbitrarily placed using a TOM/FRODO routine; however, those residues
were not included in the refinement.
Figure 3:
Difference electron density maps
calculated after 30 cycles of positional refinement of the protein
structure (not including the P and metal ions) by X-PLOR.
The contours are drawn at 3
level. The final model is
superimposed on the map. The Mg
/Co
ions (X) and the P
ions are shown in the
center of maps. The residual electron density peak of the K
ion (X) is visible in the left side of the
maps.
The structure determination has
been verified by the following procedures. The R-factor
distributions in several resolution shells are reasonably uniform (Table 3), and the free R(38) calculated using
10% reflections selected randomly is relatively low (0.26). The
structure was also checked by the computer program PROFILE
3D(39) , and no unusual connectivity has been found in the
protein structure (Fig. 4). Also, a standard protein structure
verification program, PROCHECK(40) , does not indicate any
unusual feature in the structure. The Ramachandran plot (41) indicates that nearly all residues are in allowed
conformations (Fig. 5). The root mean square deviations of bond
distances, angles, and torsion angles from the ideal geometry data are
relatively small (Table 1). As shown in Fig. 2, an omit
map calculated after 30 cycles of the positional refinement reproduces
the similar electron density distribution seen in the final
(2F - F
) map. Finally,
the correctness of the main chain connectivity is strongly supported by
the nine methionine sites determined from the SeMet-form enzyme data.
As described above, the difference electron density map calculated with
the coefficients of (F
- F
), and the phases obtained by the SIR using the
UO
derivative data showed nine peaks (>7
) at the
positions of the selenium atoms of nine selenomethionine residues (Fig. 1). There is little chance of incorrectly tracing the
peptide chain and still correctly locating the nine methionine sites.
The parameters for the structure determination and refinement are
listed in Table 1. The coordinates have been deposited in the
Protein Data Bank (code numbers 1XRA, 1XRB, and 1XRC).
Figure 4: Profile window plot of the final model of MAT. The vertical axis gives the average three-dimensional-one-dimensional score for residues in a 21-residue sliding window. In general, the negative scores suggest connectivity problems in the regions. The total score of entire residues is 169, which is quite high.
Figure 5:
Ramachandran plot of the main-chain
dihedral angles for the final atomic model. Glycine residues are
indicated as triangles. Asp is located slightly
outside of the allowed region (
= 81°,
=
137°).
The
structures of MAT-Co and SeMAT were initially refined with the
coordinates of the protein structure described above. The difference
map for SeMAT calculated after 30 cycles of the positional protocol,
showed the same residual electron densities in the active site as were
observed in the MAT structure. The residual electron density peaks in
the MAT-Co structure were also quite similar to those of the MAT
structure. However, the electron density peaks at the positions where
the Mg ions were assigned in the MAT structure were
significantly higher in the MAT-Co structure (Fig. 3). On the
basis of this difference in the residual electron density peaks, the
binding locations for the divalent ions (Mg
or
Co
) in the active site were deduced.
MAT consists of four identical subunits related by 222 symmetry (Fig. 6). The four subunits are denoted subunits A, B, C, and D.
Pairs of subunits (A and B or C and D) strongly interact with each
other to form a spherical tight dimer, and these tight dimers associate
to a peanut-shaped tetrameric enzyme. The interactions between the
tight dimers appear to be much weaker than the subunit-subunit
interactions within the tight dimer. Each tight dimer has two active
sites located between subunits A and B or C and D, with amino acid
residues from both subunits contributing to each active site (Fig. 6). We use the following convention to distinguish between
residues in subunits A and B. A residue name with no added symbol
refers to a residue of subunit A; a residue name with an appended
asterisk refers to a residue of subunit B. For example,
Glu means Glu
of subunit A, and
Arg
* means Arg
of subunit B. The convention
also applies to elements of secondary structure (
-helix: H1, H1*;
-strand: B1, B1*, etc.).
Figure 6:
Tetrameric structure of MAT. Subunits A,
B, C, and D related by 222 symmetry are marked by letters at
each carboxyl-terminal, respectively. The two P, two
Mg
, and one K
ions found in the
active site between subunits A and B are illustrated with stick
bonds and asterisks,
respectively.
Figure 7:
A, C trace of the subunit of MAT with
two P
ions, two Mg
ions (solid
circle) and one K
ion (open circle). The
three domains are illustrated with the thin, open,
and thick bonds, respectively. Selected sequence numbers are
added near the C
atoms. The P
ions (solid
bonds), Mg
ions (filled circles), and
K
ion (open circle) are illustrated. The
unresolved six amino acid residues(102-107) built arbitrarily are
drawn with dashed lines. B, ribbon representation of the
subunit.
Figure 8:
Topology diagram showing the secondary
structural similarities of the three domains of MAT. The -helices,
-strands, and 3
helices are indicated by rectangles, arrows, and circles,
respectively.
Three crystal structures of methyltransferases with bound AdoMet, catechol O-methyltransferase(44) , HhaI DNA methyltransferase(45) , and TaqI DNA methyltransferase (46) have recently been reported. The AdoMet binding domains of these methyltransferases are strikingly similar to each other, indicating that many methyltransferases may have a common structure(47) . However, the AdoMet binding pocket of the E. coli methionine repressor(48) , the only other AdoMet binding protein with a structure determined by x-ray analysis, has no resemblance to the AdoMet-binding pockets found in the methyltransferases. MAT has no structural similarity to either of these methyltransferases or to the repressor.
Figure 9:
A, interactions between two subunits in a
tight dimer. The -sheets and the core loop of subunit A are shown
by solid lines, and those of subunit B by open lines.
A K
ion located in the center of the dimer is
indicated by an open circle. The
-sheets mainly interact
by hydrophobic interactions, whereas the central region including the
central core loop and the K
ion participate in
hydrogen bonds between the subunits. B, interaction between
the tight dimers. The residues belonging to subunit D are drawn with open bonds. Hydrogen bonds between Ser
. . .
Ser
* are indicated by dashed lines. The side
chains (-CH
SH) of Cys
drawn with solid
bonds and filled atoms in the two subunits point in
opposite directions.
Interactions between the tight dimers appear to be
less extensive. There is only one residue, Ser in the B4
-strand, that is involved in polar interactions between subunit A
and subunit C. Ser
is the only residue which participates
in the 2-fold intersubunit hydrogen bonds between subunits A and D.
However, the measured dissociation constant for the equilibrium between
the tetramer and two dimers is less than 10
M(49) . It is noteworthy that upon modification
of the enzyme with the sulfhydryl reagent N-ethylmaleimide,
which reacts with both Cys
and Cys
in each
subunit, the tetramer dissociates to two dimers(49) .
Furthermore, a site-directed mutagenesis change of Cys
to
Ala
yields an enzyme that exists as a mixture of dimers
and tetramers, while changing Cys
to Ala
has no effect on the aggregation state (50) . Although
Cys
is located on the border between dimers, no strong
interaction is observed (Fig. 9B). However, Cys
is a conserved amino acid residue of MAT in various organisms
suggesting that this residue plays an important role in dimer formation
of the enzyme. Thus, the structure of the interface between the dimers
might be changed substantially by replacing Cys
with
Ala
.
Figure 10:
The final (2F - F
) map around active
site of MAT-Co complex. A, electron density peaks of two
P
, two Co
, and one K
in
the active site showing the deep cleft between the two subunits. B, electron density distribution in the bottom of the active
site showing well-defined electron density peaks for the amino acid
residues. Contours are drawn at 1.0
level for P
,
Co
, and K
ions and 1.5
level
for amino acid residues.
Figure 11:
The active site geometry with
P and metal ions. Possible polar interactions (hydrogen and
coordinate bonds) are indicated by thin lines3. P
and metal
ions are illustrated by the solid bonds and filled
circles, respectively. The hydrogen and coordination bonds are
defined as donor-acceptor distance to be less than 3.3 Å and
metal-oxygen distance to be less than 2.5 Å,
respectively.
His*(N
2) and
Lys
*(N
) hydrogen-bond to one P
, whereas
Lys
(N
) and Lys
*(N
) hydrogen-bond
to the other P
. Lys
*(N
) appears to
participate by providing the bridging hydrogen bonds between the two
P
ions. Interactions between these positively charged
residues and the negatively charged oxygen atoms of the P
ions appear to neutralize the individual charges. Some of these
positively charged residues may be involved in either the displacement
of the tripolyphosphate chain from ATP during AdoMet formation or the
subsequent hydrolysis of the tripolyphosphate to PP
and
P
.
Two Mg (or Co
)
ions are separated by 5 Å, and those ions bridge two
P
ions in a trigonal pyramidal fashion, i.e. each
divalent ion is coordinated to two oxygen atoms of one P
ion and to one oxygen atom of the other P
ion. The
negatively charged Asp
and Asp
* residues
each coordinate to one of the two Mg
(or
Co
) ions from the side opposite to the P
ions. Additionally, two oxygen atoms (O
1 of Glu
*
and O
1 of Asp
) are located within 4.0 Å from
the Co(1) ion. Similarly, the Co(2) ion is surrounded by two additional
oxygen atoms (O
1 of Glu
and O of Cys
*).
These oxygen atoms may be involved in coordination bonds to the
Co
ions. A similar environment is observed around the
Mg
ions. It is not clear whether these divalent ions
bind to the enzyme in the same fashion when ATP binds at the active
site.
Cantoni (1) reported that divalent metal ions are
essential for MAT activity. An EPR spectroscopic study with
Mn has indicated that one metal ion binds to the
enzyme in the absence of substrates and a second metal ion binds as a
complex with the substrate ATP(27) . When Mn
ions occupy both sites, the metal ions have been observed to
interact magnetically by spin exchange, suggesting that a common
ligand, such as a phosphoryl group, bridges the metal ions. In this
x-ray study, in spite of the absence of substrates, two divalent metal
ions are found in the active site, suggesting that the binding mode of
the two P
ions at the active site is somehow similar to
that of the triphosphate group of ATP. In fact, the coordination scheme
between the P
ions and the divalent metal ions found in
this study is consistent with the EPR observation described above.
Two divalent ions are found at the active site in structures of the 3`,5`-exonuclease domain of DNA polymerase I(51) . In the absence of substrate or product, the exonuclease domain of DNA polymerase I has one bound divalent metal ion (site A), whereas complexes with deoxynucleoside monophosphates show a second divalent metal ion (site B) separated by 4 Å from that in site A. Thus, MAT and DNA polymerase I may belong to an emerging group of structurally determined members of a family of nucleotide-utilizing enzymes that bind multiple divalent metal ions.
The active site
K ion is surrounded by three oxygen atoms within
coordination distance (O
1 and O
2 of Glu
and O of
Ser
), but there is no interaction with the P
ions. Thus, the monovalent ion appears to aid in construction of
the framework of the active site, a situation analogous to that
recently found for K
ion pyruvate kinase(52) .
Although there is no significant electron density peak which might
indicate water molecules around the K
ion, several
water molecules should be coordinated to the K
ion
since there are two large open spaces around the K
ion
and the coordination number of K
is often as large as
10 (53) . The binding site of the K
ion is the
same as that of one of the UO
binding sites in the heavy
atom derivative, consistent with UO
being an inhibitor of
the enzyme(54) . On the basis of this crystal structure, a
site-directed mutagenesis change of Glu
to Gln
has been carried out by McQueney and Markham(54) . The
mutation abolished the monovalent cation activation and produced an
enzyme which has an activity virtually identical with that of
K
-free wild type MAT in both the overall AdoMet
synthesis reaction and in the hydrolysis of tripolyphosphate,
indicating that the monovalent and divalent metal ions in the active
site were assigned correctly. It should be noted that all amino acid
residues involved in either hydrogen bonds or coordination bonds with
either metal ions or P
ions are conserved in the 12
reported sequences of MAT.
In summary, this crystallographic study
on MAT from E. coli has revealed the active sites of the
tetrameric enzyme which are located between the subunits forming a
spherical tight dimer. Two divalent metal ions (Mg)
and one monovalent metal ion (K
) have been found along
with two P
ions (one of the products of the reaction
catalyzed by MAT) in the active site. The Mg
binding
sites have been confirmed from the cobalt atom position found in the
MAT-Co derivative. The two Mg
ions bridge two P
ions, and Mg(1) and Mg(2) ions are surrounded by
Glu
*, Asp
, and Asp
residues,
and Asp
*, Glu
, and Cys
*
residues, respectively. The binding site (Glu
) of the
K
ion is the same as that of one of the UO
ion binding sites in the heavy atom derivative. Consequently,
UO
ion is an inhibitor of the enzyme. The K
ion bound on Glu
has been confirmed by a
site-directed mutagenesis (E42Q). The mutation abolished the monovalent
cation activation and produced an enzyme which has an activity
virtually identical with that of K
-free wild type MAT.
The positively charged amino acid residues, His
,
Lys
, Lys
, and Lys
interact
strongly with the P
ions, suggesting that some of those
amino acid residues are involved in the unusual two-step catalytic
reaction. These amino acid residues as well as the amino acid residues
interacting with the monovalent and divalent metal ions in the active
site are all conserved in the 12 reported sequences of MATs from
various species, indicating that the MATs assume quite similar
three-dimensional structures including the active site geometry.
Therefore, the central features of the mechanism of the catalytic
reactions are probably identical in the enzymes from a wide range of
organisms.
The atomic coordinates (code 1XRA, 1XRB, and 1XRC) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.