From Structural Biology and Biochemistry, Research
Institute, Hospital for Sick Children, Toronto, Ontario M5G 1X8,
Canada, the § Department of Biochemistry, Faculty of
Medicine, University of Toronto, Medical Sciences Building, Toronto,
Ontario M5S 1A8, Canada, the ¶ Department of Chemistry, Portland
State University, Portland, Oregon 97207, the
Medical Research
Service, 151-0, Veterans Affairs Medical Center, Portland, Oregon
97021, and the
Department of
Biochemistry and Molecular Biology, Oregon Health Sciences University,
Portland, Oregon 97201
Received for publication, October 22, 2002, and in revised form, December 16, 2002
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ABSTRACT |
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5'-Methylthioadenosine/S-adenosylhomocysteine
(MTA/AdoHcy) nucleosidase is a key enzyme in a number of critical
biological processes in many microbes. This nucleosidase catalyzes the
irreversible hydrolysis of the N9-C1'
bond of MTA or AdoHcy to form adenine and the corresponding thioribose. The key role of the MTA/AdoHcy nucleosidase in biological methylation, polyamine biosynthesis, methionine recycling, and bacterial quorum sensing has made it an important antimicrobial drug target. The crystal
structures of Escherichia coli MTA/AdoHcy nucleosidase complexed with the transition state analog, formycin A (FMA), and the
nonhydrolyzable substrate analog, 5'-methylthiotubercidin (MTT) have
been solved to 2.2- and 2.0-Å resolution, respectively. These are the
first MTA/AdoHcy nucleosidase structures to be solved in the presence
of inhibitors. These structures clearly identify the residues involved
in substrate binding and catalysis in the active site. Comparisons of
the inhibitor complexes to the adenine-bound MTA/AdoHcy nucleosidase
(Lee, J. E., Cornell, K. A., Riscoe, M. K., and Howell,
P. L. (2001) Structure (Camb.) 9, 941-953) structure provide evidence for a ligand-induced
conformational change in the active site and the substrate preference
of the enzyme. The enzymatic mechanism has been re-examined.
The enzyme 5'-methylthioadenosine
(MTA)1/S-adenosylhomocysteine
(AdoHcy) nucleosidase (EC 3.2.2.9) is a dual substrate specific enzyme
that irreversibly cleaves the N9-C1' bond of
5'-methylthioadenosine and S-adenosylhomocysteine to form
adenine, and 5'-methylthioribose and
S-ribosylhomocysteine, respectively (1).
MTA/AdoHcy nucleosidase has been described as an excellent target for
broad-spectrum antimicrobial drug design (2, 3). The enzyme is not
found in mammalian cells but is found in many microbial pathogens, such
as Staphylococcus aureus, Streptococcus pneumoniae, Mycobacterium tuberculosis,
Haemophilus influenza, Vibrio cholerae, and
Bacillus anthracis. Inhibition of MTA/AdoHcy nucleosidase
should selectively target the pathogenic microbes while leaving the
human host unharmed by increasing cellular levels of MTA and AdoHcy.
The buildup of these molecules will affect four major cellular
functions in microbes. First, the enzyme is important in the recycling
of methionine, an essential amino acid that is energetically expensive
to synthesize (2, 3). Second, the nucleosidase is involved in the
regulation of biological methylation, as AdoHcy is a potent negative
feedback inhibitor of methyltransferases (4). Biological methylation is
critical in all organisms and is responsible for regulating a number of
biological processes including DNA and protein metabolism (5). Third,
MTA/AdoHcy nucleosidase participates in the regulation of polyamine
biosynthesis (6, 7). The role of polyamines is not well understood but they are thought to be important in growth processes. MTA acts as a
potent negative feedback inhibitor of spermidine synthase. Finally, the
nucleosidase has recently been implicated in the quorum sensing pathway
in bacteria (8). Quorum sensing is the phenomenon whereby the
accumulation of small exported organic molecules called autoinducers
enables a bacterial cell to sense the population of bacteria. This
phenomenon was first described in the bioluminescent marine bacterium
Vibrio fischeri (9-11) and has been implicated in the
regulation of virulence factors (12-14) and biofilm formation in many
bacteria (15). When a threshold level of autoinducers is reached,
bacteria trigger a signal transduction cascade that can alter gene
expression (16). MTA/AdoHcy nucleosidase cleaves AdoHcy to generate
S-ribosylhomocysteine and adenine. The enzyme LuxS acts on
S-ribosylhomocysteine to form the autoinducer-2 furanone and
homocysteine (8, 17). Autoinducer-2 has been implicated in intra- and
interspecies communication. Inhibition of the nucleosidase is thought
to prevent the formation of the product
(S-ribosylhomocysteine) needed for LuxS to catalyze the production of autoinducer-2.
Previously we determined the 1.9-Å resolution crystal structure of
MTA/AdoHcy nucleosidase complexed with adenine bound in the active site
(18). The enzyme has a mixed This paper reports the crystal structures of MTA/AdoHcy nucleosidase
complexed with the inhibitors, formycin A (FMA) and
5'-methylthiotubercidin (MTT) (Fig. 1).
These inhibitors have a Ki of 10 and 0.75 µM, respectively (Fig. 1) (19). These are the first
nucleosidase structures complexed with a transition state and
nonhydrolyzable substrate analogs. The structures presented here reveal
the residues involved in nucleoside binding and new insight into the
identity of the catalytic base. Comparisons of the adenine-bound and
the inhibitor-bound MTA/AdoHcy nucleosidase structures provide evidence of inter- and intrasubunit conformational changes in the active site.
These conformational changes are required for substrate binding and
catalysis. Furthermore, these comparisons have allowed a structural
explanation of the substrate preference of the enzyme toward
6-aminopurine nucleosides, an intact ribose, and a 5'-neutral sulfur
atom (19). Taken together, these structures have enabled us to
re-examine the catalytic mechanism involved in the cleavage of the
N9-C1' bond.
Protein Preparation and Crystallization--
The expression and
purification of Escherichia coli MTA/AdoHcy nucleosidase was
reported earlier (20). Commercial sparse matrix screens from Hampton
Research and Emerald Biostructures were set up using purified protein
(15 mg/ml) incubated with 1 mM FMA or 1 mM MTT
on ice for 2 h. Rod-shaped crystals (0.15 × 0.15 × 1.0 mm) were grown at room temperature by the hanging drop vapor diffusion
technique over a 3-week period using 37% (w/v) PEG 200, 100 mM sodium acetate, pH 4.7, 100 mM NaCl, and 8.0 mM cobalt chloride.
Data Collection and Processing--
The FMA and MTT nucleosidase
co-crystals were frozen without use of further cryoprotectant in a
stream of nitrogen gas (100 K). Data for the nucleosidase-FMA complex
were measured on a R-AXIS-IV++ image plate and RUH3R
rotating anode x-ray generator with Osmic optics. Data for the MTT
containing crystal were measured at Station X8C at the National
Synchrotron Light Source (Brookhaven National Laboratories). All data
were processed using the program d*TREK (21). The data collection
statistics are presented in Table I.
Structural Determination and Refinement--
The program CNS
(22) was used for all structure determination and refinement steps. The
FMA-bound nucleosidase structure was determined by molecular
replacement using the adenine-bound E. coli MTA/AdoHcy
nucleosidase (Protein Data Bank code 1JYS) dimer as the search model.
Data between 12- and 5-Å resolution were included in the
cross-rotation and translation functions. The resulting model was then
refined using torsion angle simulated annealing. The interactive
computer graphics program Xfit in the XtalView crystallographic suite
(23) was used to rebuild the initial FMA-containing model. Several
rounds of torsion angle simulated annealing (24) starting at 5000 K
using all data with no
Given that the space group and cell constants of the MTT and FMA-bound
nucleosidase were isomorphous, the MTT-bound nucleosidase structure
could likely have been solved by difference Fourier methods. However,
as an additional verification of our previous result, molecular
replacement was employed with the FMA-complexed dimer as a search
model. Data between 12- and 4-Å resolution were included in the
cross-rotation and translation functions. The resulting model was
subjected to a round of rigid-body refinement using all data between 35 and 2.0 Å in CNS. Superimposition of Structures--
The MTA/AdoHcy nucleosidase
structures were aligned by nonlinear least-squares fit of selected main
chain (N-C Protein Data Bank Accession Codes--
The coordinates of the
formycin A and MTT complexes have been deposited in the Research
Collaboratory for Structural Bioinformatics (RCSB) Protein Data bank
(29), Protein Data Bank codes 1NC3 and 1NC1, respectively.
Structural Determination
The nucleosidase used for crystallization consists of 232 amino
acids and a 10-residue N-terminal extension. The 10-residue N-terminal
extension is part of a larger 31-amino acid 6-histidine fusion tag used
for purification. Chymotrypsin digestion of the enzyme results in the
cleavage of the first 21 residues of the 31-residue tag. In the FMA and
MTT nucleosidase structures, a dimer is present in the asymmetric unit.
In both structures all 232 residues of the protein were clearly visible
in the electron density map and have been modeled. The FMA and MTT
complexed structures were refined to a Rcryst = 19.8% and Rfree = 24.7%, and
Rcryst = 19.0% and Rfree = 22.3%, respectively (Table I).
Analysis of the structures in PROCHECK (28) reveal that none of the
nonglycine residues fall into the disallowed region of the Ramachandran
plot.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
/
structure with a central
9-stranded
-sheet surrounded by six
helices and a 310 helix. Although the primary sequence is not homologous
to any known proteins, its tertiary structure is similar to enzymes in
the purine nucleoside phosphorylase family.
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Fig. 1.
Structures of MTA, FMA, and MTT.
Please note that for ease of comparison, the numbering of atoms in the
purine base of the inhibitors (MTT and FMA) is based on the numbering
convention of MTA and not the IUPAC standard.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
cutoff and a bulk solvent correction were
alternated with manual rebuilding in Xfit. The progress of the
refinement was monitored by reductions in Rcryst
and Rfree factors. Difference Fourier maps
calculated after the initial round of simulated annealing revealed
strong positive electron density corresponding to FMA in the two active
sites. The FMA coordinates were generated by modifying the Protein Data
Bank file of adenosine downloaded from the HIC-UP server (25). The
topology and parameter files for FMA were generated using the XPLO2D
server (26). Water molecules were included into the model during the
later rounds of the refinement based on the presence of a 3
peak in
the
A-weighted Fo
Fc difference electron density maps and at least one hydrogen bond to a protein, inhibitor, or solvent atom. After addition
of the inhibitor and water molecules, alternating rounds of
crystallographic conjugate gradient minimization refinement and model
rebuilding in Xfit were performed.
A-Weighted difference and
2Fo
Fc Fourier maps were calculated and the model was rebuilt using Xfit. The model was subsequently refined using torsion angle-simulated annealing and conjugate gradient minimization refinement, as described above. The MTT inhibitor was
visible as the largest peak in the difference Fourier maps. The MTT
molecule was generated by modifying the coordinates of the MTA ligand
in the MTA phosphorylase structure (Protein Data Bank code 1CG6) (27).
The topology and parameter files were generated using the XPLO2D server
(26). Analysis of both structures using PROCHECK (28) reveal that none
of the nonglycine residues fall into the disallowed region of the
Ramachandran plot. The refinement statistics for the structures are
reported in Table I.
-C) atoms in the central
-sheet using the
program PROFIT (version 6.0), written by G. David Smith. The residue
ranges used in the refinement of the superposition were
Lys2-Ile6,
Glu42-Leu46,
Leu62-Ala77,
Val90-Arg96,
Pro116-Ala121, and
Val187-Val191.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Data collection and refinement statistics
Overall Structure
In the FMA and MTT complexed MTA/AdoHcy nucleosidase structures,
two monomers related by 2-fold noncrystallographic symmetry are present
in the asymmetric unit (Fig.
2a). This is consistent with
our observation that the nucleosidase elutes from an analytical fast
protein liquid chromatography size exclusion column (Superdex-75 HR
10/30) with a retention time equivalent to a 51-kDa protein (18). The
overall topology of the FMA and MTT nucleosidase complexes is similar
to that previously published for the enzyme complexed with adenine
(18). Each subunit has a central 9-stranded mixed -sheet and a
smaller 5
-stranded sheet surrounded by six
-helices and a small
310 helix (Fig. 2b). The root mean square
deviation (r.m.s.d.) between monomers A and B in the FMA- and MTT-bound structures are 0.20 and 0.22 Å, respectively. Strong electron density
was seen in both active sites of the MTA/AdoHcy nucleosidase structures
throughout refinement indicating that each active site is fully
occupied with one bound inhibitor (Fig. 2, c and
d).
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Active Site
The active site of MTA/AdoHcy nucleosidase was previously identified from the adenine-bound nucleosidase complex and was suggested to comprise two parts, an adenine- and a ribose-binding site (18). Given that only a weakly bound adenine molecule was present in the active site of the previous structure, we were only able to define the adenine-binding site with any certainty. Modeling MTA into the active site putatively identified the residues involved in the ribose-binding site. Examination of the FMA and MTT complexes now suggest that the active site can be divided into three regions: 1) the adenine, 2) the ribose, and 3) the 5'-alkylthio-binding sites. Please note that for ease of comparison, the numbering of atoms in the purine base of the inhibitors FMA and MTT will be based on the numbering convention of MTA and not the IUPAC standard (Fig. 1).
Adenine-binding Site--
The adenine-binding site is a deep
pocket formed by 10 and a loop between
8 and
4. Based on the FMA and MTT
complexed structures (Figs. 3 and 4),
this site consists of residues Phe151, Ile152,
Ser196, Asp197, and Ala199.
Phe151 makes a base-stacking interaction with the adenine
ring. An aromatic residue (Phe or Tyr) is structurally conserved
between all known MTA/AdoHcy nucleosidases and the purine nucleoside
phosphorylase family of enzymes. The main chain carbonyl oxygen and
amide nitrogen of Ile152 are in excellent hydrogen bonding
distance to the amino group (N6) and N1 of the
adenine base, respectively. Additional interactions exist between
O
2 and O
1 of Asp197 and the
N7 and N6 amino group of the adenine base,
respectively, in the FMA-bound nucleosidase structure. In the
MTT-complexed model only a hydrogen bond interaction between
O
1 of Asp197 and the N6 amino
group exists, the N7 purine base position is replaced by a
carbon in MTT. Ser196 and Ala199 do not
interact with the inhibitor directly but are involved in orienting the
side chain of the putative catalytic acid, Asp197. The
Ser196 O
and amide nitrogen of
Ala199 help anchor the Asp197 side chain by
making hydrogen bonds to the O
2 and O
1 of
Asp197, respectively. In the FMA-bound structure,
N8 makes an additional hydrogen bond to the
O
hydrogen of Ser76.
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Ribose-binding Site--
The ribose-binding site is primarily made
up of six residues, Met9, Ser76,
Met173, Glu174, Arg193, and
Phe207 (Figs. 3 and 4). Previously, only residues
Met173, Glu174, and Arg193 were
proposed to be involved in saccharide binding. The ribose in both the
FMA and MTT complexed structures are anchored by both hydrogen bonds
and van der Waals contacts. The hydrogen-bonding network consists of
four direct and one water-mediated hydrogen bond to the enzyme. Two
hydrogen bonds are made by the O1 and O
2
of Glu174 to the O3' and O2'
hydroxyls of the ribose, respectively. A third hydrogen bond is made
from the backbone amide hydrogen of Met173 to the
O2' hydroxyl of the ribose, whereas the hydrophobic side
chain of Met173 packs against the hydrophobic face of the
ribose moiety. This interaction is thought to help stabilize and
properly orient the sugar. The fourth direct hydrogen bond is made
between the O
hydrogen of Ser76 and the
O4' oxygen of the ribose. The water-mediated hydrogen bond
interactions are between WAT3 and the O2' and
O3' ribosyl hydroxyls. van der Waals contacts are made
between Met9 S
and C
and the
O4' and C4' positions of the ribose,
respectively. In addition, Phe207 C
makes a
van der Waals contact to the C4' position.
5'-Alkylthio-binding Site--
The 5' substituents of the
inhibitors are primarily coordinated by hydrophobic interactions. This
hydrophobic pocket is made up of residues Met9,
Ile50, Val102, Phe105,
Pro113, Phe151, Met173, and
Phe207 (Figs. 3 and 4). Previously, we had suggested that
residues Ile50, Val102, Phe105,
Tyr107, Pro113, Phe151, and
Phe207 would make van der Waals-type interactions with the
5'-alkylthio group, with residues Val102,
Phe105, Tyr107, and Pro113 being
donated from a neighboring subunit (Fig. 3, c and
d, in orange). The FMA- and MTT-bound structures
confirm the participation of the previously proposed residues in the
binding of the 5' moiety with three exceptions. The 5'-alkylthio
binding pocket also includes residues Met9 and
Met173, as these residues form van der Waals type
interactions to the 5'-methylthio group. The hydrophobic binding pocket
does not include Tyr107 in the presence of MTT, FMA, or
MTA. In the FMA- and MTT-complexed structures, Tyr107 is
~6 Å away from the 5'-methyl moiety. Although Tyr107 is
too far to play a major role in the binding of the methylthio group,
the O of Tyr107 may be engaged in a hydrogen
bond with the carboxylic acid or amino group of the homocysteinyl
moiety of AdoHcy.
Conformation of the Nucleosides--
In solution, MTA and AdoHcy
are found predominantly in the anti-conformation and exhibit
a glycosidic torsion angle of 45° (C8-N9-C1'-O4')
(30). The FMA and MTT nucleosides bound to MTA/AdoHcy nucleosidase are
found in a high syn-conformation relative to the glycosidic bond. This energetically unfavorable orientation of the substrate puts
strain on the substrate and presumably favors the cleavage of the
N9-C1' bond. In the human MTAP structure, the
bound MTA also exhibited a high syn-conformation with a
glycosidic torsion angle of 54° (27). In the FMA- and MTT-bound
structures, FMA and MTT have glycosidic torsion angles of
68° and
64°, respectively. The superimposition of the enzyme-bound MTA,
FMA, and MTT structures confirm the similarity in the glycosidic
torsion angle (Fig. 5). However, the
superimposition also reveals differences in the torsion angles of the
5'-alkylthio tail. MTA bound in the human MTAP structure has a
5'-alkylthio torsion angle
(C4'-C5'-S5-CS) of
39°, whereas MTT in the nucleosidase has a torsion angle of
83°
(Fig. 5).
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Conformational Changes
Three structures of E. coli MTA/AdoHcy nucleosidase
have now been determined, and the protein complexed with adenine, FMA, and MTT. A detailed comparison of these structures has been carried out
to determine whether any conformational changes occur on substrate binding. The structures were superimposed using residues in the central
-sheet as described under "Experimental Procedures." A
superimposition of the dimeric FMA and MTT inhibitor complexes based on
the
-sheets from both subunits revealed an overall r.m.s.d. of 0.3 Å for the main chain atoms (N-C
-C). The two
structures are virtually identical and for the purpose of the following
discussion, we have used the MTT complex as the representative model to
describe the structural changes observed. A r.m.s.d. plot of all
C
atoms in the monomer of the adenine- and MTT-complexed
structures highlights six major regions of the structure that deviate
by more than 2.0 Å (labeled 1-6 in Fig.
6a). However, these six
regions can be simplified into three areas of conformational change, as changes in region 1 are propagated to regions 4 and 6 and region 2 affects region 5. As an additional check on the validity of our
superimposition, the adenine-bound structure was superimposed onto the
MTT-complexed structure based on the adenine moiety. The r.m.s.d. plot
of the C
positions shows similar regions of
conformational change. Stereo van der Waals diagrams before (Fig.
6b) and after binding MTT (Fig. 6c) reveal the
closure of the active site and the formation of a well defined
hydrophobic pocket on substrate/inhibitor binding.
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Adenine-binding Site Conformational Changes--
A comparison of
the adenine and MTT-bound structures of MTA/AdoHcy nucleosidase reveals
numerous backbone and side chain movements (Fig.
7). The largest movement is a loop to
helix transition in residues 199-207. In the adenine-bound
nucleosidase structure (Protein Data Bank code 1JYS), the electron
density for residues 202-205 was either weak or missing. These
residues were disordered in a solvent-exposed region of the enzyme.
Upon binding of FMA or MTT, residues 200-211 form a N-terminal
extension to the pre-existing 6 helix (residues 206-229). This
extended
6 helix is not continuous as it has a ~45° kink at
Leu211 (Fig. 7b). This conformational change
results in a closure of the active site and the movement of residues
199-201 into the adenine-binding site where they make key hydrogen
bonds to the enzyme, as described above. The amide nitrogen of
Ala199 moves ~2 Å to make a hydrogen bond to
Asp197 O
2. The hydrogen bond together with
one made between Asp197 O
1 and
Ser196 O
are proposed to reposition the side
chain of Asp197 closer to the N7 atom of the
purine ring. The N-terminal extension of the
6 helix and the
additional hydrogen bonds made by Ala199 are likely
important in closing the adenine-binding cavity. Thus, the hydrogen
bond formed between the carbonyl oxygen of Ala199 and the
amide nitrogen of Gly154 may serve to pull in residues
150-152 and hence shrink the adenine-binding cavity. Residues 150-152
shift ~1.2 Å, thus positioning Phe151 closer to
the purine ring (Fig. 7c).
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The adenine molecule seen in the 1JYS structure is loosely bound as
evident from the long hydrogen bond between the Asp197 and
the N7 of adenine (~3.9 Å), and by the higher than
average B-factors for the adenine (~55 Å2)
versus the rest of the protein (33.7 Å2). The
hydrogen bond between the N7 of the purine ring and the
O hydrogen of Asp197 in the FMA-complexed
structure is now 2.7 Å (Fig. 4a). The N-terminal extension
of the
6 helix (residues 200-211) also allows Phe207 to
move ~7.5 Å toward the 5'-alkylthio group to help complete the
hydrophobic pocket. Phe151 and Phe207 make a
herringbone interaction to Phe105 of the neighboring subunit.
Intramolecular 5'-Alkylthio-binding Site Conformational
Changes--
A major intrasubunit conformational change involves a
movement of residues 7-42. Residues Met9 and
Glu12 in the 1 helix undergo a movement into the active
site upon ligand binding and this conformational change is propagated
to residues in
-strands,
2,
3, and
4 (Fig. 7d).
Met9 moves toward the 5'-alkylthio-binding site. The side
chain of Met9 packs against the 5'-methylthio group of MTT
and helps create part of the hydrophobic pocket. Glu12 does
not bind the substrate but hydrogen bonds a water molecule (WAT3) that
is in close proximity to the C1' position of MTT (~3.5
Å). The conformational change of Glu12 creates hydrogen
bonds to the amide hydrogen of Met9 and Ser76,
and the O
of Ser76. We propose that
Glu12 is critical for the deprotonation of the nucleophilic
water required for the hydrolysis of the
N9-C1' bond (see later discussion).
The importance of the N-terminal residues of MTA/AdoHcy nucleosidase
was previously analyzed with a truncation mutant of the first 8 residues (19). A high degree of sequence similarity exists with the
first 8 residues of the human MTAP (VKIGIIGG) and the E. coli MTA/AdoHcy nucleosidase (MKIGIIGA). Cornell et al.
(19) postulated that this region is involved in MTA or AdoHcy binding.
Although the inhibitor-enzyme complexed structures show that residues
1-8 do not play a direct role in the binding of the substrate,
elimination of these residues may affect the local structural stability
of this region and therefore hinder the conformational changes required
in the 1 helix for catalysis, particularly residues Met9
and Glu12.
Intermolecular 5'-Alkylthio-binding Site Conformational Changes-- MTA/AdoHcy nucleosidase is thought to be a dimer based on the donation of residues in the 100s loop from a neighboring subunit. At the 5'-alkylthio tail, residues Val102, Phe105, Tyr107, and Pro113 were proposed to help make hydrophobic interactions to the 5'-methylthio or homocysteinyl group. The identity of these residues was determined using a superimposed model of MTA taken from the human MTAP structure (27). However, the modeled MTA molecule revealed that Tyr107, Pro113, and Val102 are ~8.7, 7.7, and 6.9 Å, respectively, away from the nearest atom in the 5'-methylthio group. This led us to suggest that an inter-subunit conformational change was probably needed to bring the residues into van der Waals contacts. The only residue modeled close enough to the 5' position of the substrate was Phe105 (4.4 Å).
The C superimposition of the adenine and MTT-bound dimer
reveals a conserved dimer interface (Fig.
8a). Upon further inspection, the 100s loop containing the donated residues was not found to undergo
a major conformational change but rather a smaller loop translation by
only some of the donated residues (Fig. 8b). Residues Val102 and Pro113 do not seem to undergo a
conformational change but residues Phe105 and
Tyr107 move ~1.5 Å closer to the methylthio group in
both structures. This result is not surprising given that the
5'-methylthio group in the enzyme complexed with MTT is in a different
conformation than the previously modeled MTA molecule. Residues
Val102, Phe105, and Pro113 are
actually in good van der Waals proximity to the methylthio tail of MTT.
Even with the conformational changes seen in Tyr107, this
residue is still too far removed (5.8 Å) to make van der Waals
contacts with the methylthio group. However, as noted above Tyr107 O
could potentially hydrogen bond
with the carboxyl moiety of the longer homocysteinyl group from
AdoHcy.
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Implications for Catalysis
MTA/AdoHcy nucleosidase is an inverting hydrolase that irreversibly cleaves the N9-C1' bond. The reaction catalyzed is similar to the N-ribohydrolase class of enzymes, which includes N-ribosyl hydrolases and N-ribosyltransferases, as well as the purine nucleoside phosphorylases. Given the similarities in structure and the similarities in the reactions catalyzed by the purine nucleoside phosphorylases, the nucleosidase has been proposed to share a similar two-step catalytic mechanism (18, 27, 31-34). The first step of the common catalytic mechanism requires a proton donation to the N7 atom of the adenine base. The proton donation allows a hydrogen bond to be formed between the N7 and enzyme. The purine base is electron withdrawing (electron pull) and is stabilized by a flow of electrons from the ribosyl O4' (electron push). This migration of electrons favors the elongation and the cleavage of the N9-C1' bond to form the oxocarbenium-like intermediate. The existence of the oxocarbenium intermediate is supported by kinetic isotope effect experiments (33). Following the formation of the oxocarbenium intermediate, an SN1-type attack by an activated nucleophilic water molecule occurs at the anomeric carbon to liberate the thioribose. Although the adenine-bound nucleosidase complex provided valuable structural insight into the catalytic mechanism, many specific details remain unanswered. In light of the inhibitor-enzyme complexes, we have re-examined the catalytic mechanism and have identified the catalytic base and nucleophilic water.
Identifying the Catalytic Base and Water-- Two water molecules (WAT2 and WAT3) were seen in the active site in proximity to the C1' atom of the modeled MTA molecule. WAT2 was originally proposed to be the nucleophilic water because of its proximity to the anomeric carbon (~4.2 Å). WAT2 was also hydrogen bonded to Glu172, which could act as a catalytic base. WAT3 was also in close proximity (~4.0 Å) but was not chosen because there was no neighboring catalytic base to activate the water. Analysis of the FMA- and MTT-bound structure reveals the displacement of WAT2 by the side chain of Arg193 and clearly identifies WAT3 as the catalytic water. In the MTT-bound structure, WAT3 is 3.6 Å from the anomeric carbon and 2.6 Å from Glu12 (Fig. 4b). In addition, WAT3 is in an excellent position for a rear attack on the ribosyl C1'. Glu12 is proposed to be the catalytic base because of its proximity to the nucleophilic water. Future site-directed mutagenesis experiments will confirm the role of Glu12 in catalysis.
Role of the 5' Thio Group--
In the purine nucleoside
phosphorylases, sandwiching of the ribosyl O4' by two
electron-rich groups, the negatively charged O4 phosphate
and the 5' hydroxyl, is thought to push the flow of electrons from the
ribosyl moiety to the purine base. When we superimposed MTA into the
adenine-bound nucleosidase structure (18), our model identified the
5'-thio and the Ser76 O groups as being the
electron-rich atoms in proximity to the O4' ribose. This
observation seemed consistent with the biochemical data because
modification of the sulfur to a less electronegative group such as
selenium was known to decrease catalytic activity (19). However, the
MTT complexed nucleosidase structure reveals that the 5'-thio group is
in a different conformation than that seen in the human MTAP structure
(Fig. 5) and likely does not play a role in the electron push. The two
closest electron-rich atoms to the ribosyl O4' are
Ser76 O
(3.5 Å) and the nucleophilic water
(3.7 Å). However, these groups do not sandwich the O4'
ribose and instead are found on the same side of the ribose. In
addition, Ser76 is not conserved in all nucleosidase
species (Fig. 9) and the negatively
charged nucleophilic water seems to be too far (3.7 Å) from the
ribosyl O4' to facilitate an electron push. The current
structures suggest that MTA/AdoHcy nucleosidase does not require the
ribosyl O4' to be sandwiched between two electron-rich
groups.
|
Although the 5' sulfur atom is likely not involved in the electron push
and pull mechanism, it may have another role in catalysis. The FMA- and
MTT-bound nucleosidases reveal structural differences that may provide
an alternative explanation for the importance of the 5'-alkylthio
sulfur. The FMA-bound structure reveals a different Glu12
conformation than the MTT-bound structure (Figs. 3 and 4). In the
MTT-bound structure, Glu12 O1 makes hydrogen
bond interactions to the amide nitrogen of Ala8 and
Met9 and the nucleophilic water, and Glu12
O
2 hydrogen bonds with the amide nitrogen and
O
of Ser76 and the nucleophilic water. A
total of five hydrogen bonds are made to Glu12. The
positioning of Glu12 in the MTT-bound structure is thought
to be the catalytically active state with Glu12 in a
position to activate the nucleophilic water. Unlike the MTT-complexed
structure, Glu12 in the FMA-bound structure has a different
conformation in each of the two active sites. In monomer A,
Glu12 O
1 is hydrogen bonded to
Thr74 O
1, Ser218
O
, and Ser219 N and Glu12
O
2 is hydrogen bonded to Gly75 N,
Thr74 O
1, and Ser218
O
. Glu12 lacks the crucial hydrogen bonds to
the nucleophilic water (WAT3) and Ser76. In monomer B,
Glu12 hydrogen bonds to the nucleophilic water (WAT3), the
amide nitrogen of Met9 and Glu12 but it lacks a
hydrogen bond to the Ser76 O
.
Glu12 also makes three less hydrogen bonds and is farther
away from the nucleophilic water (3.1 Å) than in the MTT-bound complex
(2.6 Å). The binding of ligands with 5' hydroxyl groups seems to
disrupt the proper positioning of the catalytic base,
Glu12. This would account for the decrease in nucleosidase
activity for 5'-hydroxyl containing nucleosides such as adenosine
(19).
Substrate Specificity--
The inhibitor MTT
(Ki = 0.75 µM) resembles the natural
substrate MTA (Fig. 1) (Km = 0.5 µM)
and has a very similar binding constant (19). Replacement of the
methylthio group with a hydroxyl in FMA causes a ~20-fold decrease in
the binding affinity (Ki = 10 µM)
(19). The FMA- and MTT-inhibitor complexed structures reveal evidence
to account for the lowered FMA binding affinity. FMA has a weaker
binding constant because of the loss of van der Waals interactions made
in the 5'-alkylthio hydrophobic pocket. In the MTT-complexed
nucleosidase, the methylthio group makes van der Waals interactions to
Met9, Ile50, Phe105,
Phe151, Met173, and Phe207. In the
FMA-bound nucleosidase, the 5'-hydroxyl has only van der Waals
interactions to residues Ile50 and Met173. In
addition, the unsatisfied hydrogen bond of the 5'-hydroxyl makes it
further energetically unfavored. The decrease in FMA affinity may be
partially offset by an additional hydrogen bond between
Ser76 O and the N8 position of
the purine.
Order of Product Release-- The order of substrate binding and product release in MTA/AdoHcy nucleosidase has not been experimentally characterized. The structure reveals that the adenine-binding site is deeply buried and the ribose- and 5'-alkylthio-binding sites are solvent exposed (Fig. 6c). This architecture suggests that the thioribose product is released first followed by the purine base. The purine base may not be released immediately as adenine is known to be a weak competitive inhibitor of E. coli MTA/AdoHcy nucleosidase (Ki = 300 µM) (19). In the adenine-complexed nucleosidase structure, the loosely bound adenine was salvaged from the cell and remained in the active site throughout purification (18). The residual adenine is likely only displaced when a new substrate molecule (MTA or AdoHcy) binds.
Conservation of Active Site Residues--
A sequence alignment of
nucleosidases from 8 different organisms was performed. The residues
involved in catalysis and substrate binding have varying degrees of
conservation but their overall chemical nature is generally maintained
(Fig. 9). Not surprisingly, the catalytic residues (Glu12
and Asp197) are invariant as are the residues involved in
coordinating the catalytic water (Arg193 and
Glu174) and orienting the catalytic acid
(Ser196). The catalytic acid (Asp197) is also
held in place by the main chain amide of Ala199. In
H. influenzae Ala199 is replaced by a glycine.
This substitution is not expected to perturb the local structure or
affect the positioning of Asp197. Ile152 and
Met173 also make main chain hydrogen bonds to the
substrate. Whereas some variation is observed at position 152 with
substitutions to leucine and valine, Met173 is strictly
conserved in keeping with the additional role its side chain plays in
stabilizing and orienting the ribose moiety of the substrate. With the
exception of Ser76, the other adenine- and ribose-binding
site residues that interact through their side chains are fairly well
conserved. The hydrophobic nature of Phe151 and the
5'-alkylthio binding residues Met9, Ile50, and
Phe207 are all maintained in the aligned nucleosidase
species. The hydrophobic residues donated from the neighboring subunit
(Val102, Phe105, Tyr107, and
Pro113) are, however, more variable. Val102
maintains its hydrophobic nature with substitutions to alanine or
leucine, but Phe105 and Tyr107 have been
substituted in certain species with aspartate and histidine, respectively. It is unclear what role the aspartate may play in substrate binding. Although the replacement of tyrosine to histidine may look like a fairly drastic substitution, the proposed hydrogen bond
between Tyr107 and the homocysteinyl tail of AdoHcy could
potentially be maintained with a histidine. Pro113 is well
conserved, with the exception of an alanine in S. pneumoniae.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. G. David Smith for the program PROFIT v6.0 and help and insight into the analysis of the structures and Dr. Yuri D. Lobsanov for assistance at the Hospital for Sick Children x-ray diffraction facility. Fig. 1 was kindly prepared by Tom Rodinger.
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FOOTNOTES |
---|
* This work was supported in part by Canadian Institutes for Health Research Grant 43998 (to P. L. H.) and a doctoral research award (to J. E. L.). Station X8-C was supported by the United States Department of Energy and a Multiuser maintenance grant from the Canadian Institutes for Health Research and the Natural Science and Engineering Research Council of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1NC3 and 1NC1 for the formycin A and MTT complexes) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
** Supported by the United States Veterans Affairs Medical Research Program.
§§ To whom correspondence should be addressed. Tel.: 416-813-5378; Fax: 416-813-5022; E-mail: howell@sickkids.ca.
Published, JBC Papers in Press, December 20, 2002, DOI 10.1074/jbc.M210836200
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ABBREVIATIONS |
---|
The abbreviations used are: MTA, 5'-methylthioadenosine; AdoHcy, S-adenosylhomocysteine; FMA, formycin A; MTT, 5'-methylthiotubercidin; MTAP, 5'-methylthioadenosine phosphorylase; r.m.s.d., root mean square deviation.
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