|
|
PLoS Biol. 2008 August; 6(8): e206. Published online 2008 August 26. doi: 10.1371/journal.pbio.0060206. | PMCID: PMC2525682 |
Copyright : © 2008 Min et al. This is an
open-access article distributed under the terms of the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are credited. An Enzymatic Atavist Revealed in Dual Pathways for Water Activation Donghong Min,1 Helen R Josephine,2 Hongzhi Li,1 Clemens Lakner,3 Iain S MacPherson,2 Gavin J. P Naylor,3 David Swofford,3 Lizbeth Hedstrom,24* and Wei Yang1567* 1 School of Computational Science, Florida State University, Tallahassee, Florida, United States of America 2 Department of Biochemistry, Brandeis University, Waltham, Massachusetts, United States of America 3 Department of Biological Sciences, Florida State University, Tallahassee, Florida, United States of America 4 Department of Chemistry, Brandeis University, Waltham, Massachusetts, United States of America 5 Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida, United States of America 6 Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida, United States of America 7 College of Life Science, Nankai University, Tianjin 300071, China Daniel Herschlag, Academic Editor Stanford University, United States of America Received October 30, 2007; Accepted July 15, 2008. Inosine monophosphate dehydrogenase (IMPDH) catalyzes an essential step in the
biosynthesis of guanine nucleotides. This reaction involves two different
chemical transformations, an NAD-linked redox reaction and a hydrolase reaction,
that utilize mutually exclusive protein conformations with distinct catalytic
residues. How did Nature construct such a complicated catalyst? Here we employ a
“Wang-Landau” metadynamics algorithm in hybrid quantum
mechanical/molecular mechanical (QM/MM) simulations to investigate the mechanism
of the hydrolase reaction. These simulations show that the lowest energy pathway
utilizes Arg418 as the base that activates water, in remarkable agreement with
previous experiments. Surprisingly, the simulations also reveal a second pathway
for water activation involving a proton relay from Thr321 to Glu431. The energy
barrier for the Thr321 pathway is similar to the barrier observed experimentally
when Arg418 is removed by mutation. The Thr321 pathway dominates at low pH when
Arg418 is protonated, which predicts that the substitution of Glu431 with Gln
will shift the pH-rate profile to the right. This prediction is confirmed in
subsequent experiments. Phylogenetic analysis suggests that the Thr321 pathway
was present in the ancestral enzyme, but was lost when the eukaryotic lineage
diverged. We propose that the primordial IMPDH utilized the Thr321 pathway
exclusively, and that this mechanism became obsolete when the more sophisticated
catalytic machinery of the Arg418 pathway was installed. Thus, our simulations
provide an unanticipated window into the evolution of a complex enzyme. Many enzymes have the remarkable ability to catalyze several different
chemical transformations. For example, IMP dehydrogenase catalyzes both an
NAD-linked redox reaction and a hydrolase reaction. These reactions utilize
distinct catalytic residues and protein conformations. How did Nature
construct such a complicated catalyst? While using computational methods to
investigate the mechanism of the hydrolase reaction, we have discovered that
IMP dehydrogenase contains two sets of catalytic residues to activate water.
Importantly, the simulations are in good agreement with previous
experimental observations and are further validated by subsequent
experiments. Phylogenetic analysis suggests that the simpler, less efficient
catalytic machinery was present in the ancestral enzyme, but was lost when
the eukaryotic lineage diverged. We propose that the primordial IMP
dehydrogenase utilized the less efficient machinery exclusively, and that
this mechanism became obsolete when the more sophisticated catalytic
machinery evolved. The presence of the less efficient machinery could
facilitate adaptation, making the evolutionary challenge of the IMPDH
reaction much less formidable. Thus our simulations provide an unanticipated
window into the evolution of a complex enzyme. Textbooks extol the extraordinary catalytic power and specificity of enzymes, yet the
ability of many enzymes to promote several different chemical transformations is
even more remarkable. In examples such as the polyketide synthases, the substrate is
tethered to a flexible linker and swings gymnastically between separate active sites
[ 1]. The evolutionary path to the assembly of such enzymes seems
reasonably straightforward: gene duplication and recombination, followed by
optimization of a promiscuous activity [ 2– 6]. In contrast, enzymes such as IMP
dehydrogenase (IMPDH) move around a stationary substrate, restructuring the active
site to accommodate different transition states [ 7]. Such enzymes pose an evolutionary
conundrum: it seems unlikely that Nature could simultaneously install multiple sets
of catalytic machinery into the ancestral protein. IMPDH controls the entry of
purines into the guanine nucleotide pool, which suggests that the origins of IMPDH
are primordial, so the ancestral IMPDH probably utilized a simpler catalytic
strategy. IMPDH catalyzes two very different chemical transformations: (1) a dehydrogenase
reaction between IMP and NAD + that produces a Cys319-linked
intermediate E-XMP* and NADH, and (2) a hydrolysis reaction that
releases XMP (A)
[ 7, 8]. A mobile flap is
open during the hydride transfer reaction, permitting the association of
NAD +. After NADH departs, this flap occupies the
dinucleotide site, carrying Arg418 and Tyr419 into the active site and converting
the enzyme into a hydrolase (B). Thus, the dehydrogenase and hydrolase reactions utilize mutually
exclusive conformations of the active site. All enzymes that catalyze hydrolysis reactions have some strategy to activate water.
This strategy has been difficult to recognize in IMPDH because the hydrolytic water
interacts with three residues that are usually protonated at physiological pH:
Thr321, Arg418, and Tyr419 (C) [ 9]. The rate of the hydrolysis step decreases by a factor of
10 3 when Arg418 is substituted with Ala or Gln, whereas a decrease of
approximately 20 is observed when Tyr419 is substituted with Phe [ 10, 11]. Neither Arg418 nor Tyr419 is
involved in the dehydrogenase reaction, as expected, given their position on the
mobile flap. In contrast, Thr321 is found on the same loop as the catalytic Cys319,
and both the dehydrogenase and hydrolysis reactions are decreased by a factor of 20
when this residue is substituted [ 11]. These observations suggest that
Arg418 is the most likely candidate for the role of general base in the IMPDH
reaction [ 11, 12]. We performed a series of hybrid quantum mechanical/molecular mechanical (QM/MM)
simulations to further investigate the mechanism of the hydrolysis reaction of
IMPDH. Surprisingly, these simulations find that IMPDH possesses two mechanisms to
activate water: the Arg418 pathway as previously proposed, and a second pathway
utilizing Thr321. Phylogenetic analysis indicates that the Thr321 pathway was
present in the ancestral enzyme. These observations suggest that the primordial
IMPDH used the Thr321 pathway exclusively, and elimination of the Arg418 pathway by
mutation of modern IMPDH creates an enzymatic atavist. We have applied computational methods to further investigate the mechanism of water
activation in IMPDH, employing a “Wang-Landau” metadynamics
algorithm [ 13] in hybrid quantum mechanical/molecular mechanical (QM/MM)
simulations [ 14– 21]. The Wang-Landau recursion procedure adaptively updates the
height of the basis Gaussian, which allows the metadynamics algorithm to be realized
in a more robust and efficient fashion. The simulation models were derived from the
crystal structure of Tritrichomonas
foetus IMPDH in complex with mizoribine monophosphate (MZP)
(Protein Data Bank [PDB] accession code 1PVN), which
describes the closed conformation of the hydrolysis reaction [ 9].
E-XMP* was modeled based on the guanine residue topology and parameters
in CHARMM 22 [ 22]. The atoms in the reaction centers (colored in red in –) were treated quantum
mechanically using the self-consistent charge density-functional tight-binding
(SCCDFTB) potential [ 23], and the rest of the system (colored in blue in –) was treated classically using
the CHARMM 22 force field [ 22]. The molecular dynamics simulations were performed with
generalized solvent boundary conditions (GSBP) [ 24, 25]. Further details on the simulations are provided in
Figure
S1. Simulation of the Hydrolysis Reaction When Arg418 Is Neutral When Arg418 is deprotonated in the starting condition, the lowest energy pathway
for the hydrolysis reaction involves the transfer of a proton to the neutral
Arg418 (the Arg418 pathway, A–C). Proton transfer is virtually complete at the transition state, and
the developing hydroxide is stabilized by interactions with Tyr419, Thr321, and
another water molecule. Importantly, a stable hydroxide intermediate is not
observed; the developing hydroxide instantaneously reacts with
E-XMP*. These results are in remarkable agreement with experimental
observations: solvent isotope effects (SIE) demonstrate that proton transfer is
rate limiting (SIE = ~2 [ 11]), and
Bronsted analysis indicates that proton transfer is virtually complete in the
transition state (β = ~1 [ 12]). However,
the calculated energy barrier is only 8.0 kcal/mol, much less than the
experimentally observed barrier of 16 kcal/mol [ 10]. This
difference may reflect uncertainties in the calculation, but we believe this is
unlikely. A more intriguing source of discrepancy arises from the starting
condition of neutral Arg418; if only a small fraction of the enzyme exists in
this state, the energy barrier will be correspondingly increased. Indeed, if the
pK a of Arg418 is 12.5, as for a typical Arg residue, the barrier
would be increased by approximately 6 kcal/mol. Tyr419 May Be a Surrogate General Base in the Absence of Arg418 The pK a of a Tyr residue is usually two units lower than an Arg, which
suggests that a deprotonated Tyr419 might activate water while Arg418 remains
protonated. Further simulations argue against such a mechanism; instead, the
deprotonated, negatively charged Tyr419 interacts strongly with positively
charged Arg418 and cannot interact with water. Therefore, Tyr419 is unlikely to
play the role of general base in the wild-type enzyme. However, the situation
changes when Arg418 is substituted with Gln: now the Tyr419 phenolate can accept
a proton from water. The barrier is approximately 17 kcal/mol (). Assuming a
pK a of 10, as is usual for a Tyr residue, then deprotonation of
Tyr419 will further increase the barrier to 21–22 kcal/mol, which is
very similar to the barrier observed in the reactions of the Arg418Gln and
Arg418Ala variants (~20 kcal/mol [ 10, 11]). As above, the simulations
suggest that proton transfer is rate limiting and essentially complete in the
transition state. Whereas the landscape contains a shallow valley suggesting the
presence of a hydroxide intermediate, the barriers are less than a kT, so the
intermediate would not have a finite lifetime. This simulation is generally
consistent with experiments, where SIEs of 3–5 are observed when
Arg418 is substituted [ 11]. However, the magnitude of these SIEs is greater
than expected if the transition state is indeed late as suggested by the
simulations. Interestingly, no activity is observed in the Arg418Ala/Tyr419Phe
double mutant, though this fact may equally well be attributed to the inability
to form the closed conformation required for the hydrolysis reaction as to the
loss of the general base catalyst [ 12]. Together, the simulations
and experiments suggest Tyr419 may act as a surrogate general base in the
absence of Arg418. Similar surrogate residues have been invoked to explain residual activity in
other enzyme systems [ 26]. In RNase T1, His40 residue assumes the role the
general base when Glu58 is substituted with Ala [ 27]. Similarly,
in ketosteroid isomerase, Asp99 may catalyze proton transfers in the Asp38Ala
variant [ 28]. Water or buffer molecules can also replace the function
of missing catalytic residues [ 29, 30]. These examples illustrate
the resilience and plasticity of enzyme catalysis. Simulation of the Hydrolysis Reaction When Arg418 Is Protonated Surprisingly, the simulations suggest a second pathway for water activation when
the starting condition is protonated Arg418: Thr321 abstracts a proton from
water while simultaneously transferring its own proton to Glu431 (). As in the Arg418
pathway, the developing hydroxide is stabilized by Tyr419 and another water
molecule, and the hydroxide attack occurs instantaneously; the protonated Arg418
also stabilizes the developing hydroxide by 1–2 kcal/mol. The
calculated free energy barrier for the Thr321 pathway is approximately 20
kcal/mol. The proton transfers are simultaneous and rate limiting. When a
simulation was performed with Glu431 treated as a molecular mechanical (MM)
residue, which eliminates the possibility of proton transfer while maintaining
electrostatic interactions, the energy barrier increases to at least 35 kcal/mol
( Figure
S2). Likewise, when Glu431 is substituted with Gln, the barrier increases
to at least 38 kcal/mol. Therefore, the presence of Glu431 is essential for the
operation of the Thr321 pathway. Experimental Verification of the Thr321 Pathway The simulations suggest that the Thr321 pathway is favored at low pH, whereas the
Arg418 pathway becomes dominant at high pH, which predicts that the pH-rate
profile will shift to the right when the Thr321 pathway is disrupted by the
Glu431Gln mutation. This prediction was confirmed experimentally (): the Glu431Gln
mutation shifts the pK a from 7.2 ± 0.1 to 7.6
± 0.1, but has only a small effect on the pH-independent value of
kcat ( kcat
= 2.2 and 1.4 s −1 for wild type and
Glu431Gln, respectively; these values are in good agreement with previous
reports [ 31, 32]). Assuming that the pK a shift is entirely
attributable to the loss of the Thr321 pathway, the barrier for the Thr321
pathway is approximately 19 kcal/mol, as predicted by the simulations (see Figure
S3). When Arg418 is substituted with Gln, the barrier for the Thr321 pathway is
approximately 21 kcal/mol, which is similar to the barrier observed
experimentally in the Arg418Ala and Arg418Gln variants [ 10, 11]. Therefore, both the Thr321
pathway and the Tyr419 pathway can account for the residual activity of the
Arg418Ala and Arg418Gln variants. However, since the Thr321 pathway involves the
simultaneous transfer of two protons, this pathway can account for the large
solvent isotope effects observed in the Arg418 variants (SIE =
3–5 [ 10, 11]). Therefore, we constructed the Arg418Gln/Glu431Gln
variant, which should disrupt the Thr321 pathway but leave the Tyr419 pathway
intact. The simulations predict that the activity of this variant should be
approximately the same as the Arg418Gln, but that the solvent isotope effect
should be reduced. These predictions were confirmed in subsequent experiments:
(1) the value of kcat for Arg418Gln/Glu431Gln is
decreased by 50% relative to that of Arg418Gln, as expected if the
Thr321 pathway was lost (0.0020 ± 0.0002 s −1
and 0.0040 ± 0.0004 s −1, respectively); and
(2) though the errors on the SIE are larger than ideal, nonetheless, a smaller
SIE is observed in the reaction of Arg418Gln/Glu431Gln, consistent with the loss
of the Thr321 pathway (SIE = 2.1 ± 0.3 and 2.3
± 0.4 for Arg418Gln/Glu431Gln in two independent determinations
versus 2.9 ± 0.5 for Arg418Gln and 5 ± 2 for Arg418Ala
[ 10, 11]). These
experiments confirm the operation of the Thr321 pathway in IMPDH. GMP Reductase and the Evolutionary Origins of the Thr321 and Arg418 Pathways To the best of our knowledge, the presence of dual mechanisms for water
activation in an enzyme active site is unprecedented. Why would an enzyme have
two pathways to accomplish the same task? We believe the Thr321 pathway may be
vestige of evolution, and phylogenetic analysis is consistent with this
hypothesis (; see
Figure
S4 for the complete phylogenetic tree). The closest relative of IMPDH is
GMP reductase (GMPR), which catalyzes the conversion of GMP to IMP and ammonia
with concomitant oxidation of NADPH () [ 33]. Cys319, Thr321, and Glu341
are also conserved in GMPR, which suggests that these residues were present in
the IMPDH/GMPR ancestor. X-ray crystal structures show that the conserved Cys,
Thr, and Glu display similar interactions in both GMPR and IMPDH (), suggesting that
these residues may have similar functions in both enzymes. To confirm that GMPR activity depends on the presence of Cys186, Thr188, and
Glu289, we tested the effect of mutations of these residues on the activity of
Escherichia coli
GMPR in a complementation assay (). E.
coli H1173 requires both adenosine and guanosine for growth due
to mutations in both purA and guaC
[ 34] (). Growth on guanosine alone is restored with plasmid pGS682, which
carries the wild-type E. coli guaC gene [ 35]. However,
mutations in Cys186, Thr188, and Glu289 clearly compromise the ability of pGS682
to restore GMPR activity, demonstrating that selective pressure exists to
conserve these residues. Although the mechanism of the GMPR reaction has not been characterized, some
clear parallels can be drawn with the IMPDH reaction, and E-XMP* may
well be an intermediate. Importantly, if E-XMP* forms as proposed,
the active site must be constructed to prevent the hydrolysis reaction. Kinetic
and structural experiments clearly indicate that the reaction only proceeds when
NADPH is bound in the active site and can block the access of water
[ 33, 36, 37]. Moreover, GMPR does not
contain the Arg418-Tyr419 dyad, and the flap is truncated relative to the
corresponding region of IMPDH, as expected, given that the hydrolysis of
E-XMP* must be avoided during the GMPR reaction. Therefore, the
Arg418-Tyr419 dyad could have been installed as IMPDH optimized. Alternatively,
the dyad may have been present in the ancestral IMPDH/GMPR, but was subsequently
remodeled in the GMPR lineage; since the flap binds in the same site as
NAD +, this scenario suggests that the ancestral
IMPDH/GMPR was a hydrolase. While we cannot rule out the latter scenario, we
note that IMPDH is a member of the FMN oxidoreductase superfamily of
(β/α) 8 barrel proteins (unfortunately,
none of these proteins is sufficiently similar to permit rooting of the tree)
[ 38– 40]. Therefore, it seems more likely that the ancestral
enzyme was a promiscuous dehydrogenase, and the flap carrying the hydrolase
activity was the later addition. In contrast, the Thr321 pathway was likely present in the ancestral IMPDH/GMPR.
All IMPDHs and GMPRs contain Thr321 ( and S4, and
Text
S1). As noted above, Thr321 also plays a role in the dehydrogenase
reaction of IMPDH [ 11], which suggests that Thr321, like Cys319, was inherited
from the ancestral redox enzyme. Glu431 is conserved among GMPRs, suggesting
that the Thr321 pathway has a crucial function in this reaction, perhaps
operating in the reverse to protonate the ammonia leaving group. Curiously,
although Glu431 is highly conserved among IMPDHs, it is substituted with Gln in
the eukaryotic branch as well as in a few prokaryotic IMPDHs. We suggest that
the ancestral IMPDH/GMPR utilized the Thr321 pathway exclusively, but this
pathway became expendable once the Arg418 pathway was established. Phylogenetic
analysis is consistent with this view: maximum likelihood analysis indicates
that the ancestral enzyme almost certainly contained Glu at position 431
(probability = 0.87) [ 41]. Why then is Glu431 conserved in the majority of prokaryotic IMPDHs? The presence
of the Thr pathway increases turnover, which may be important in maintaining the
high concentration of guanine nucleotides required to support the rapid
proliferation of prokaryotes. More intriguingly, Glu431 provides
5–10-fold resistance to mycophenolic acid, a natural product that
specifically inhibits IMPDH [ 32]. Approximately 5%
of microorganisms contain some mechanism to modify mycophenolic acid, which
suggests that this compound is reasonably prevalent in the environment
[ 42]. Indeed, the extraordinary divergence of the adenosine
subsite of IMPDH may be a response to the assault of natural product inhibitors
such as mycophenolic acid and mizoribine [ 43]. This divergence occurs
despite the multiple functional constraints imposed by interactions with both
NAD +/NADH and the flap. The presence of the Thr
pathway could facilitate this adaptation, making the evolutionary challenge of
the IMPDH reaction much less formidable. Materials. Plasmid pGS682, a pUC plasmid carrying the 1.4-kb guaC insert
from pGS89 [ 35], was a generous gift from Simon Andrews (University of
Sheffield). E. coli
strain H1173 was obtained from the E. coli Genetic Stock Center (Yale University). Computational methods. Atoms within a radius of 22 Å around the reaction center were treated
as the dynamic region; this region was propagated with regular Newtonian
dynamics by applying leapfrog integrator and 1-fs time step. The atoms in the
layer between the radii of 22 Å and 25 Å were treated as
the buffer region; the heavy atoms in this region were harmonically restrained
with the force constants scaled linearly with the distance from the sphere
center. The force constants around the boundary of the 25 Å sphere
were set as implied by the B factors of the crystal structure. In the buffer
region, Langevin dynamics were applied with the friction coefficients also
linearly scaled with the distance from the sphere center. The friction
coefficients around the boundary 25 Å sphere were set as 60. CHARMM
22 force fields [ 22] were utilized as the molecular mechanical potentials in
these simulations (colored in blue in –) and SCCDFTB (self-consistent charge
density-functional tight-binding) method was applied as the quantum mechanical
potential on the atoms involved in the chemical reactions (colored in red in
–).
For the nonbonded interactions, an extended electrostatic treatment was applied
with the electrostatic interactions within 12 Å described by
group-based coulombic interactions. Enzyme assays. IMP, acetylpyridine adenine dinucleotide (APAD +), Tris,
and MES were purchased from Sigma. DTT was purchased from Research Organics.
Wild-type and Glu431Gln IMPDH from T. foetus were expressed in E. coli and purified as
described previously [ 10, 32]. All assays were performed at 25 °C. The
release of NADH is partially rate limiting [ 11, 31]. Therefore, to ensure that
hydrolysis is completely rate limiting, these experiments used
APAD + [ 31]. Pre-steady-state experiments
were performed to demonstrate that hydride transfer and APADH are rapid over the
entire pH range ([ 11] and unpublished data). Standard IMPDH assays contained
saturating concentrations of IMP (2 mM) and varying concentrations of
APAD + in 100 mM KCl, 1 mM DTT, and 50 mM of the
appropriate buffer (MES for pH 5.0–7.0, and Tris-HCl for pH
7.3–9.3). Activity was measured by monitoring the absorbance of
APADH at 363 nm on a Hitachi U-2000 UV-visible spectrophotometer. Steady-state
parameters with respect to APAD + were derived at
saturating IMP concentrations by plotting the initial velocity against
APAD + concentration and fitting to an equation
describing uncompetitive substrate inhibition using SigmaPlot (SPSS):
where ( kcat) app are the
apparent values at each pH, ( kcat) indep
are the pH-independent values, and Ka is the acid
dissociation constant for the most acidic ionization. Phylogenetic analysis. IMPDH/GMPR amino acid sequences (IMPDH IPR005990, GMPR1 IPR005993, and GMPR2
IPR005994) were retrieved from the InterPro database ( http://www.ebi.ac.uk/interpro/). Additionally, BLAST
[ 44] searches with the T. foetus IMPDH ( P50097) and
human GMPR1 ( P36959) amino acid sequences were performed. Sequences from the
BLAST search that were already part of the InterPro dataset were removed, and an
initial multiple sequence alignment was performed with MUSCLE [ 45]. A neighbor
joining tree (unpublished data) was constructed in PAUP* 4.0b10
[ 46], and 95 sequences were selected for a Bayesian
phylogenetic analysis. The sequences of this subset were realigned with Espresso
[ 47, 48]. A Bayesian
phylogenetic analysis was performed with the parallel version of MrBayes 3.1.2
[ 49, 50]. Amino acid
substitution rates and state frequencies were fixed to the WAG parameters
[ 51]. A uniform (0.0, 200.0) prior was assumed for the shape
parameter of the gamma distribution of substitution rates [ 52], an
unconstrained exponential prior with rate 10.0 for branch lengths, and all
labeled topologies were a priori equally probable. Two independent MCMC analyses
were run, each with one cold chain and three heated chains, with the incremental
heating schema implemented in MrBayes (λ=0.2).
Convergence was assumed after the topology samples from the two cold chains had
reached an average standard deviation of split frequencies of less than 0.01
(after 1,610,000 generations). Accession numbers, detailed results, and the full
tree are found in Text S1. Complementation assay for GMPR activity. E. coli strain H1173
(F-, guaC23, tonA2,
proA35, lacY1, tsx-70,
supE44?, gal-6, l-,
trp-45, tyrA2, rpsL125,
malA1 (lR), xyl-7, mtl-2,
thi-1, purH57) contains mutations in
purH and guaC, and therefore requires both
adenosine and guanosine for growth. Bacteria were transformed with pGS682
carrying either the wild-type guaC gene or variants containing
C186A, T188A, and E289Q mutations. Cultures were grown overnight in LB or
LB/ampicillin and 5 μl of 1/20 serial dilutions were plated on M9
minimal media containing 0.5% casamino acids, 100 μg/ml
l-tryptophan, 0.1% thiamin, 50 μg/ml
guanosine, and/or 50 μg/ml adenosine.
Figure S1: Optimization of the Wang-Landau Metadynamics Conditions
In order to optimize the Wang-Landau metadynamics conditions, three setups
with the final Gaussian heights 1.0 kcal/mol, 0.06 kcal/mol, and 0.01
kcal/mol were executed. The 1.0 kcal/mol simulation yielded a result with
large uncertainties and gave the free energy barrier of 14 kcal/mol at the
end of a 1-ns simulation. The 0.01 kcal/mol simulation yielded a nicely
converged free energy diagram with a barrier of 8 kcal/mol, but required
more than 20 ns. The 0.06 kcal/mol also yielded a free energy barrier of 8
kcal/mol with acceptable fluctuations, but required only 5 ns. Based on
these benchmark results, 0.06 kcal/mol was utilized as the final Gaussian
height throughout all the simulations.
(3.5 MB TIF)
Figure S2: Simulations of the Thr Pathway with Glu431 Treated MM.
(1.75 MB TIF)
Figure S3: Experimental Estimation of the Contribution of the Thr Pathway
Assuming that E431Q mutation disables the Thr pathway, but has no effect on
the pH dependence of the Arg pathway, then the pH-rate profile of the
wild-type enzyme is described by: where Ka =
10 −7.6 as determined from the pH dependence of
E431Q. The pH-rate profile of the wild-type enzyme could be reasonably
described with the above equation when the value of
kThr is 0.15 s −1,
which corresponds to an energy barrier of approximately 19 kcal/mol. For
easier visualization, the pH-rate profiles of A were normalized so that the
pH-independent values of kcat = 1
for both wild type and E431Q, where x becomes the
normalized value of kThr. (2.05 MB TIF)
Figure S4: Phylogenetic Tree of IMPDH and GMPR
The unrooted tree was inferred with MrBayes (including posterior
probabilities) [ 49]. Organism names are followed by their sequence
accession codes. IMPDH, GMPR1, and GMPR2 refer to members of InterPro
accession codes IPR005990, IPR005993, and IPR005994, respectively. (4.39 MB TIF)
Text S1: Phylogenetic Analysis
Detailed description of the derivation of Figure
S4. (101 KB PDF)
We thank Karen Allen, Margaret Phillips, and Gregory Petsko for comments on the
manuscript. Jeffrey Boucher constructed the GMPR mutants.
|
| GMPR |
GMP reductase |
|
| IMPDH |
IMP dehydrogenase, MZP, mizoribine monophosphate |
|
| SIE |
solvent isotope effect |
-
Schwarzer D,
Marahiel MA.
2001.
Multimodular biocatalysts for natural product assembly
Naturwissenschaften
88
93
101
[PubMed]
-
Hopwood DA.
1997.
Genetic contributions to understanding polyketide synthases
Chem Rev
97
2465
2498
[PubMed]
-
Jenke-Kodama H,
Borner T,
Dittmann E.
2006.
Natural biocombinatorics in the polyketide synthase genes of the
actinobacterium Streptomyces avermitilis
PLoS Comput Biol
2
e132
doi:10.1371/journal.pcbi.0020132
[PubMed]
-
Gerlt JA,
Raushel FM.
2003.
Evolution of function in (beta/alpha)8-barrel enzymes
Curr Opin Chem Biol
7
252
264
[PubMed]
-
Wise EL,
Yew WS,
Akana J,
Gerlt JA,
Rayment I.
2005.
Evolution of enzymatic activities in the orotidine
5′-monophosphate decarboxylase suprafamily: structural basis for
catalytic promiscuity in wild-type and designed mutants of 3-keto-L-gulonate
6-phosphate decarboxylase
Biochemistry
44
1816
1823
[PubMed]
-
Bergthorsson U,
Andersson DI,
Roth JR.
2007.
Ohno's dilemma: evolution of new genes under continuous selection
Proc Natl Acad Sci U S A
104
17004
17009
[PubMed]
-
Hedstrom L,
Gan L.
2006.
IMP dehydrogenase: structural schizophrenia and an unusual base
Curr Opin Chem Biol
10
520
525
[PubMed]
-
Hedstrom L.
1999.
IMP dehydrogenase: mechanism of action and inhibition
Curr Med Chem
6
545
560
[PubMed]
-
Gan L,
Seyedsayamdost MR,
Shuto S,
Matsuda A,
Petsko GA,
et al.
2003.
The immunosuppressive agent mizoribine monophosphate forms a
transition state analog complex with IMP dehydrogenase
Biochemistry
42
857
863
[PubMed]
-
Guillén Schlippe YV,
Riera TV,
Seyedsayamdost MR,
Hedstrom L.
2004.
Substitution of the conserved Arg-Tyr dyad selectively disrupts
the hydrolysis phase of the IMP dehydrogenase reaction
Biochemistry
43
4511
4521
[PubMed]
-
Guillén Schlippe YV,
Hedstrom L.
2005.
Is Arg418 the catalytic base required for the hydrolysis step of
the IMP dehydrogenase reaction
Biochemistry
44
11700
11707
[PubMed]
-
Guillén Schlippe YV,
Hedstrom L.
2005.
Guanidine derivatives rescue the Arg418Ala mutation of
Tritrichomonas foetus IMP dehydrogenase
Biochemistry
44
16695
16700
[PubMed]
-
Min D,
Liu Y,
Carbone I,
Yang W.
2007.
On the convergence improvement in the metadynamics simulations: a
Wang-Landau recursion approach
J Chem Phys
126
194104
[PubMed]
-
Warshel A,
Levitt M.
1976.
Theoretical studies of enzymic reactions: dielectric,
electrostatic and steric stabilization of the carbonium ion in the reaction
of lysozyme
J Mol Biol
103
227
249
[PubMed]
-
Brooks BR,
Bruccoleri RE,
Olafson BD,
States DJ,
Swaminathan S,
et al.
1983.
CHARMM: a program for macromolecular energy, minimization, and
dynamics calculations
J Comp Chem
4
187
217
.
-
Field MJ,
Bash PA,
Karplus M.
1990.
A combined quantum mechanical and molecular mechanical potential
for molecular dynamics simulations
J Comp Chem
11
700
733
.
-
Madura JD,
Jorgensen WL.
1986.
Ab initio and Monte Carlo calculations for a nucleophilic
addition reaction in the gas phase and in aqueous solution
J Am Chem Soc
108
2517
2527
.
-
Monard G,
Merz KM., Jr
1999.
Combined quantum mechanical/molecular mechanical methodologies
applied to biomolecular systems
Acc Chem Res
32
904
911
.
-
Gao J.
1996.
Hybrid quantum and molecular mechanical simulations: an
alternative avenue to solvent effects in organic chemistry
Acc Chem Res
29
298
305
.
-
Laio A,
Parrinello M.
2002.
Escaping free-energy minima
Proc Natl Acad Sci U S A
99
12562
12566
[PubMed]
-
Ensing B,
De Vivo M,
Liu Z,
Moore P,
Klein ML.
2006.
Metadynamics as a tool for exploring free energy landscapes of
chemical reactions
Acc Chem Res
39
73
81
[PubMed]
-
MacKerell AD, Jr,
Bashford D,
Bellott M,
Dunbrack RL,
Evanseck JD,
et al.
1998.
All-atom empirical potential for molecular modeling and dynamics
studies of proteins
J Phys Chem B
102
3586
3616
.
-
Elstner M,
Porezag D,
Jungnickel G,
Elsner J,
Haugk M,
et al.
1998.
Self-consistent-charge density-functional tight-binding method
for simulations of complex materials properties
Phys Rev B
58
7260
7268
.
-
Im W,
Berneche S,
Roux B.
2001.
Generalized solvent boundary potential for computer simulations
J Chem Phys
114
2924
2937
.
-
Schaefer P,
Riccardi D,
Cui Q.
2005.
Reliable treatment of electrostatics in combined QM/MM simulation
of macromolecules
J Chem Phys
123
14905
14918
.
-
Peracchi A.
2001.
Enzyme catalysis: removing chemically ‘essential'
residues by site-directed mutagenesis
Trends Biochem Sci
26
497
503
[PubMed]
-
Steyaert J,
Hallenga K,
Wyns L,
Stanssens P.
1990.
Histidine-40 of ribonuclease T1 acts as base catalyst when the
true catalytic base, glutamic acid-58, is replaced by alanine
Biochemistry
29
9064
9072
[PubMed]
-
Henot F,
Pollack RM.
2000.
Catalytic activity of the D38A mutant of 3-oxo-Delta 5-steroid
isomerase: recruitment of aspartate-99 as the base
Biochemistry
39
3351
3359
[PubMed]
-
Carter P,
Wells J.
1988.
Dissecting the catalytic triad of a serine protease
Nature
332
564
568
[PubMed]
-
An H,
Tu C,
Duda D,
Montanez-Clemente I,
Math K,
et al.
2002.
Chemical rescue in catalysis by human carbonic anhydrases II and
III
Biochemistry
41
3235
3242
[PubMed]
-
Digits JA,
Hedstrom L.
1999.
Kinetic mechanism of Tritrichomonas foetus
inosine-5′-monophosphate dehydrogenase
Biochemistry
38
2295
2306
[PubMed]
-
Digits JA,
Hedstrom L.
1999.
Species-specific inhibition of inosine
5′-monophosphate dehydrogenase by mycophenolic acid
Biochemistry
38
15388
15397
[PubMed]
-
Li J,
Wei Z,
Zheng M,
Gu X,
Deng Y,
et al.
2006.
Crystal structure of human guanosine monophosphate reductase 2
(GMPR2) in complex with GMP
J Mol Biol
355
980
988
[PubMed]
-
De Haan PG,
Hoekstra WP,
Verhoef C,
Felix HS.
1969.
Recombination in Escherichia coli. 3. Mapping by the gradient of
transmission
Mutat Res
8
505
512
[PubMed]
-
Andrews SC,
Guest JR.
1988.
Nucleotide sequence of the gene encoding the GMP reductase of
Escherichia coli K12
Biochem J
255
35
43
[PubMed]
-
Spector T,
Jones T,
Miller R.
1979.
Reaction mechanism and specificity of human GMP reductase
J Biol Chem
254
2308
2315
[PubMed]
-
Deng Y,
Wang Z,
Ying K,
Gu S,
Ji C,
et al.
2002.
NADPH-dependent GMP reductase isoenzyme of human (GMPR2).
Expression, purification, and kinetic properties
Int J Biochem Cell Biol
34
1035
1050
[PubMed]
-
Bork P,
Gellerich J,
Groth H,
Hooft R,
Martin F.
1995.
Divergent evolution of a beta/alpha-barrel subclass: detection of
numerous phosphate-binding sites by motif search
Protein Sci
4
268
274
[PubMed]
-
Nagano N,
Orengo CA,
Thornton JM.
2002.
One fold with many functions: the evolutionary relationships
between TIM barrel families based on their sequences, structures and
functions
J Mol Biol
321
741
765
[PubMed]
-
Copley RR,
Bork P.
2000.
Homology among (betaalpha)(8) barrels: implications for the
evolution of metabolic pathways
J Mol Biol
303
627
641
[PubMed]
-
Pupko T,
Pe'er I,
Hasegawa M,
Graur D,
Friedman N.
2002.
A branch-and-bound algorithm for the inference of ancestral
amino-acid sequences when the replacement rate varies among sites:
application to the evolution of five gene families
Bioinformatics
18
1116
1123
[PubMed]
-
Jones DF,
Moore RH,
Crawley GC.
1970.
Microbial modification of mycophenolic acid
J Chem Soc (C)
1725
1737
.
-
Kohler GA,
Gong X,
Bentink S,
Theiss S,
Pagani GM,
et al.
2005.
The functional basis of mycophenolic acid resistance in Candida
albicans IMP dehydrogenase
J Biol Chem
280
11295
11302
[PubMed]
-
Altschul SF,
Gish W,
Miller W,
Myers EW,
Lipman DJ.
1990.
Basic local alignment search tool
J Mol Biol
215
403
410
[PubMed]
-
Edgar RC.
2004.
MUSCLE: multiple sequence alignment with high accuracy and high
throughput
Nucleic Acids Res
32
1792
1797
[PubMed]
-
Swofford DL.
2003.
PAUP*. Phylogenetic analysis using parsimony
(*and other methods). Version 4
Sunderland (Massachusetts):
Sinauer Associates;
-
Notredame C,
Higgins DG,
Heringa J.
2000.
T-Coffee: a novel method for fast and accurate multiple sequence
alignment
J Mol Biol
302
205
217
[PubMed]
-
Armougom F,
Moretti S,
Poirot O,
Audic S,
Dumas P,
et al.
2006.
Expresso: automatic incorporation of structural information in
multiple sequence alignments using 3D-Coffee
Nucleic Acids Res
34
W604
608
[PubMed]
-
Ronquist F,
Huelsenbeck JP.
2003.
MrBayes 3: Bayesian phylogenetic inference under mixed models
Bioinformatics
19
1572
1574
[PubMed]
-
Altekar G,
Dwarkadas S,
Huelsenbeck JP,
Ronquist F.
2004.
Parallel Metropolis coupled Markov chain Monte Carlo for Bayesian
phylogenetic inference
Bioinformatics
20
407
415
[PubMed]
-
Whelan S,
Goldman N.
2001.
A general empirical model of protein evolution derived from
multiple protein families using a maximum-likelihood approach
Mol Biol Evol
18
691
699
[PubMed]
-
Yang Z.
1993.
Maximum-likelihood estimation of phylogeny from DNA sequences
when substitution rates differ over sites
Mol Biol Evol
10
1396
1401
[PubMed]
-
Pettersen EF,
Goddard TD,
Huang CC,
Couch GS,
Greenblatt DM,
et al.
2004.
UCSF Chimera—a visualization system for exploratory
research and analysis
J Comp Chem
25
1605
1612
[PubMed]
|
See "
