The role of phosphate in a multistep enzymatic reaction: Reactions of the substrate and intermediate in pieces

Article abstract of DOI:10.1021/ja512911f

Several mechanistically unrelated enzymes utilize the binding energy of their substrate's nonreacting phosphoryl group to accelerate catalysis. Evidence for the involvement of the phosphodianion in transition state formation has come from reactions of the substrate in pieces, in which reaction of a truncated substrate lacking its phosphorylmethyl group is activated by inorganic phosphite. What has remained unknown until now is how the phosphodianion group influences the reaction energetics at different points along the reaction coordinate. 1-Deoxy-d-xylulose-5-phosphate (DXP) reductoisomerase (DXR), which catalyzes the isomerization of DXP to 2-C-methyl-d-erythrose 4-phosphate (MEsP) and subsequent NADPH-dependent reduction, presents a unique opportunity to address this concern. Previously, we have reported the effect of covalently linked phosphate on the energetics of DXP turnover. Through the use of chemically synthesized MEsP and its phosphate-truncated analogue, 2-C-methyl-d-glyceraldehyde, the current study revealed a loss of 6.1 kcal/mol of kinetic barrier stabilization upon truncation, of which 4.4 kcal/mol was regained in the presence of phosphite dianion. The activating effect of phosphite was accompanied by apparent tightening of its interactions within the active site at the intermediate stage of the reaction, suggesting a role of the phosphodianion in disfavoring intermediate release and in modulation of the on-enzyme isomerization equilibrium. The results of kinetic isotope effect and structural studies indicate rate limitation by physical steps when the covalent linkage is severed. These striking differences in the energetics of the natural reaction and the reactions in pieces provide a deeper insight into the contribution of enzyme-phosphodianion interactions to the reaction coordinate.

Full text of DOI:10.1021/ja512911f

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Article
The Role of Phosphate in a Multistep Enzymatic Reaction:
Reactions of the Substrate and Intermediate in Pieces
Svetlana A. Kholodar, C. Leigh Allen, Andrew M. Gulick, and Andrew S. Murkin
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The Role of Phosphate in a Multistep Enzymatic Reaction: Reactions
of the Substrate and Intermediate in Pieces
Svetlana A. Kholodar,† C. Leigh Allen,‡ Andrew M. Gulick,‡ and Andrew S. Murkin†,*
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† Department of Chemistry, University at Buffalo, Buffalo, New York 14260-3000, United States.
‡ Hauptman-Woodward Institute and Department of Structural Biology, University at Buffalo, Buffalo, New York 14203-1102, United
States.
ABSTRACT: Several mechanistically unrelated enzymes utilize the binding energy of their substrate’s nonreacting phosphoryl group to
accelerate catalysis. Evidence for the involvement of the phosphodianion in transition state formation has come from reactions of the sub-
strate in pieces, in which reaction of a truncated substrate lacking its phosphorylmethyl group is activated by inorganic phosphite. What has
remained unknown until now is how the phosphodianion group influences the reaction energetics at different points along the reaction coor-
dinate. 1-Deoxy-D-xylulose-5-phosphate (DXP) reductoisomerase (DXR), which catalyzes the isomerization of DXP to 2-C-methyl-D-
erythrose 4-phosphate (MEsP) and subsequent NADPH-dependent reduction, presents a unique opportunity to address this concern. Previ-
ously, we have reported the effect of covalently linked phosphate on the energetics of DXP turnover. Through the use of chemically synthe-
sized MEsP and its phosphate-truncated analogue, 2-C-methyl-D-glyceraldehyde, the current study revealed a loss of 6.1 kcal/mol of kinetic
barrier stabilization upon truncation, of which 4.4 kcal/mol was regained in the presence of phosphite dianion. The activating effect of phos-
phite was accompanied by apparent tightening of its interactions within the active site at the intermediate stage of the reaction, suggesting a
role of the phosphodianion in disfavoring intermediate release and in modulation of the on-enzyme isomerization equilibrium. The results of
kinetic isotope effect and structural studies indicate rate limitation by physical steps when the covalent linkage is severed. These striking dif-
ferences in the energetics of the natural reaction and the reactions in pieces provide a deeper insight into the contribution of enzyme–
phosphodianion interactions to the reaction coordinate.
INTRODUCTION
significantly different characteristics from those described above.
DXR performs a multistep reaction that proceeds through retro-
aldol/aldol isomerization of the ketone DXP to the branched alde-
hyde intermediate 2-C-methyl-D-erythrose 4-phosphate (MEsP),
followed by NADPH-dependent reduction to form 2-C-methyl-D-
erythritol 4-phosphate (MEP) (Scheme 1).17 Importantly, a phos-
Phosphorylated biomolecules play a central role in metabolism of
all living species. The phosphate group as a part of a small molecule
is often nonreacting but essential for the recognition and catalysis
by enzymes. Previous studies by Richard and co-workers have
demonstrated that mechanistically unrelated enzymes including
triosephosphate isomerase (TIM),6-8 orotidine-5ʹ-monophosphate
decarboxylase (OMPDC),10 and glycerol-3-phosphate dehydro-
genase (GPDH)11 can bind and turnover truncated versions of
their corresponding substrates lacking the terminal phosphorylme-
thyl group with catalytic efficiencies decreased dramatically by
factors of 108–1010. These reactions could be rescued by factors of
up to 80000 by the inclusion of inorganic phosphite dianion. These
curious results have been the subject of several reviews8,12-14 and
have been rationalized with respect to the classical paradigm intro-
duced by Jencks,15 in which binding interactions between a nonre-
acting determinant of the substrate and the active site serve a signif-
icant role in transition state stabilization. Specifically, this binding
energy is argued to drive thermodynamically unfavorable confor-
mational changes that pre-organize the enzyme’s active site for
catalysis. In agreement, structural data suggest that phosphodian-
ion binding in the active site serves as a trigger for closure of the
flexible loop over the active site of each of these phosphite-
activated enzymes.13 This critical conformational change is impli-
cated in sequestration of the active site from bulk solvent, thus
creating a lower dielectric environment poised for catalysis.
SCHEME 1. Mechanism for DXR-catalyzed Conversion
of DXP to MEP and DE to 2MG
phodianion group is critical for substrate turnover18,19 and for inhi-
bition by the antibiotics fosmidomycin and FR-900098. In our
previous study, we demonstrated that 1-deoxy-L-erythrulose (DE),
a DXP analogue lacking the terminal phosphorylmethyl group, can
be converted by Mycobacterium tuberculosis DXR (MtDXR) to
In earlier work we probed the generality of phosphodianion-
assisted catalysis by extending the approach to 1-deoxy-D-xylulose-
5-phosphate (DXP) reductoisomerase (DXR),16 an enzyme with
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the corresponding MEP analogue 2-C-methylglycerol (2MG) with hyde), 3.63 (d, J = 12.1 Hz, 1H, aldehyde), 3.60 (d, J = 11.7 Hz,
a kcat/Km that is 106 lower than that for DXP.16 As with the enzymes
mentioned above, phosphite dianion was found to be a non-
essential activator; however, only a 5-fold increase in kcat/Km and
kcat was observed at saturating concentrations. This low activation
factor can be rationalized by (1) the much lower catalytic efficiency
of MtDXR, suggestive of intrinsically suboptimal transition state
stabilization by the phosphodianion, (2) the distribution of total
binding energy between distal portions of the substrate (i.e., the
phosphodianion at C-5 and the divalent metal-binding groups at C-
2 and C-3), and (3) the multistep nature of the reaction, in which
the degree of transition state stabilization by the phosphodianion
may vary along the reaction coordinate.
1H, hydrate), 3.53 (d, J = 11.7 Hz, 1H, hydrate), 1.27 (s, 3H, alde-
hyde), 1.14 (s, 3H, hydrate). ESI-MS calcd for [2M+Na+]
C18H16O6Na: 231.08, found: 231.10. ee = 94%. As steady state ki-
netic parameters of 2MGA turnover by MtDXR were found to be
stock-dependent, the synthetic route to 2MGA was altered to im-
prove product purity. Details of the altered synthesis of 2MGA,
enantiomeric excess determination and quantification of the final
stocks can be found in the Supporting Information.
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Synthesis of MEsP. 2,3-O-Isopropylidene-2-C-methyl-D-
erythrofuranose was synthesized from D-arabinose (7) as reported
previously1 and converted to dibenzyl 2,3-O-isopropylidene-2-C-
methyl-D-erythrose 4-phosphate (8) according to the published
procedure.2 The resulting product was deprotected and quantified
according to procedures described in the Supporting Information.
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In this work we report a complete investigation of the role of the
covalent linkage to the phosphodianion in modulation of the ener-
gy landscape of the MtDXR-catalyzed reaction. The reaction in-
termediate MEsP and its truncated version, 2-C-metyl-D-
glyceraldehyde (2MGA), were synthesized and kinetically charac-
terized to reveal a possible role of the phosphodianion in reducing
of the energy gap between enzyme-bound substrate and intermedi-
ate. On the basis of X-ray structural data and kinetic isotope effects
(KIEs) for the natural compounds and their corresponding “piec-
es”, we suggest a function of the covalently-linked phosphodianion
in the productive orientation of the substrate and intermediate in
the catalytic site and/or in induction of conformational changes
required for catalysis.
1
The H NMR spectrum of the product was in agreement with that
previously reported.26
Synthesis
of
(2-13C;3,4,4-2H3)DE.
(1,2,2-
2H3)Glycolaldehyde was prepared enzymatically starting from
(1,1,2,2-2H4)ethylene glycol as described for non-labeled glycolal-
dehyde with certain modifications.27 To a 1 M solution (final vol-
ume 1 mL) of (1,1,2,2-2H4)ethylene glycol in 0.8 M Tris-HCl, pH
9.0, and in D2O (87% v/v final) was added 100 U P. pastoris alco-
hol oxidase and 3000 U bovine liver catalase. The reaction mixture
was stirred at 5 °C for 60 h resulting in 70% conversion to (1,2,2-
2H3)glycolaldehyde-Tris imine. (2-13C;3,4,4-2H3)DE was subse-
quently synthesized enzymatically by DXS-catalyzed condensation
of (1,2,2-2H3)glycolaldehyde with sodium (2-13C)pyruvate and
purified as described previously for unlabeled DE.16 1H NMR (500
MHz, D2O) δ 2.27 (d, J = 5.9 Hz, 3H).13C NMR (125 MHz, D2O)
δ 215.4.
EXPERIMENTAL PROCEDURES
Materials. All chemicals were of analytical or reagent grade and
were used without further purification unless otherwise stated.
Escherichia coli DXP synthase (DXS) was expressed and purified
as reported.20 MtDXR was cloned, expressed, purified, and quanti-
fied as reported previously.21 Pichia pastoris alcohol oxidase was
purchased from MP Biomedicals. Bacterial glucose dehydrogenase
was from Toyobo. Bovine liver catalase was from Calbiochem.
(2H4)Ethylene glycol and deuterium oxide were obtained from
Cambridge Isotope Laboratories, Inc. Sodium (2-13C)pyruvate and
(1-13C)glycine were obtained from Icon Isotopes. (4S)-(4-
2H1)NADPH was synthesized and purified using published proce-
dures.22,23 DXP was synthesized and purified as described previous-
ly.24 1H and 13C NMR spectra were acquired on a Varian INOVA
Synthesis of (2-13C)DE. (2-13C)DE was prepared as de-
scribed above using unlabeled glycolaldehyde. 1H NMR (500 MHz,
D2O) δ 4.42 (dd, J = 7.3, 3.5 Hz, 1H), 3.95 (ddd, J = 12.3, 4.2, 1.5
Hz, 1H), 3.89 (ddd, J = 12.3, 5.0, 3.4 Hz, 1H), 2.27 (d, J = 5.9 Hz,
3H). 13C NMR (125 MHz, D2O) δ 215.5.
Steady-State Kinetics. Measurement of initial velocities of
DE and 2MGA turnover was performed using an Applied Photo-
physics SX-20 stopped flow spectrophotometer fit with a 20 μL
flow cell (1 cm path length). The stopped-flow instrument was
employed in preference to a conventional spectrophotometer pri-
marily to reduce reactant quantities; unlike the reactions with the
natural substrate DXP, no additional kinetic events were observed
within the initial second of detection. Final assay mixtures with DE
as a substrate contained 0–50 mM sodium phosphite buffer (pH
7.5), 25 mM Tris-HCl buffer (pH 7.5), 10 mM MgCl2, 10 mM
DTT, 200 μM NADPH, 1.5–40 mM DE, and 2.5 μM MtDXR at an
ionic strength of 0.2 M adjusted with NaCl. Final assay mixtures
with 2MGA as a substrate contained 0–50 mM sodium phosphite
buffer (pH 7.5), 25 mM HEPES buffer (pH 7.5), 10 mM MgCl2,
200 μM NADPH, 1.5–35 mM 2MGA (aldehyde and hydrate
form), and 2.5 μM MtDXR at an ionic strength of 0.2 M adjusted
with NaCl. 2MGA solutions were pre-incubated overnight at room
temperature to minimize dimer formation. Measurement of initial
velocities of MEsP and DXP turnover was performed using Varian
Cary 3E UV-VIS spectrophotometer. Final assay mixtures for
measurement of inhibition of MtDXR-catalyzed DXP turnover by
phosphite dianion contained 0–50 mM sodium phosphite buffer
(pH 7.5), 25 mM Tris-HCl buffer (pH 7.5), 10 mM MgCl2, 10 mM
DTT, 200 μM NADPH, 0.05-2 mM DXP, and 50 nM MtDXR at
1
500 spectrometer equipped with a H,13C,15N probe using default
pulse sequences.
E. coli DXR Expression and Purification. BL21(DE3) E.
coli cells previously transformed with a plasmid resulting from the
insertion of the gene encoding E. coli DXR (EcDXR) into
pCA24N were obtained from the NationalBioResource Project,
Japan (NBRP-E. coli at NIG).25 Cultures were inoculated and
grown at 37 °C to an OD600 of 0.5−0.6 in LB-Miller broth contain-
ing 25 μg/L chloramphenicol. Optimal expression was achieved via
induction with 500 μM IPTG at 31 °C for 4 h. Cell lysis and protein
purification and storage were performed as described for MtDXR.
Synthesis of 2MGA. (2S)-2,3-Dihydroxy-N-methoxy-2,N-
dimethylpropionamide (3) was synthesized as reported previously3
and reduced for 1 h in THF at 0 °C with 1.25 equiv of LiAlH4. The
resulting product was hydrolyzed in the presence of 1 M NaHSO4
and desalted upon treatment with Amberlite IRA-400 (OH–) and
Amberlite IR-120 (H+) ion-exchange resins to yield colorless oil
1
(75% isolated yield). H NMR (500 MHz, D2O) δ 9.61 (s, 1H,
1
aldehyde), 4.92 (s, H, hydrate), 3.89 (d, J = 12.1 Hz, 1H, alde-
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duction of the Weinreb amide by LiAlH4 followed by hydrolysis
an ionic strength of 0.2 M adjusted with NaCl. For MtDXR, final
1
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5
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7
8
assay mixtures with MEsP as a substrate contained 100 mM
HEPES buffer (pH 7.5), 10 mM MgCl2, 150 μM NADPH, 0–300
μM MEsP (aldehyde form), and 50 nM MtDXR. For EcDXR, final
assay mixtures with DXP or MEsP as a substrate contained 50 mM
triethanolamine-HCl (pH 7.7), 3 mM MgCl2, 2 mM DTT, 150 μM
NADPH, 0–300 μM DXP or MEsP (aldehyde form), and 10 nM
EcDXR. Monitoring of the NADPH absorbance decay at 340 nm
(ε340 = 6.22 mM-1 cm-1) was performed at 25 °C for MtDXR and 37
°C for EcDXR.
with mild acid produced 2MGA with 94% ee. Attempts to purify
2MGA by flash chromatography were hampered by its instability in
the presence of silica, which was found to catalyze its quantitative
rearrangement to DE (or its enantiomer). To facilitate purification,
chiral Weinreb amide precursor 3 was converted to the acetonide,9
reduced by LiAlH4 to the corresponding aldehyde, and distilled
under vacuum. Pure 2MGA was obtained following cleavage of the
acetonide in the presence of Amberlite (H+ form). To assess the
effect of truncation on kinetics and KIEs for the reaction interme-
9
2-
diate, we synthesized MEsP (Scheme 1, R = CH2OPO3 ), initially
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Steady state kinetics data were analyzed by analytical nonlinear
regression using Pro-Data Viewer (Applied Photophysics),
GraphPad Prism (GraphPad Software, La Jolla, CA), and Sig-
maPlot Version 12.5 (Systat Software, San Jose, CA), and standard
errors associated with fitting are reported. Kinetic parameters pre-
sented in Table 1 were obtained by global fitting of eq 2 to the data.
following the procedure reported by Hoeffler and co-workers;26
however, because of the formation of significant impurities, an
alternative synthetic sequence was adapted from protocols for the
synthesis of MEsP precursor dibenzyl 2,3-O-isopropylidene-2-C-
methyl-D-erythrose 4-phosphate (8) (Scheme 3).1,2
SCHEME 3. Synthesis of 2-C-Methyl-D-erythrose 4-
Phosphate (MEsP)
KIE Determinations. NADPH primary deuterium KIEs were
measured by the direct comparison method (i.e., determination of
V and V/K for NADPH and NADPD separately) by global fitting
of steady-state data to eq 1,
v0
kcat[S]
=
(1)
[E]t Km (1+ F EV /K ) +[S](1+ F EV )
i
i
where EV/K and EV are the isotope effects minus 1 on V/K and V,
respectively, and Fi is the fraction of deuterium. The 2,3,3-2H3 KIE
was measured by competitive reaction of (2-13C)- and (2-13C;3,4,4-
Reaction conditions: (a) From Refs. 1,2; (b) H2, Pd/C, MeOH; (c) 37
°C, H2O (9 h); (d) 1 M NaOH.
13
2H3)DE monitored by C NMR, similar to literature methods.28,29
Steady-State Kinetics. The kinetics of MEsP reduction by
MtDXR was measured in the presence of saturating concentrations
of NADPH and Mg2+ and varying MEsP (0–200 μM final, as alde-
hyde) (Table 1). To permit comparison with literature,26 steady-
state kinetic parameters were also measured for EcDXR in the pre-
viously reported conditions. While our value of kcat of 9.8 ± 0.3 s–1
agrees favorably with the reported value of ~8 s–1 (calculated from
specific activity), the Km value of 54 ± 5 μM (aldehyde form) was
found to be three times less than the reported value of 158 μM.26
Since the concentration of MEsP in the previous study was deter-
mined enzymatically, the reported value of Km likely reflects the
total concentration of MEsP (aldehyde and hydrate forms). Con-
sequently, the Km value could have been overestimated by the fac-
tor of (1 + Khyd), where Khyd = 1.0 at 37 ºC.
Samples (700 µL) contained 200 μM MtDXR, 5 mM (2-13C)DE, 5
mM (2-13C;3,4,4-2H3)DE, 25 mM Tris-HCl, pH 7.8, 10 mM
MgCl2, 50 mM sodium phosphite, pH 7.8, 0.2 mM NADPH, and
10% v/v D2O. (1-13C)Glycine (20 mM) was used as an internal
standard for calculation of the fraction of conversion, F1. D-Glucose
(15 mM) and glucose dehydrogenase (2 U) were added to regen-
erate NADPH. Acquisition, processing, and data analysis are de-
scribed in the Supporting Information.
RESULTS
Synthesis of Truncated and Complete Reaction In-
termediates. Previously, 2MGA was synthesized in racemic form
to serve as a potential alternative substrate of fructose-1,6-
diphosphate aldolase.30 To avoid ambiguity in interpretation of the
kinetics with MtDXR, we developed an enantioselective synthetic
scheme (Scheme 2) leading predominantly to the D isomer. This
In this work we have assumed that isomerization of the truncated
substrate DE results in formation of the truncated intermediate
2MGA, which undergoes reduction to the final product 2MG
(Scheme 1, R = H).16 Similar to observations with the complete
intermediate, incubation of 2MGA and NADPH with MtDXR in
the presence of phosphite dianion was found to produce 2MG.
Interestingly, when 2MGA was incubated with NADP+ and
MtDXR in the presence of phosphite dianion, only 2% of the start-
ing material was converted to DE; the remaining 98% of 2MGA
was observed to disproportionate to 2MG and 2-C-methyl-D-
glyceric acid (Figure S1). When monitored spectrophotometrical-
ly, transient formation of NADPH was observed upon incubation
of 2MGA with NADP+ and MtDXR (Figure S2), suggesting direct
oxidation of the hydrate form of 2MGA to 2-C-methyl-D-glyceric
acid, followed by reduction of the aldehyde form of 2MGA by the
generated NADPH. In contrast, the complete intermediate MEsP
was found to predominantly partition towards DXP in the presence
of NADP+ and MtDXR, with only 24% undergoing disproportiona-
tion (Figure S3).
SCHEME 2 . Synthesis of 2-C-Methyl-D-glyceraldehyde
(2MG A)
Reaction conditions: (a) (i) SOCl2, CH2Cl2; (ii) CH3ONHCH3·HCl,
pyridine;3 (b) Modified α-AD mix;4,5 (c) Me2C(OMe)2, p-
MeC6H4SO3H;9 (d) LiAlH4, THF; (e) Amberlite (H+ form), H2O.
approach features asymmetric dihydroxylation of Weinreb amide
2,3 which was previously reported to proceed with 96.5% ee.4 Re-
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Table 1. Steady-state kinetic parameters and kinetic isotope effects on the hydride transfer step for DXP, MEsP,
1
DE, and 2MGA reactions catalyzed by MtDXR
2
3
4
5
Substrate
DE + HPO3
Kinetic
parametera
DXPb
MEsP
DEc
2MGA
2MGA + HPO3
2–c
2–
6
7
8
9
kcat (s-1)
5.25 ± 0.19
18 ± 1d
0.32 ± 0.04
9.8 ± 0.3d
0.040 ± 0.004
0.054 ± 0.005d
N/A
(1.55 ± 0.12)·10–3
(8.6 ± 0.6)·10–3
53 ± 5
(1.90 ± 0.40)·10–3 (4.42 ± 0.20)·10–2
Km (mM)
0.115 ± 0.007
0.115 ± 0.014 d
N/A
53 ± 5
6.6 ± 0.4
6.6 ± 0.4
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KHPi (mM)
N/A
26 ± 4e
N/A
13 ± 1e
kcat/Km (M-1 s-1)
(4.6 ± 0.3)·104
(8.1 ± 0.3)·103
0.0292 ± 0.0018
N/Af
0.163 ± 0.010
1.1 ± 0.1
1.0 ± 0.2
c
0.29 ± 0.07
1.08 ± 0.06
1.11 ± 0.16
6.7 ± 0.2
1.09 ± 0.05
1.05 ± 0.06
d
D(V/K)
2.2 ± 0.2
1.0 ± 0.1
DV
1.35 ± 0.04
1.71 ± 0.04
b
N/Af
a
Errors are standard errors from global fitting. From Liu and Murkin21 except as noted. From Kholodar and Murkin.16 Measured with
e
–
EcDXR at 37 ºC. Concentration of dianion form calculated at pH 7.5, pKa (H2PO3 ) = 6.31 (determined from the pH of a 50 mM solution of
sodium salt at 25 °C and I = 0.2 M (NaCl) at a 1:1 monoanion:dianion ratio). f Not calculated due to the high error associated with measurement
of rates.
To investigate the effect of truncation on the kinetic parameters
of intermediate turnover, the kinetics of MtDXR-catalyzed reduc-
tion of 2MGA was assayed by monitoring NADPH absorbance
upon mixing a 1:1 volume ratio of solutions containing enzyme
(2.5 µM final) and varying 2MGA (0–5.9 mM final, as aldehyde),
respectively. Both solutions also contained saturating NADPH
(200 µM) and varying phosphite dianion (0–50 mM), and a con-
stant ionic strength of 0.2 M was maintained by addition of NaCl.
As reported in our previous work, due to the much lower Km of
NADPH (9.8 µM), an ordered sequential mechanism with
NADPH preceding 2MGA was assumed.20,21 The data revealed
hyperbolic dependences on both ligands (Figure 1), consistent
with non-essential activation by phosphite (Scheme 4).31 In this
SCHEME 4. Non-essential activation model for phos-
phite-activated 2MG A turn over by MtDXRa
a E = MtDXR•NADPH.
scheme, kcat and k′cat are the respective first-order rate constants in
the absence and presence of phosphite, k1, k–1, k2, and k–2 are the
rate constants for 2MGA binding to form its ternary
(MtDXR•NADPH•2MGA)
and
quaternary
2–
(MtDXR•NADPH•2MGA•HPO3 ) complexes, and K3 and K4 are
the respective dissociation constants for phosphite from its ternary
and quaternary complexes. Eq 2 describes the kinetics of activation
under assumption of either (1) rapid equilibrium ligand binding
and independence of dissociation constants on the presence of the
other ligand (i.e., k–1/k1 = k–2/k2 = Km and K3 = K4 = KHPi), or (2)
rapid equilibrium binding of phosphite where KHPi = K4 and steady-
state binding of 2MGA under conditions that provide a constant
Km (i.e., (kcat + k–1)/k1 = (k′cat + k–2)/k2; refer to the Discussion for
details). The first- and second-order rate constants as well as Km
Figure 1. Dependence of the rate of MtDXR-catalyzed 2MGA turno-
2-
ver on the concentrations of (A) 2MGA ([HPO3 ] from bottom to
top: 0, 4.7, 14.1, 23.5, 32.9, and 47.0 mM) and (B) phosphite dianion
([2MGA] from bottom to top: 0, 0.5, 0.8, 1.2, 1.7, 2.5, 4.2, and 5.9
mM) at pH 7.5, 25 °C (I = 0.2 M, NaCl). Curves are the result of non-
linear regression performed globally using eq 2.
and KHPi (Table 1) were obtained by global fitting of the rate equa-
tion (eq 2)31 to the data.
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(kcat KHPi + kc′at[HPO32− ])[2MGA]
[2MGA][HPO32− ]+ Km[HPO32− ]+ KHPi[2MGA]+ Km KHPi
The final model for chain A contains residues Arg12-Met389 has
v0
(2)
=
1
2
3
4
5
6
7
8
better density and shows clear density for all three ligands (Figure
S8). This chain was used for all analysis. The active site loop,
Ala189 through Gly206, is disordered and not included in the final
density. There is weak density for Ser204 and Met205 from this
loop however it was not of sufficient quality to be included in the
final model. The active site of chain A shows clear density for the
[E]t
Kinetic Isotope Effects. To study the effect of covalently
linked phosphodianion on the contribution of hydride transfer step
to the reaction coordinate, primary deuterium KIEs originating
from deuterium substitution at the C-4 pro-S position of the dihy-
dronicotinamide ring of NADPH were measured. KIEs on parame-
ters kcat, symbolized DV, and kcat/Km, symbolized D(V/K), were
measured using a direct comparison method for the reactions of
substrate and intermediate in pieces (Figure S5) and compared
with the corresponding KIEs determined for the reactions of DXP
(reported in literature) and MEsP (this work, Figure S4) (Table 1).
2-
Mn2+ ion, NADPH, a molecule of DE, and one HPO3 ion. Unlike
the natural substrate, the DE ligand coordinates the metal ion in a
tridentate fashion. Cleavage of the linkage between the phos-
phate/phosphite and the remainder of the molecule allows the
deoxy sugar to form a more compact orientation that donates three
oxygens to the Mn2+ ion.
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10
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To test whether the carbon-skeleton rearrangement is a signifi-
cant contributor to the reaction coordinate for the substrate in
pieces, a deuterium KIE on this step was determined using trideu-
terated DE. Labeling of the three hydrogen atoms at C-3 and C-4
was chosen based on the ease of synthesis and the expected in-
creased magnitude of the resultant KIE, due to the multiplicative
DISCUSSION
Chemical Competence of Intermediates. The aldehyde
intermediate of the DXR-catalyzed interconversion between DXP
and MEP, MEsP (Scheme 1, R = CH2OPO3 ), was proposed in
2-
early studies of the MEP pathway33 but its generation has never
been directly observed.19,26,34 Our studies revealed that synthetic
MEsP is converted to MEP by EcDXR and MtDXR in the presence
of NADPH, confirming MEsP’s chemical competence reported
previously.26 Likewise, 2MGA was exclusively converted to 2MG in
the presence of the reduced coenzyme. When the oxidized coen-
zyme was used, however, both intermediates were observed to
undergo varying degrees of disproportionation in addition to the
expected rearrangement to their respective ketone isomers. This
aldehyde dehydrogenase activity by DXR had not been reported
previously. The extent of disproportionation is considerably sup-
pressed with MEsP, suggesting that its covalently linked phosphate
group plays a role in the enzyme’s ability to discriminate between
aldehyde and hydrate forms of the intermediate.
effect of the three sp3→sp2 hybridization changes associated with
retro-aldolization. Labeling was accomplished by a chemoenzymat-
ic synthesis (Scheme S1). Commercial (2H4)ethylene glycol was
selectively oxidized by P. pastoris alcohol oxidase to form (1,2,2-
2H3)glycolaldehyde, as reported in literature.27 In order to prevent
α-deuterium washout from the product,12 the reaction was con-
ducted in 87% D2O. Double oxidation to glyoxal was circumvented
by the inclusion of Tris base, which readily forms an imine with
glycoladehyde as it is released from the oxidase. This reversible
adduct was competent in subsequent decarboxylative condensation
with (2-13C)pyruvate catalyzed by DXS. (2-13C)- and (2-13C;3,4,4-
2H3)DE were prepared by this route using unlabeled and deuterat-
ed ethylene glycol, respectively, and purified by flash column silica
gel chromatography as described previously.16 An internal competi-
tion experiment based on the 13C NMR technique of Bennet and
coworkers28 was employed to measure the KIE. In this procedure,
the 13C signal at C-2 serves as a reporter for the neighboring isotope
substitution, appearing as resolved singlets for (2-13C)DE and (2-
13C;3,4,4-2H3)DE whose chemical shifts differ by ~0.1 ppm due to
an isotope effect on the nuclear shielding.32 The ratio of heavy to
light isotopologue (R) was calculated by integration and plotted as
a function of the fraction of conversion of light isotopologue (F1)
(Figure S6), to which eq 3 was fit to yield a KIE of 1.25 ± 0.02.29
Kinetic Competence of Intermediates. A candidate in-
termediate is considered kinetically competent when it reacts at a
rate equal to or exceeding that of the substrate through the com-
plete reaction pathway. The complete intermediate MEsP was
previously demonstrated to be kinetically competent with EcDXR,
being turned over as fast as the substrate DXP.26 Although we have
independently reproduced the reported kcat (reported as specific
activity) for turnover of MEsP by EcDXR, the kcat for DXP under
the same conditions was found to be twofold larger in our hands.
An even larger difference was observed with MtDXR, in which case
MEsP exhibited a 16-fold lower first-order rate constant than that
for DXP. Do these new results imply that MEsP is not an interme-
diate in the natural DXR reaction?
⎛
1
⎞
−1
⎜
⎟
⎠
⎝ KIE
R = R 1− F
(
)
0
1
(3)
Determination of the Structure of MtDXR with the Sub-
strate in Pieces. The structure of MtDXR bound to NADPH,
The observation of kinetic competence supports a compound’s
designated role as a reaction intermediate. A lack of kinetic compe-
tence, however, does not necessary imply that the compound can-
not be a reaction intermediate. As noted by Cleland,35 an exoge-
nously added intermediate must first bind to the enzyme, a step
that is absent when the intermediate is generated transiently in the
active site. To be productive catalysts, most enzymes do not readily
permit dissociation of their intermediates. Because excessively tight
binding of the intermediate, defined by the ratio of dissociation and
association rate constants, would preclude efficient turnover, it is
not unusual for the slow dissociation to be accompanied by compa-
rably slow association. Additionally, once bound, the intermediate
might not adopt the same orientation as when produced transiently
during the natural reaction. Hence, only if the rates of association
and reorientation significantly exceed the substrate’s kcat will an
intermediate appear kinetically competent.
2-
Mn2+, HPO3 , and DE was solved at 2.3 Å resolution. The electron
density revealed differences between the two chains in the asym-
metric unit and will be described separately. The electron density
for Chain B is of poorer overall quality. The chain contains Gly11-
Met389 in the final model. Density for the active site loop is of
modest quality; however all but His200 and Pro201 are present in
the final model. While density for the metal ion was clear, no densi-
ty is present for the truncated substrate. Additionally, the density
for the nucleotide cofactor showed partial occupancy. The
NADPH molecule was not present at full occupancy and was built
containing the only 2ʹ-phosphoribose and the diphosphate linker
between the two nucleotides.
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SCHEME 5. Simplified Kinetic Model for Turnover of DXP an d MEsPa
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
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a The isotope (H or D) and isotope-sensitive step are in red.
First, consider that MEsP binds to the E•NADPH complex to
⎛
⎞ ⎛
⎞
k7k11
1
1
cvf
=
+
(9)
form a ternary complex E*•NADPH•MEsP (Scheme 5, lower
pathway) distinct from that formed transiently during turnover of
DXP (i.e., E•NADPH•MEsP; Scheme 5, upper pathway). Two
possibilities can be envisioned from this point: (1) hydride transfer
occurs with a rate constant k10 much lower than k7, or (2) a slow
conformational change converts E*•NADPH•MEsP to
E•NADPH•MEsP, which then undergoes hydride transfer. The
magnitude of the primary deuterium KIE on kcat for reduction of
MEsP can distinguish between these possibilities.
KIEs for DXP turnover using (4S)-(4-2H)NADPH (NADPD)
were previously measured by the direct comparison method.20,24,36
This method provides KIEs on both kcat/Km and kcat, given by eqs 4
and 5, which reflect different portions of the kinetic mechanism.
⎜
⎟ ⎜
⎟
k + k
k
k8
⎝
−11 ⎠ ⎝
⎠
11
11
These two cases differ in that the slow step for case 1, k10, appears in
the numerator, while that for case 2, k11, appears in the denomina-
tor. The consequence of this is that eq 8 predicts a larger V than
D
D
that observed for DXP while eq 9 predicts a smaller V. The larger
measured value of 1.7 (Table 1) is clearly inconsistent with case 2.
Thus, we conclude that exogenous MEsP lacks kinetic competence
because it binds and reacts in a conformation that is distinct from
that adopted during turnover of DXP.
Rate-Limiting Steps for Substrates and Intermediates.
KIEs are invaluable tools in establishing the mechanism of enzyme-
catalyzed reactions, as exemplified in the preceding experiments
with MEsP. The magnitudes of observed KIEs not only reveal the
degree of rate limitation by the isotope-sensitive step, but also pro-
vide insights on the contribution of other steps, chemical or physi-
cal, to the reaction coordinate.
Primary deuterium KIEs20 together with 13C KIEs for C-2, C-3,
and C-4 of DXP previously measured with MtDXR29 indicate that
isomerization and reduction are jointly rate-limiting processes with
respect to kcat/Km for DXP. With removal of the rearrangement step
and in light of the V of 1.7, one might expect (V/K) for MEsP to
surpass the value of 2.2 measured for DXP (Table 1); interestingly,
this was found to be unity, indicating that hydride transfer is com-
pletely masked by the forward commitment factor. According to
Scheme 5, cf is given by eq 10.
D
Dk + cf + cr Keq
D
V K =
(4)
(
)
1+ cf + cr
Dk + cvf + cr Keq
D
DV =
(5)
1+ cvf + cr
The intrinsic KIE, Dk, that would be observed if the isotope-
sensitive step were completely rate-limiting is mitigated by the
forward (cf for D(V/K) and cvf for DV) and reverse (cr) commitment
factors and the equilibrium isotope effect for the overall reaction
D
D
D
D
(DKeq). With DXP as the varied substrate, (V/K) and V were
found to be 2.2 and 1.3, respectively (Table 1), indicating that re-
duction is partially rate limiting. Suppression of DV relative to
D(V/K) is reflective of a larger cvf. According to Scheme 5, the ex-
pressions for cf and cvf are described by eqs 6 and 7.24
k10
cf =
(10)
k−9
⎛
⎞
k7
k6
k7
The absence of a KIE on kcat/Km suggests that MEsP is a “sticky”
substrate in which it preferentially partitions from the Michaelis
complex through the chemical reaction rather than dissociating
(i.e., k–9 < k10). Given that dissociation of the transiently formed
intermediate has never been observed during DXP turnover, it can
be expected to be similarly sticky.
cf =
1+
≈
(6)
(7)
⎜
⎟
k−6
k−5
k−6
⎝
⎠
⎛
⎜
⎞ ⎛
⎞
k6k7
1
1
cvf
=
+
⎟ ⎜
⎟
k + k
k
k8
⎝
−6 ⎠ ⎝
⎠
6
6
D
The increased value of cvf and associated suppression of V have
been attributed to the slow release of the second product, MEP,
under control of k8.21,24
A similar KIE analysis was performed with the truncated substrate
and intermediate in order to explore the energetic consequences of
severing the phosphodianion’s covalent linkage and of its removal
from the reaction. Unlike the reactions of DXP and MEsP, the reac-
tions of the substrate and intermediate in pieces were found to
exhibit KIEs of unity on both kcat and kcat/Km. To test if the absence
of KIEs in the reaction of the substrate in pieces is the result of fully
rate-limiting isomerization, a combined α-secondary 3,4,4-2H3 KIE
was measured for DE. The observed value of 1.25 represents an
If exogenously added MEsP proceeds through direct, slow hy-
dride transfer (case 1 above), cvf is given by eq 8; case 2 (slow con-
formational change) would provide cvf according to eq 9.
k10
cvf
=
(8)
k8
2
average of 1.08 per H, which is smaller than the typical values of
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binding of phosphite and steady-state binding of 2MGA, and we
1.15–1.3537,38 for such sp3→sp2 rehybridization. Thus, although the
1
2
3
4
have required phosphite binding to be twofold tighter to
E•NADPH•2MGA than to E•NADPH (i.e., K3 = 2K4). The ther-
modynamic box therefore also requires k–1/k1 = 2k–2/k2. Further, in
order to maintain a Km independent of phosphite concentration,
this scheme requires satisfaction of eq 11.
isomerization step is rate contributing in DE turnover, a non-
chemical step such as a conformational change likely also contrib-
utes to rate limitation. In the case of the intermediate in pieces, the
absence of KIEs during reduction also implicates a rate-limiting
D
D
5
6
7
8
physical step. Since both V and (V/K) are suppressed, this step
must occur after formation of the Michaelis complex with 2MGA
and before the first irreversible step (presumably release of the first
product). Thus, a mechanism consistent with the above KIE exper-
iments is identical to Scheme 5 but with an additional ternary com-
plex between isomerization and reduction steps (Scheme S2).
kcat + k−1 kc′at + k−2
Km =
=
(11)
k1
k2
If k1 = k2, then k–1 = 2k–2, allowing an estimation of k–1 = 0.084 s-1
and k–2 = 0.042 s-1 using the values of kcat and kʹcat from Table 1. The
rate equation for such a kinetic model is identical to eq 2 (see deri-
vation in Supporting Information). From this analysis we conclude
that the rate of 2MGA release from the enzyme must be of similar
magnitude to its turnover. However, such a scenario would predict
release and detectable accumulation of 2MGA during transient
turnover of DE. Because we have never observed such accumula-
tion in the absence or presence of phosphite dianion, the kinetic
pathways involving exogenous and transient 2MGA are likely dis-
tinct, not unlike the situation with MEsP. Nevertheless, the reac-
tion of exogenous 2MGA is a useful model system, revealing tight-
ening of the interactions between the enzyme and phosphodianion
when the active site is occupied by the intermediate. Additionally,
this model suggests slower release of the truncated intermediate
from the phosphite dianion-bound active site. One may reasonably
propose similar effects to take place during the formation of transi-
ent 2MGA. Interestingly, according to the thermodynamic box in
Scheme 6, tightening of phosphite–enzyme interactions at the in-
9
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11
12
13
14
15
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60
Kinetic Role of the Phosphodianion Group. In our pre-
vious study we demonstrated that removal of the terminal phos-
phorylmethyl group of DXP results in a 106-fold decrease in turno-
ver rate.39 Therefore, according to the Eyring equation (eq S2), the
nonreacting phosphodianion group provides ∆GPi = 8.4 ± 0.1
kcal/mol of the average kinetic barrier stabilization. Interestingly,
the same analysis applied to the reaction of the intermediate re-
vealed a substantially lower ∆GPi of 6.1 ± 0.1 kcal/mol. Although
this result may appear to indicate that the phosphodianion provides
more stabilization during isomerization than reduction, the fac-
tor(s) responsible for the apparent “kinetic incompetence” of
MEsP likely suppresses the actual stabilization energy.
The kinetic barrier destabilization resulting from substrate and
intermediate truncation was partially relieved by inorganic phos-
phite dianion. While the reaction of truncated substrate experi-
enced only 5-fold activation by saturating phosphite, the reaction of
exogenous truncated intermediate was activated by a factor of 23.
Importantly, in each case an equivalent increase in both the first-
and second-order rate constants was observed, indicating that the
observed activation is the result of a catalytic effect rather than a
binding effect. Accordingly, interactions of the active site with
phosphite dianion provided ∆GHPi = 3.2 ± 0.1 kcal/mol and ∆GHPi
= 4.4 ± 0.2 kcal/mol of kinetic barrier stabilization for turnover of
DE and 2MGA, respectively (eq S3). Since DE turnover is partially
limited by chemistry and 2MGA turnover is entirely limited by
physical steps, the greater phosphite binding energy observed dur-
ing 2MGA turnover suggests an increased role of phosphodianion–
enzyme interactions in acceleration of the physical steps.
SCHEME 6. Thermodynam ic box describing conver-
sion of DE to 2MGA in the absence and presence of
phosphite dianiona
a E = MtDXR•NADPH.
The increase in the activation factor observed for 2MGA turnover
was also accompanied by a twofold decrease in KHPi, the concentra-
tion of phosphite dianion required to reach half-maximal activation.
Recall that the data for each substrate treated separately is con-
sistent with a random binding mechanism (Scheme 4). The inde-
pendence of KHPi with respect to 2MGA concentration (Figure 1B)
suggests that the affinities of phosphite dianion to E•NADPH and
E•NADPH•2MGA are equal. Because the non-essential activation
models for both substrates share a common binding of phosphite
dianion to E•NADPH (K3 in Scheme 4), the observed KHPi should
be identical for DE and 2MGA under equilibrium binding condi-
tions. To evaluate this apparent discrepancy, we independently
determined K3 by measuring a Ki of 28 ± 4 mM for competitive
inhibition by phosphite dianion with respect to DXP. This Ki favor-
ably agrees with the KHPi of 26 mM determined with DE as sub-
strate,16 thus validating the rapid-equilibrium random non-essential
activation model previously invoked with this substrate; the lower
value of KHPi in the case of 2MGA turnover therefore suggests a
change in the kinetic mechanism with the truncated intermediate.
To account for this change we have assumed rapid equilibrium
termediate stage of the reaction (i.e., K15 < K14) would require a
proportional increase in the equilibrium constant for the conver-
sion of the substrate to the intermediate (i.e., K13 = K12 K14/ K15).
Structural Rationale for the Role of the Phosphodian-
ion. To directly assess the nature of physical steps implicated in
rate limitation of the reaction of the substrate in pieces, we co-
crystallized MtDXR with the complete set of ligands involved in
this reaction. The structure resolved in this work is the first example
of DXR crystallized as a catalytically active complex containing a
substrate, NADPH, and divalent metal. Contrary to our previous
hypothesis that phosphite dianion bound in the active site stabilizes
the closed conformation of the enzyme,16 we did not observe elec-
tron density for the flexible catalytic loop 189–206 in the enzyme
2–
subunit bound to DE, NADPH, and HPO3 . The absence of elec-
tron density suggests disorder in the loop, thus supporting our
proposal that the covalent linkage to the phosphodianion is re-
quired to assist induction of productive enzyme conformations.
With the exception of this loop, the overall enzyme conformation is
conserved in structures of MtDXR bound with the substrate in
pieces and with inhibitor FR-90009840 (Figure S8). The active-site
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Journal of the American Chemical Society
cleft located between N- and C-terminal domains is visibly more CONCLUSION
Page 8 of 10
1
2
3
4
5
6
7
8
closed in the structure with inhibitor, thus making it a more-
compact and tight complex.
The deconstruction of ligands in order to determine binding
modes and activities of the resulting fragments lies at the core of
fragment-based drug design, an approach rapidly eclipsing classical
structure-based drug design.42 However, a recent systematic study
demonstrated that application of the same approach to substrate
discovery is particularly challenging and system dependent.43 The
work in this manuscript addresses the energetic and structural ef-
fects of removal of the covalent linkage to the phosphoryl group in
the substrate and intermediate of a multistep enzymatic reaction.
The orientation of the truncated substrate likely reflects a non-
productive binding mode, as the molecule is positioned with the C-
1 methyl group proximal to the dianion (Figure 2), unlike FR-
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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25
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In summary, we have demonstrated the following functional rela-
tionships between the nonreacting phosphodianion and catalysis at
different stages of the reaction. (1) The absence of the covalent
linkage to the phosphate compromises the enzyme’s specificity for
the true (aldehyde) form of the reaction intermediate. A promiscu-
ous conformation of MtDXR induced by separate phosphodianion
exhibits a novel aldehyde dehydrogenase activity. (2) Highly or-
chestrated progress of the reaction does not allow the release of
transiently formed intermediate in the absence or presence of the
covalently linked phosphodianion. This assertion is reinforced by
the apparent inability of the enzyme to adopt the transiently
formed conformation during turnover of the exogenous intermedi-
ates. (3) While the rate of the reaction of the phosphorylated sub-
strate is limited by both chemical steps, reaction of the substrate in
pieces is predominantly limited by physical steps, displaying only
partial rate limitation from isomerization. (4) The loss of interac-
tions with the covalently linked phosphate result in 8.4 and 6.1
kcal/mol destabilization of the kinetic barriers to turnover of sub-
strate and exogenous intermediate, respectively. Part of this energy
(3.2 kcal/mol and 4.4 kcal/mol, respectively) is recovered by inclu-
sion of phosphite dianion, which thereby serves as a non-essential
activator of both reaction steps. (5) At the intermediate stage of the
reaction, there is a tightening of the interactions between the active
site and the phosphodianion, which results in a corresponding in-
crease in the equilibrium between substrate and intermediate. (6)
The structure of the MtDXR complex with the substrate in pieces
underscores the role of the covalently linked phosphate in promot-
ing the productive orientation of the substrate and induction of the
loop-closed conformation associated with efficient catalysis.
Figure 2. Superposition of the crystal structures of MtDXR with
inhibitor FR-900098, Mn2+, and NADPH (not shown) (PDB entry
4A03, purple) and MtDXR with DE, Mn2+, NADPH (not shown), and
HPO3 (PDB entry 4RCV, green). Residues of the flexible loop, phos-
phodianion binding pocket, and metal-binding pocket are shown. Loop
189–206 is absent in the structure with the substrate in pieces (green).
2-
900098 and DXP. The retro-aldol step requires deprotonation of
the alcohol at C-4 to generate glycolaldehyde phosphate (from
DXP) or formaldehyde (from DE) (Scheme 1). Although the en-
zymatic base has not yet been identified, it is likely positioned prox-
imal to C-4, which occupies the same space as the C-1 methyl
group of DE in the crystal structure; thus, reorientation of DE
would be expected for reaction to take place, an action that likely
requires dissociation, rotation, and re-association. Further, all three
oxygen atoms in DE coordinate the catalytic divalent metal. The C-
4 hydroxyl group may need to sever its coordinate bond in order to
assume a conformation poised for proton transfer to the catalytic
base. The covalently linked phosphodianion of DXP restricts rota-
tion along the carbon backbone making tridentate coordination
highly unlikely for the complete substrate, assuming its binding
mode resembles that of FR-900098; thus, the attached phosphoryl
group may facilitate adoption of a reactive conformation in DXP.
ASSOCIATED CONTENT
Supporting Information. 2MGA and MEsP synthesis; H-NMR
1
spectra of reactions with 2MGA, DE, and MEsP; A340 transient for time
course of the incubation of 2MGA with NADP+ and MtDXR; Michae-
lis-Menten plots for primary deuterium KIEs; determination of the
(3,4,4-2H3)DE KIE; crystallographic details and refinement statistics;
electron density for ligands of MtDXR in its crystal structure with sub-
strate in pieces, comparison of conformations of FR-900098-bound
and substrate in pieces-bound MtDXR subunits. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
The position of phosphite in the phosphate-binding pocket par-
tially overlaps the position of the phosphonate group of FR-900098
(Figure 2). Both groups are in the proximity to establish hydrogen
bonds with Lys219, Asn218, and Ser213. A hydrogen bond (or salt
bridge) with His200 is considered crucial for the closure of the
flexible loop upon binding of the phosphodianion of the ligand.41 It
is possible that a similar interaction exists with phosphite dianion.
* amurkin@buffalo.edu
Funding Sources
This work was supported by a DuPont Young Professor Grant and
NSF CAREER Award CHE1255136 (to A.S.M.) and NIH Grant GM-
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank Jeffrey Marvin and Margaret Moynihan for expression and
purification of EcDXR, and Dr. Gregory Tombline for preparation of
DXP.
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