Substrate orientation and specificity in xanthine oxidase: Crystal structures of the enzyme in complex with indole-3-acetaldehyde and guanine

Article abstract of DOI:10.1021/bi401465u

Xanthine oxidase is a molybdenum-containing hydroxylase that catalyzes the hydroxylation of sp²-hybridized carbon centers in a variety of aromatic heterocycles as well as aldehydes. Crystal structures of the oxidase form of the bovine enzyme in complex with a poor substrate indole-3-acetaldehyde and the nonsubstrate guanine have been determined, both at a resolution of 1.6 A. In each structure, a specific and unambiguous orientation of the substrate in the active site is observed in which the hydroxylatable site is oriented away from the active site molybdenum center. The orientation seen with indole-3-acetaldehyde has the substrate positioned with the indole ring rather than the exocyclic aldehyde nearest the molybdenum center, indicating that the substrate must rotate some 30 in the enzyme active site to permit hydroxylation of the aldehyde group (as observed experimentally), accounting for the reduced reactivity of the enzyme toward this substrate. The principal product of hydroxylation of indole-3-acetaldehyde by the bovine enzyme is confirmed to be indole-3-carboxylic acid based on its characteristic UV-vis spectrum, and the kinetics of enzyme reduction are reported. With guanine, the dominant orientation seen crystallographically has the C-8 position that might be hydroxylated pointed away from the active site molybdenum center, in a configuration resembling that seen previously with hypoxanthine (a substrate that is effectively hydroxylated at position 2). The ～180 reorientation required to permit reaction is sterically prohibited, indicating that substrate (mis)orientation in the active site is a major factor precluding formation of the highly mutagenic 8-hydroxyguanine.

Full text of DOI:10.1021/bi401465u

Article
pubs.acs.org/biochemistry
Substrate Orientation and Specificity in Xanthine Oxidase: Crystal
Structures of the Enzyme in Complex with Indole-3-acetaldehyde
and Guanine
Hongnan Cao,† James Hall, and Russ Hille*
Department of Biochemistry, University of California, Riverside, California 92521, United States
ABSTRACT: Xanthine oxidase is a molybdenum-containing hydroxylase that catalyzes the
hydroxylation of sp2-hybridized carbon centers in a variety of aromatic heterocycles as well as
aldehydes. Crystal structures of the oxidase form of the bovine enzyme in complex with a poor
substrate indole-3-acetaldehyde and the nonsubstrate guanine have been determined, both at a
resolution of 1.6 Å. In each structure, a specific and unambiguous orientation of the substrate in the
active site is observed in which the hydroxylatable site is oriented away from the active site
molybdenum center. The orientation seen with indole-3-acetaldehyde has the substrate positioned with
the indole ring rather than the exocyclic aldehyde nearest the molybdenum center, indicating that the
substrate must rotate some 30° in the enzyme active site to permit hydroxylation of the aldehyde group
(as observed experimentally), accounting for the reduced reactivity of the enzyme toward this substrate.
The principal product of hydroxylation of indole-3-acetaldehyde by the bovine enzyme is confirmed to
be indole-3-carboxylic acid based on its characteristic UV−vis spectrum, and the kinetics of enzyme
reduction are reported. With guanine, the dominant orientation seen crystallographically has the C-8
position that might be hydroxylated pointed away from the active site molybdenum center, in a configuration resembling that
seen previously with hypoxanthine (a substrate that is effectively hydroxylated at position 2). The ∼180° reorientation required
to permit reaction is sterically prohibited, indicating that substrate (mis)orientation in the active site is a major factor precluding
formation of the highly mutagenic 8-hydroxyguanine.
MoS group of the molybdenum coordination sphere,
anthine oxidase is the eponymous member of a large
Xfamily of molybdenum-containing enzymes that catalyze yielding a key intermediate that can be formulated as
LMoIVO(SH)(OR), where OR represents the now-hydroxy-
the hydroxylation of carbon centers in a broad range of
aromatic heterocycles and aldehydes.1−4 The bovine enzyme
lated product coordinated to molybdenum via the catalytically
introduced hydroxyl group.8−10 The structure of this
has been crystallized and found to be a homodimer, with each
intermediate has been confirmed by X-ray crystallography of
the bovine enzyme in reaction with the slow substrate 2-
hydroxy-6-methylpurine.11 Finally, the bound product is
displaced from the molybdenum coordination sphere by
solvent hydroxide to regenerate the Mo-OH ligand in
preparation for a subsequent catalytic cycle.
Several active site residues are highly conserved in the
xanthine-hydroxylating enzymes of many species, including two
phenylalanine residues that constrain the bound substrate to a
plane perpendicular to the equatorial plane of the molybdenum
center, as well as Glu 1261, Glu 802, and Arg 880. Glu 1261 is
believed to function as the catalytic base in both the
heterocycle- and aldehyde-utilizing molybdenum hydroxy-
lases.7,12−14 The oxygen atom of the Mo-OH is within
hydrogen bonding distance of this glutamate in both the
bovine and bacterial enzymes,5,15 and an E730A variant of the
Rhodobacter capsulatus enzyme is essentially inactive toward
xanthine, conservatively exhibiting an at least 107-fold reduction
in the limiting rate of enzyme reduction relative to that of the
subunit containing four redox-active sites in separate, well-
defined domains of the polypeptide: an active site molybdenum
center, a pair of spinach ferredoxin-like [2Fe-2S] clusters, and
FAD.5 The overall catalytic sequence consists of a reductive
half-reaction in which the substrate is oxidatively hydroxylated
at the molybdenum center [reducing it from Mo(VI) to
Mo(IV)] and, after intramolecular electron transfer involving
the intervening Fe−S centers, an oxidative half-reaction in
which the reducing equivalents are removed from the enzyme
via its FAD (with O2 and NAD+ serving as the electron
acceptor for the oxidase and dehydrogenase forms of the
enzyme, respectively).1,4 As shown in Figure 1, the oxidized
molybdenum center has an LMoVIOS(OH) structure in a
square-pyramidal coordination geometry, with the MoO
group occupying the apical position; L represents the
enedithiolate ligand of a pyranopterin cofactor common to all
mononuclear molybdenum- and tungsten-containing enzymes.
In the reductive half-reaction, substrates are hydroxylated at a
specific carbon position in a reaction initiated by nucleophilic
attack of the equatorial Mo-OH group of the metal center upon
deprotonation involving a conserved glutamate residue (Glu
1261 in the bovine enzyme), as shown in Figure 1.6,7 Hydride is
then transferred from the carbon being hydroxylated to the
Received: October 28, 2013
Revised: December 18, 2013
Published: January 3, 2014
© 2014 American Chemical Society
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Figure 1. Reaction mechanism for xanthine oxidase. Catalysis is initiated by base-assisted nucleophilic attack of the equatorial Mo-OH on the C-8
atom of xanthine with concomitant hydride transfer from C-8 to MoS, resulting in reduction of Mo(VI) to Mo(IV). Reoxidation of the
molybdenum center occurs by the transfer of an electron to the other redox-active centers of the enzyme, accompanied by deprotonation of the Mo-
SH bond and displacement of the bound product by hydroxide from solvent to regenerate the Mo-OH group.
wild-type enzyme.6 Arg 880, which is conserved only in the
heterocycle-oxidizing enzymes and is usually a methionine in
those molybdenum-containing hydroxylases that act on
aldehydes, has been proposed to stabilize the accumulation of
negative charge on the heterocycle in the course of nucleophilic
attack via electrostatic interactions with the C-6 carbonyl
oxygen of xanthine.16 The guanidinium group of this arginine is
within hydrogen-bonding distance of the C-6 carbonyl oxygen
of xanthine bound at the active site,17 and its mutation to
methionine in the R. capsulatus enzyme results in a 2 × 104-fold
decrease in the reaction rate with xanthine as the substrate.16
Comparable results in steady-state experiments have been seen
for the homologous mutations of the human enzyme.18
structures of compounds relevant to this work are shown in
Figure 2.
We have previously shown that hypoxanthine binds to
bovine xanthine oxidase in two different orientations, only one
of which is catalytically competent.19 This illustrates that the
substrate can assume alternate orientations in the active site and
that this can influence reactivity. Here we report crystal
structures for bovine xanthine oxidase in complex with indole-
3-acetaldehyde (a poor substrate for the bovine enzyme, but the
physiological substrate for the closely related aldehyde oxidase
AOX III from Arabidopsis thaliana, for which the product
carboxylic acid is an important hormone) and guanine (a
purine upon which the xanthine oxidase is known not to act
effectively), both at 1.6 Å resolution. Consistent with their poor
reactivities, we find that both substrates show a dominant
orientation in the active site of the enzyme that is catalytically
nonproductive. In the case of indole-3-acetaldehyde, a modest,
sterically allowed rotation juxtaposes the aldehyde group with
the molybdenum center that allows hydroxylation to take place.
With guanine, the substrate must rotate approximately 180° to
present its 8 position to the active site molybdenum center for
hydroxylation, and this rotation is sterically prohibited. The
observed nonproductive orientation seen with guanine provides
a steric explanation for how the enzyme discriminates against
guanine as the substrate, thereby avoiding in vivo formation of
the highly mutagenic 8-hydroxyguanine.
Figure 2. Chemical structures and numbering scheme of compounds
used in this work.
Purification of Bovine Xanthine Oxidase. Bovine
xanthine oxidase was purified from fresh, unpasteurized bovine
milk (Scott Brothers Dairy, Chino, CA) as described
elsewhere.20 To eliminate possible problems caused by genetic
heterogeneity in the dairy herd, the enzyme used for
crystallographic work was isolated from milk obtained from
an individual cow. The purified enzyme exhibited a ratio of
absorbance at 280 and 450 nm of 6.0−6.8 and a percentage of
functionality of 30−60% due to variability in the amount of the
nonfunctional desulfo form of the enzyme present (in which a
catalytically essential MoS group has been replaced by a
second MoO group in the molybdenum coordination
sphere). The percentage of functionality was determined as
described previously by Massey and Edmondson.21 A folate
affinity column procedure22 was used as a final purification step
to enrich the functionality to >80%. The purified enzyme was
made 1.0 mM in sodium salicylate and stored in liquid nitrogen
and passed down a Sephadex G-25 column (to remove
salicylate) prior to being used.
Crystallization, Data Acquisition, and Structure
Determination. Xanthine oxidase crystals were grown using
the batch method according to previously published methods
using microbridges to hold batch solutions in sealed wells of a
24-well tray.11,19 The batch solutions consisted of 10 μL of a
34.5 μM (5 mg/mL) enzyme solution mixed with 5 or 6 μL of
a 12% PEG 8000 precipitant solution. Throughout this work,
the enzyme concentration refers to the total monomer
EXPERIMENTAL PROCEDURES
■
Materials. Magnetic bases for use with the automated
crystallography system at the LRL-CAT beamline of Argonne
National Laboratory (Argonne, IL) were obtained from MAR-
USA (Evanston, IL). Mounted cryoloops, magnetic cryovials,
and crystal growth materials were obtained from Hampton
Research (Aliso Viejo, CA). Polyethylene glycol (PEG) 200
and PEG 8000 stock solutions were obtained from Hampton
Research. All other chemicals and reagents, including indole-3-
acetaldehyde, indole-3-acetate, and guanine, were obtained at
the highest quality or purity available from Sigma-Aldrich or
Fisher Scientific and used without further purification. The
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concentration based on an extinction coefficient at 450 nm of
37.8 mM−1 cm−1.20
flushing with O2-scrubbed argon over the course of ≥1 h.
Substrate solutions were made anaerobic by being bubbled with
argon in airtight glass syringes for 10−15 min. The reaction
conditions included 0.1 M pyrophosphate and 0.3 mM EDTA
(pH 8.5) at 25 °C. Reactions were followed at 450 nm,
monitoring the decrease in absorbance due to enzyme
reduction. The initial reaction mix typically contained ∼4 μM
functional enzyme and 25−500 μM substrate (after mixing).
For each substrate concentration used, the reaction was
repeated in triplicate with kinetic transients fit to a single-
exponential equation using manufacturer’s software to obtain
kobs values, which were then averaged for each substrate
concentration. The kinetic parameters kred and Kd were
obtained from hyperbolic fits to plots of kobs versus [S] using
eq 1:
Crystals were grown in the dark at 25 °C for 2−3 days before
being harvested. A 42% solution of PEG 200 was used as a
cryoprotectant. Indole-3-acetaldehyde or guanine was intro-
duced to xanthine oxidase crystals via soaking to reach a final
substrate concentration of 1 mM in the crystal well. After
soaking in indole-3-acetaldehyde for 2−5 min or soaking in
guanine for 5−8 min, crystals were mounted and flash-frozen in
liquid nitrogen. Diffraction data were collected at Argonne
National Laboratory on the LRL-CAT beamline using a
wavelength of 0.9793 Å and a MARCCD 165 detector. Data
sets were collected to a resolution of 1.6 Å for both structures
reported here.
Crystallographic data were processed using the MOSFLM
package of the CCP4 program suite.23 The structure of each
complex was determined by molecular replacement using the
MOLREP package of CCP4 with the previously reported
crystal structure of xanthine oxidase of Enroth et al. as the
search model [Protein Data Bank (PDB) entry 1FIQ5]. The
output structure was refined first by rigid body refinement and
subsequently by restrained refinement using the REFMAC
program of the CCP4 suite.24−28 The weighting term for
geometric restraints was adjusted in REFMAC to minimize
Rcryst while at the same time minimizing the difference between
Rcryst and Rfree. No noncrystallographic symmetry restraints
were used during refinement of the structures. Water molecules
were added to both the crystal structures (using REFMAC)
prior to building in the substrates.
Structure files for indole-3-acetaldehyde and guanine were
constructed using the PRODRG2 server,29 and the respective
Protein Data Bank entries were built into the corresponding
2F0 − Fc and F0 − Fc omit electron density maps observed in
the active sites using COOT.30 After the substrate structure
files had been merged with those for the refined protein
structures, the results were refined again using the restrained
refinement mode in REFMAC. The final structure files were
deposited into the PDB as entries 3NVZ and 3NVW for the
structures of xanthine oxidase in complex with indole-3-
acetaldehyde and guanine, respectively. All structures in the
figures have been rendered using PyMol.31
UV−Visible Spectroscopic Analysis of Products of
Hydroxylation of Indole-3-acetaldehyde by Bovine
Xanthine Oxidase. To confirm the product of hydroxylation
of indole-3-acetaldehyde by bovine xanthine oxidase as the
corresponding carboxylic acid, the following experiments were
performed. With the bovine enzyme, which was effective, 50
μM indole-3-acetaldehyde was incubated aerobically with a
catalytic amount (118 nM) of enzyme in 1 mL of 0.1 M sodium
pyrophosphate, 0.3 mM EDTA buffer (pH 8.5) at 4 °C in the
dark for 24 h. The UV−visible absorption spectrum of the
reaction product was recorded directly and was corrected for
the relatively small absorbance due to the enzyme. The UV−
visible absorption spectrum of the eluate was then recorded,
and the dominant hydroxylation product of indole-3-
acetaldehyde was identified as indole-3-carboxylic acid based
on a comparison of the observed UV−vis spectra of the
reaction products with that authentic indole-3-carboxylic acid.
Reductive Half-Reaction Experiments. Rapid kinetic
experiments with indole-3-acetaldehyde were performed using
an Applied Photophysics SX-18MV stopped-flow instrument
equipped with a diode array detector. Enzyme solutions in glass
tonometers were made anaerobic by alternately evacuating and
kobs = (kred[S])/(Kd + [S])
(1)
RESULTS
■
Crystal Structure of Xanthine Oxidase in Complex
with Indole-3-acetaldehyde. The crystal structure of
xanthine oxidase in complex with indole-3-acetaldehyde was
determined at 1.6 Å resolution by molecular replacement using
the previously reported crystal structure of xanthine oxidase of
Enroth et al. as the search model (PDB entry 1FIQ5). Final R
factors were an Rcryst of 21.5% and an Rfree of 24.6% (Table 1).
Table 1. Statistics for Data Collection and Refinement of the
Crystal Structures of Xanthine Oxidase in Complex with
Indole-3-acetaldehyde and Guanine
XO with indole-3-
acetaldehyde
XO with guanine
PDB entry
3NVZ
P21
3NVW
space group
P21
cell dimensions
a, b, c (Å)
133.4, 73.7, 138.9 132.7, 73.4, 138.1
α, β, γ (deg)
90.0, 97.1, 90.0
90.0, 97.0, 90.0
resolution (Å)
wavelength (Å)
no. of unique reflections (test set)
45.2−1.6
0.9793
131.7−1.6
0.9793
339741 (17094)
96.5 (93.3)
334928 (16908)
96.6 (94.3)
completeness (%) [highest-
resolution shell (Å)]
a
I/σ (highest-resolution shell)
6.1 (2.2)
18.2 (2.4)
18.4 (23.8)
21.2 (26.8)
Rcryst (highest-resolution shell)
Rfree (highest-resolution shell)
21.5 (37.9)
24.6 (44.3)
b
Ramachandran statistics (%)
90.4, 9.0, 0.3, 0.3 90.0, 9.7, 0.1, 0.2
mean coordinate error based on the 0.103
0.091
0.059
0.008
1.2
free R value (Å)
mean coordinate error based on the 0.076
maximum likelihood (Å)
root-mean-square deviation for
bond lengths (Å)
0.011
root-mean-square deviation for
bond angles (deg)
average B value (Å2)
1.3
21.6
18.0
no. of non-hydrogen atoms in
refinement
20672
21495
no. of waters
1600
2173
a
I/σ is defined as the ratio of the averaged value of the intensity to its
b
standard deviation. Ramachandran statistics indicate the percentage
of residues in the most favored, additionally allowed, generously
allowed, and disallowed regions of the Ramachandran diagram as
defined by PROCHECK.34
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Figure 3. Electron density maps of xanthine oxidase complexed with indole-3-acetaldehyde. Both active sites of the assymmetric unit (A and B, and
C and D) have the same single dominant orientation with C-2 proximal to the molybdenum center. Panels A and C show F0 − Fc omit maps
contoured at 3.0σ, while panels B and D show 2F0 − Fc omit maps contoured at 1.0σ, all within 2.0 Å of all atoms shown. All omit maps were
constructed prior to addition of indole-3-acetaldehyde in the refinement model and subsequently overlaid with the final model. The atoms were
color-coded as follows: cyan for molybdenum, white for carbon, blue for nitrogen, red for oxygen, yellow for sulfur, and orange for phosphorus. All
structures were rendered with PyMOL.31 Abbreviation: I3A, indole-3-acetaldehyde.
There was a single homodimer in the asymmetric unit, with an
overall structure very similar to that seen previously for the
oxidized bovine enzyme5 and our own previously reported
structures of the enzyme in complex with 2-hydroxy-6-
methylpurine, xanthine, lumazine, and hypoxanthine,11,17,19
including the observation of finite electron density for the C-
terminal residues (residues 1316−1325) of one monomer of
the homodimer, and these were included during the refinement
process.
Figure 3 shows F0 − Fc and 2F0 − Fc omit electron density
maps for indole-3-acetaldehyde bound in each of the two active
sites in the asymmetric unit, contoured at 3.0σ and 1.0σ,
respectively. In each active site, a single dominant orientation of
indole-3-acetaldehyde is observed. The aromatic indole ring is
sandwiched between phenylalanine residues Phe 1009 and Phe
914 (Figure 4), with the indole nitrogen within hydrogen
bonding distance of Glu 802 (at 2.78 and 2.86 Å in the two
active sites) and the exocyclic carbonyl oxygen within hydrogen
bonding distance of Arg 880 (2.89 and 3.15 Å from the two
guanidinium nitrogens in one active site and 2.99 and 3.36 Å in
the other). C-2 of the substrate is within 3.96 Å (and 4.07 Å) of
the equatorial oxygen of the Mo-OH group and 5.76 Å (and
5.97 Å) of the molybdenum. No bridging electron density is
observed between the equatorial Mo-OH group and the
proximal carbon of the substrate, indicating that catalysis has
not progressed to any significant degree in either active site of
the asymmetric unit, presumably because of the low reactivity
of the substrate.
very similar vertebrate aldehyde oxidases (where a methionine
is most commonly found at the corresponding position32). To
become hydroxylated, as observed experimentally (see below),
it is necessary that the indole-3-aldehyde rotate within the plane
defined by the two phenylalanine residues by some 30°, a
motion that disrupts the interaction between the aldehyde
functionality of the substrate and Arg 880 (Figure 4) but is
otherwise minimally restricted from a steric standpoint.
Although not quantified directly in this study, we note that
the energetics of this motion must be small compared to kT at
room temperature because the indole-3-aldehyde is in fact
experimentally observed to be hydroxylated.
While the two phenylalanines of the active site constrain the
substrate to a plane approximately perpendicular to the base
plane of the molybdenum coordination sphere, the substrate
can assume multiple orientations within this plane. We have
previously observed, for example, two alternate orientations for
both hypoxanthine and 6-mercaptopurine binding in bovine
xanthine oxidase,19 with one orientation being appropriate for
hydroxylation and the other not, having the site of
hydroxylation (C-2) oriented away from the molybdenum
center rather than toward it. More generally, for any given
substrate, an equilibrium distribution of orientations may be
considered to exist with a distribution of intrinsic reactivities, in
which case the overall catalytic throughput would reflect the
weighted average of the catalysis for the ensemble of substrate
orientations, the individual contribution of each being
determined by the product of its fractional population in a
given orientation and its intrinsic catalytic rate, as given in eq 2.
It is evident from the crystal structure that the substrate is
not optimally oriented for hydroxylation of the exocyclic
carbonyl group, whose carbon is some 5.4 Å from the oxygen of
the Mo-OH group. Arg 880 appears to be principally
responsible for the unfavorable orientation, because of its
interaction with the exocyclic aldehyde group, and it is
noteworthy that this residue is not conserved in the otherwise
kred = ∑n(qiki)
(2)
where kred is the rate constant for enzyme reduction, qi is the
fractional population of orientation i, and ki is the intrinsic
catalytic rate for orientation i. The situation is an example of
the principal of near-attack conformers, in which thermally
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Figure 4. Structures of xanthine oxidase complexed with indole-3-acetaldehyde. Panels A and B show one active site, while panels C and D show the
other active site in the asymmetric unit. Panels B and D are rotated approximately 90° about the vertical from panels A and C. Atoms are colored as
in Figure 3. (E) Indole-3-acetaldehyde bound in the active site with Phe 914 and Phe 1009 shown, with the hydrogen bonding distances indicated.
All structures were rendered with PyMOL.31
populated configurations of the Michaelis complex play an
important role in facilitating the transition state and
accelerating the reaction rate.33−35 At the extreme limit, a
strictly nonproductive orientation (ki = 0) will have zero
contribution to the overall rate of enzyme reduction. In the case
of hypoxanthine,15,19 the nonproductive orientation observed in
the crystal structure represents an extreme case of this situation,
having the wrong carbon (C-8 rather than C-2) oriented
toward the molybdenum center for hydroxylation to yield
xanthine (the known physiological product). The lack of
reactivity of this orientation has been confirmed by the failure
to detect the expected 6,8-dihydroxypurine product in the
course of the reaction of the enzyme with hypoxanthine.19 In
the crystal structure with bound indole-3-acetaldehyde
presented here, the observed orientation of the substrate
suggests a single dominant but nonproductive orientation with
C-2 proximal to the molybdenum center for hydroxylation,
consistent with the observed low reactivity of the enzyme
toward this substrate (see below).
reactivity of indole-3-acetaldehyde as a substrate was next
examined. The bovine enzyme is reported to slowly hydroxylate
indole-3-acetaldehyde to the corresponding carboxylic acid (a
reaction analogous to that of the bona fide molybdenum-
containing aldehyde oxidase in plants that catalyzes the
synthesis of this plant hormone under physiological con-
ditions), which is confirmed here, the crystallographically
observed orientation notwithstanding. Figure 5 shows the UV−
visible absorption spectra of indole-3-acetaldehyde, authentic
indole-3-carboxylic acid, and the product of hydroxylation of
indole-3-acetaldehyde by the enzyme. It is clear that the UV−
visible spectra of indole-3-acetaldehyde and indole-3-carboxylic
acid are distinct, with local maxima at 244, 260, and 300 nm for
the former and 278 nm for the latter. The product of the
reaction of indole-3-acetaldehyde with xanthine oxidase has a
normalized spectrum identical to that of authentic indole-3-
carboxylic acid. We conclude that bovine xanthine oxidase is
indeed able to hydroxylate indole-3-acetaldehyde and that the
product of the reaction is indole-3-carboxylic acid.
Analysis of the Products of Hydroxylation of Indole-
3-acetaldehyde by Bovine Xanthine Oxidase. The
Reductive Half-Reaction Experiments with Indole-3-
acetaldehyde. In the orientation of indole-3-acetaldehyde
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Figure 5. UV−visible absorption spectra of indole-3-acetaldehyde,
indole-3-carboxylic acid, and the products of hydroxylation of indole-
3-acetaldehyde by bovine xanthine oxidase. Spectra shown are for
indole-3-acetaldehyde, indole-3-carboxylic acid, and the product of the
reaction of indole-3-acetaldehyde with xanthine oxidase. The
extinction coefficients of the reaction products are normalized at
278 nm where the local maximum is observed for the characteristic
UV−vis absorption spectrum of indole-3-carboxylic acid. For the
reaction with bovine xanthine oxidase, 118 nM enzyme was added
prior to incubation for 1 day, and its absorption was subtracted as the
baseline from that of the final reaction mix. Abbreviations: XO,
xanthine oxidase; XDH, xanthine dehydrogenase; WT, wild type; I3A,
indole-3-acetaldehyde; I3C, indole-3-carboxylic acid.
Figure 6. Plot of kobs vs indole-3-acetaldehyde concentration for the
reaction with xanthine oxidase. The reaction conditions included 0.1
M pyrophosphate buffer, 0.3 mM EDTA, pH 8.5, and 25 °C. The
hyperbolic fit to the data indicated yields a kred of 0.13 s−1 and a Kd of
95 μM.
Crystal Structure of Xanthine Oxidase in Complex
with Guanine. The overall protein structure of guanine-
complexed xanthine oxidase at 1.6 Å resolution is again very
similar to that previously reported for the oxidized enzyme,
with all differences limited to the active site. Table 1
summarizes the structural refinement statistics, and Figure 7
shows the two active sites of the asymmetric unit overlaid with
the F0 − Fc omit map contoured at 3σ and the 2F0 − Fc omit
map contoured at 1σ (with guanine omitted from the latter
map). The strong positive electron density in the F0 − Fc omit
map in each active site of the enzyme clearly indicates a single
dominant orientation for the bound guanine with C-8 pointing
away from and the C-2 amino group oriented toward the active
site molybdenum center. Again, no electron density is observed
connecting the Mo-OH group and guanine that might suggest
partial progression through the catalytic sequence.
The interactions stabilizing the observed orientation of
guanine include an approximately planar hydrogen bonding
network involving the C-2 amino group of guanine with the
side chain carboxylate group of Glu 802 (at 2.78 and 2.84 Å in
the two active sites) and the Mo-OH oxygen (2.74 and 2.78 Å);
Glu 1261 remains within hydrogen bonding distance of the
Mo-OH group (at 3.11 and 3.06 Å, respectively) but does not
interact directly with bound guanine in either active site of the
asymmetric unit. The C-6 keto group are also within hydrogen
bonding distance of the guanidinium group of Arg 880 (3.11
and 3.10 Å, respectively), and N-3 is within hydrogen bonding
distance of Glu 802 (2.72 and 2.74 Å, respectively). This
network is shown in Figure 7E, with the distances indicated.
The two active site phenylalanine residues are again in van der
Waals contact with the bound guanine (but are shown only in
Figure 7E for the sake of clarity). The complex strongly
resembles that seen with hypoxanthine that leads to
hydroxylation at C-2 with that substrate,15,19 but which is
impossible with guanine given the presence of the C-2 amino
group. The orientation of guanine is the opposite of that
required for hydroxylation of C-8 of guanine. This unfavorable
orientation, with the substrate misoriented by ∼180°, is
undoubtedly a major reason for the lack of reactivity of the
enzyme toward guanine.
seen crystallographically with the bovine enzyme, hydroxylation
should occur at C-2 of the indole ring rather than the exocyclic
carbonyl, which we observed experimentally as described above.
Given the low intrinsic reactivity of xanthine oxidase toward
indole-3-acetaldehyde and the inherent uncertainty in steady-
state assays, to quantify the reactivity of the enzyme with
indole-3-acetaldehyde we have undertaken a reductive half-
reaction analysis rather than conventional steady-state experi-
ments. The experiment involves mixing the oxidized enzyme
with varying concentrations of indole-3-acetaldehyde, yielding a
rate constant for enzyme reduction from the observed
exponential decrease in absorbance as the substrate reduces
the enzyme (see Experimental Procedures). Figure 6 shows a
plot of the observed rate constant for reduction of xanthine
oxidase under anaerobic conditions by varying concentrations
of indole-3-acetaldehyde (following the loss of absorbance at
450 nm due to reduction of the enzyme’s FAD and iron−sulfur
centers). A hyperbolic fit to the data yields a limiting kred of
0.13 s−1 and a Kd of 95 μM. It is evident that enzyme is
compromised in terms of transition-state stabilization (kred is
less than 1% of the value of 18 s−1 observed with xanthine as
the substrate36) but that the affinity of the enzyme for the
substrate is decreased by a factor of only 6 (Kd for xanthine
under the reaction conditions of 15 μM).
Given that indole-3-acetate is the sole product of the
enzyme-catalyzed reaction, it is evident that the substrate
aldehyde is able to reorient in the active site to bring the
aldehyde group into the proximity of the molybdenum center
for reaction to occur. This involves an ∼30° rotation of the
indole ring between the two active site phenylalanine residues
Phe 914 and Phe 1009 of the bovine enzyme, a motion that
appears to be sterically allowed on the basis of the crystal
structure (Figure 4), if thermodynamically unfavorably in that it
ruptures the hydrogen bond with Arg 880.
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Figure 7. Structures of xanthine oxidase complexed with guanine. Panels A and B show one active site, while panels C and D show the other active
site in the asymmetric unit. Panels A and C show F0 − Fc omit maps contoured at 3.0σ, and panels B and D show 2F0 − Fc omit maps contoured at
1.0σ, all within 1.8 Å of all atoms shown. All the omit maps were constructed before the introduction of guanine for the refinement of the model and
overlaid with the final model. Molybdenum is colored teal, carbon white, nitrogen blue, oxygen red, sulfur yellow, and phosphorus orange. (E)
Guanine bound in the active site with Phe 914 and Phe 1009 shown, with the hydrogen bonding distances indicated. All structures were rendered
with PyMOL.31
Even if guanine were able to transiently adopt a more
favorable orientation (as indole-3-acetaldehyde is apparently
able to do), the less effective stabilization of negative charge on
the heterocycle in the course of the reaction due to the
presence of the C-2 amino group substitution might well
reduce the intrinsic susceptibility of the guanine C-8 to
nucleophilic attack.19 To quantitatively assess this effect, we
have performed partial charge calculations for the neutral form
of stable N-7-H tautomers of guanine and xanthine using the
MOPAC 2009 software package.37 We find that the positive
partial charges at C-8 of guanine and xanthine are 0.026 and
0.065, respectively, and the partial charges on the C-8-H groups
are 0.256 and 0.199, respectively. These results indicate that the
overall polarity of the C-8-H bond and its susceptibility to
nucleophilic attack is somewhat greater in xanthine that in
guanine, but not sufficiently so to account for the vast
difference in reactivity. We conclude that the unfavorable
orientation guanine assumes upon binding to the enzyme is a
principal reason it fails to serve as an effective substrate for
xanthine oxidase.
DISCUSSION
■
In this work, we observe a single dominant orientation for
binding of both indole-3-acetaldehyde and guanine in the active
site of bovine xanthine oxidase, both of which are expected to
be catalytically unproductive on the basis of the distance of the
site to be hydroxylated from the catalytically labile equatorial
Mo-OH group of the enzyme’s molybdenum center. In the case
of indole-3-acetaldehyde, while the substrate is readily
accommodated between Phe 914 and Phe 1009 that define
the substrate binding pocket, Arg 880 forms hydrogen bonds to
the exocyclic aldehyde group that is hydroxylated in such a way
that the carbonyl carbon is held approximately 5.4 Å from the
oxygen of the Mo-OH group. To orient more appropriately for
catalysis, the substrate must rotate some 30° to bring the
exocyclic carbonyl into juxtaposition with the Mo-OH group.
This is a relatively modest rearrangement, and the motion
appears to be readily allowed sterically in the active site;
although it involves disruption of the hydrogen bond with Arg
880, the hydrogen bond with Glu 802 is maintained (as are the
539
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enzymes in higher organisms. Coord. Chem. Rev. 255, 1179−1205.
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M. Y., LeGall, J., Hille, R., Archer, M., and Romao, M. J. (1996) A
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hydrophobic contacts with Phe 914 and Phe 1009). It is likely
that kT is sufficient to populate the catalytically effective
orientation at least a portion of the time, in which case the
situation is equivalent to the near-attack conformer formalism
developed by Hur and Bruice,33−35 which explicitly considers
the majority of catalytic throughput occurring through
configurations in the Michaelis complex that may lie far from
the equilibrium mean. Such an interpretation is consistent with
the small but finite value seen for kred with indole-3-
acetaldehyde (0.13 s−1).
In the case of the complex of the enzyme with guanine, a
catalytically ineffective orientation is again observed in the
crystal structure, with the C-8 position that might otherwise
become hydroxylated oriented essentially directly away from
the Mo-OH group. The principal interactions that hold guanine
in this unfavorable orientation are with Glu 802 (interacting
with N3 and the C-2 amino group) and Arg 880, hydrogen
bonding to the C-6 keto group. A rotation of guanine between
the two phenylalanines of ∼180° is required to juxtapose its C-
8 position with the molybdenum center, so large a rotation is
sterically prevented by Glu 802 and Arg 880, both of which
would have to move considerably to accommodate such a
rotation. Although a computational analysis indicates that the
C-8 position of guanine is indeed somewhat less reactive than
that of xanthine, it appears that it is the orientation of guanine
when bound to the enzyme that is the principal factor that
allows the enzyme to discriminate against this potential
substrate, preventing formation of the mutagenic 8-hydrox-
yguanine.
AUTHOR INFORMATION
Corresponding Author
■
*University of California, 1463 Boyce Hall, Riverside, CA
92521. E-mail: russ.hille@ucr.edu. Telephone: (951) 827-6354.
Fax: (951) 827-2364.
(14) Cao, H. N., Pauff, J., and Hille, R. (2011) Substrate orientation
and the origin of catalytic power in xanthine oxidoreductase. Indian J.
Chem., Sect. A: Inorg., Bio-inorg., Phys., Theor. Anal. Chem. 50, 355−362.
(15) Dietzel, U., Kuper, J., Doebbler, J. A., Schulte, A., Truglio, J. J.,
Leimkuhler, S., and Kisker, C. (2009) Mechanism of Substrate and
Inhibitor Binding of Rhodobacter capsulatus Xanthine Dehydrogenase.
J. Biol. Chem. 284, 8759−8767.
Present Address
†H.C.: Department of Biochemistry and Cell Biology, Rice
University, Houston, TX 77251-1892.
Funding
This work was supported by the U.S. Department of Energy
(DE-SC0010666).
(16) Pauff, J. M., Hemann, C. F., Junemann, N., Leimkuhler, S., and
Hille, R. (2007) The role of arginine 310 in catalysis and substrate
specificity in xanthine dehydrogenase from Rhodobacter capsulatus. J.
Biol. Chem. 282, 12785−12790.
(17) Pauff, J. M., Cao, H., and Hille, R. (2009) Substrate orientation
and catalysis at the molybdenum site in xanthine oxidase crystal
structures in complex with xanthine and lumazine. J. Biol. Chem. 284,
8751−8758.
(18) Yamaguchi, Y., Matsumura, T., Ichida, K., Okamoto, K., and
Nishino, T. (2007) Human xanthine oxidase changes its substrate
specificity to aldehyde oxidase type upon mutation of amino acid
residues in the active site: Roles of active site residues in binding and
activation of purine substrate. J. Biochem. 141, 513−524.
(19) Cao, H., Pauff, J. M., and Hille, R. (2010) Substrate orientation
and catalytic specificity in the action of xanthine oxidase: The
sequential hydroxylation of hypoxanthine to uric acid. J. Biol. Chem.
285, 28044−28053.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Takeshi Nishino for advice in use of the folate
column procedure and for helpful discussions. Use of the
Advanced Photon Source at Argonne National Laboratory was
supported by the U.S. Department of Energy, Office of Science,
Office of Basic Energy Sciences, under Contract DE-AC02-
06CH11357. Use of the Lilly Research Laboratory Collabo-
rative Access Team (LRL-CAT) beamline at Sector 31 of the
Advanced Photon Source was provided by Eli Lilly & Co.,
which operates the facility.
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