Stereoselective hydride transfer by aryl-alcohol oxidase, a member of the GMC superfamily

Article abstract of DOI:10.1002/cbic.201100709

Primary alcohol oxidation by aryl-alcohol oxidase (AAO), a flavoenzyme providing H₂O₂ to ligninolytic peroxidases, is produced by concerted proton and hydride transfers, as shown by substrate and solvent kinetic isotope effects (KIEs). Interestingly, when the reaction was investigated with synthesized (R)- and (S)-α-deuterated p-methoxybenzyl alcohol, a primary KIE (≈6) was observed only for the R enantiomer, revealing that the hydride transfer is highly stereoselective. Docking of p-methoxybenzyl alcohol at the buried crystal active site, together with QM/MM calculations, showed that this stereoselectivity is due to the position of the hydride- and proton-receiving atoms (flavin N5 and His502 Nε, respectively) relative to the alcohol Cα-substituents, and to the concerted nature of transfer (the pro-S orientation corresponding to a 6 kcalmol^-1 penalty with respect to the pro-R orientation). The role of His502 is supported by the lower activity (by three orders of magnitude) of the H502A variant. The above stereoselectivity was also observed, although activities were much lower, in AAO reactions with secondary aryl alcohols (over 98% excess of the R enantiomer after treatment of racemic 1-(p-methoxyphenyl)ethanol, as shown by chiral HPLC) and especially with use of the F501A variant. This variant has an enlarged active site that allow better accommodation of the α-substituents, resulting in higher stereoselectivity (S/R ratios) than is seen with AAO. High enantioselectivity in a member of the GMC oxidoreductase superfamily is reported for the first time, and shows the potential for engineering of AAO for deracemization purposes.

Full text of DOI:10.1002/cbic.201100709

DOI: 10.1002/cbic.201100709
Stereoselective Hydride Transfer by Aryl-Alcohol Oxidase,
a Member of the GMC Superfamily
Aitor Hernꢀndez-Ortega,[a] Patricia Ferreira,[b] Pedro Merino,[c] Milagros Medina,[b]
Victor Guallar,[d] and Angel T. Martꢁnez*[a]
Primary alcohol oxidation by aryl-alcohol oxidase (AAO), a fla-
voenzyme providing H2O2 to ligninolytic peroxidases, is pro-
duced by concerted proton and hydride transfers, as shown by
substrate and solvent kinetic isotope effects (KIEs). Interesting-
ly, when the reaction was investigated with synthesized (R)-
and (S)-a-deuterated p-methoxybenzyl alcohol, a primary KIE
(ꢀ6) was observed only for the R enantiomer, revealing that
the hydride transfer is highly stereoselective. Docking of p-me-
thoxybenzyl alcohol at the buried crystal active site, together
with QM/MM calculations, showed that this stereoselectivity is
due to the position of the hydride- and proton-receiving
atoms (flavin N5 and His502 Ne, respectively) relative to the al-
cohol Ca-substituents, and to the concerted nature of transfer
(the pro-S orientation corresponding to a 6 kcalmolÀ1 penalty
with respect to the pro-R orientation). The role of His502 is
supported by the lower activity (by three orders of magnitude)
of the H502A variant. The above stereoselectivity was also ob-
served, although activities were much lower, in AAO reactions
with secondary aryl alcohols (over 98% excess of the R enan-
tiomer after treatment of racemic 1-(p-methoxyphenyl)ethanol,
as shown by chiral HPLC) and especially with use of the F501A
variant. This variant has an enlarged active site that allow
better accommodation of the a-substituents, resulting in
higher stereoselectivity (S/R ratios) than is seen with AAO. High
enantioselectivity in a member of the GMC oxidoreductase su-
perfamily is reported for the first time, and shows the potential
for engineering of AAO for deracemization purposes.
Introduction
Aryl-alcohol oxidase (AAO, EC 1.1.3.7) is a secreted flavooxidase
involved in the extracellular degradation of lignin by some
white-rot basidiomycetes (including Pleurotus and Bjerkandera
species) through provision of the H2O2 required by ligninolytic
peroxidases.[1] Removal of the recalcitrant lignin polymer is the
key step for carbon recycling in land ecosystems and is also a
central issue for the industrial use of renewable plant biomass
in the sustainable production of chemicals, materials, and
fuels.[2,3]
and cholesterol oxidase, among others) has been investigated.
The consensus mechanism involves substrate activation by a
catalytic base and oxidation of the resulting alkoxide to the
aldehyde (and eventually to the acid in a second step) by the
FAD cofactor (N5 atom), which is then reoxidized by O2.[10]
Stereoselective (or stereospecific) redox reactions have been
described in oxidases, dehydrogenases, and other oxidoreduc-
tases, often being associated with active site architectures.[11–17]
The reducing power for O2 activation to H2O2 comes from ar-
omatic alcohols that AAO oxidizes to the corresponding alde-
hydes, with p-methoxybenzyl alcohol being one of the pre-
ferred reducing substrates of Pleurotus eryngii AAO.[4] This
enzyme also oxidizes some aromatic aldehydes to acids, acting
on their gem-diol forms.[5] In this and related white-rot fungi, a
continuous supply of H2O2 for lignin degradation is available
through the redox-cycling of secreted p-methoxybenzaldehyde
(p-anisaldehyde), the main extracellular aromatic metabolite of
Pleurotus, in a process involving intracellular aromatic dehydro-
genases together with AAO.[6,7]
[a] A. Hernꢀndez-Ortega,+ A. T. Martꢁnez
Centro de Investigaciones Biolꢂgicas (CIB)
Consejo Superior de Investigaciones Cientꢁficas (CSIC)
Ramiro de Maeztu 9, 28040 Madrid (Spain)
E-mail: ATMartinez@cib.csic.es
[b] P. Ferreira,+ M. Medina
Department of Biochemistry and Molecular and Cellular Biology and
Institute of Biocomputation and Physics of Complex Systems
University of Zaragoza
50009 Zaragoza (Spain)
[c] P. Merino
Department of Organic Chemistry and
Instituto de Sꢁntesis Quꢁmica y Catꢀlisis Homogꢃnea (ISQCH)
CSIC, University of Zaragoza
To be a good AAO substrate, an alcohol must have a primary
hydroxy group conjugated with a planar double bond system
(benzylic, b-naphthylmethyl, and aliphatic polyunsaturated al-
cohols[8] are all included) establishing a stacking interaction
with an active-site tyrosine.[9] Substrate oxidation by other
members of the glucose/methanol/choline oxidase (GMC) su-
perfamily, a large group of oxidoreductases including well-
known flavoenzymes (such as glucose oxidase, choline oxidase,
50009 Zaragoza (Spain)
[d] V. Guallar
ICREA Joint BSC-IRB research programme in Computational Biology
Barcelona Supercomputing Center
Jordi Girona 29, 08034 Barcelona (Spain)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201100709.
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427

A. T. Martꢁnez et al.
Microbial and, more recently, en-
zymatic transformations are
being actively investigated for a
variety of asymmetric redox re-
actions of increasing commercial
interest.[18] These include drug
synthesis, because more than
half of all drug candidates in-
clude chiral centers.[19] Although
hydrolases are more frequently
used in chiral synthesis, oxidore-
ductases are also valuable tools
for the production of com-
pounds of industrial interest by
asymmetric transformations.[18]
In these reactions, both enantio-
selective synthesis and resolu-
tion of chiral mixtures are possi-
Table 1. Steady state and transient state kinetic constants for alcohol (Al) and O2 (Ox) in AAO oxidation of a-
2
deuterated and normal (a-protiated) p-methoxybenzyl alcohol (the latter in H2O or H2O buffer).[a]
Deuterated alcohol (in H2O)
Normal alcohol
2H2O
(R)-[a-2H]
(S)-[a-2H]
[a-2H2]
H2O
Steady state
kcat
Km(Al)
kcat/Km(Al)
Km(Ox)
kcat/Km(Ox)
Transient state
kred
38Æ1
34Æ1
144Æ2
45Æ1
25Æ1
196Æ2
131Æ1
25Æ1
1020Æ16
32Æ1
49Æ1
3980Æ113
159Æ5
38Æ1
2510Æ103
114Æ4
1110 Æ17
47Æ1
3190Æ101
134Æ5
808Æ18
1070Æ38
785Æ20
1236Æ38
1150Æ38
25Æ1
36Æ2
77Æ1
33Æ1
12Æ1
23Æ6
139Æ16
26Æ6
79Æ2
23Æ3
Kd
[a] Maximum steady state constants [catalytic constant (kcat, sÀ1), Michaelis–Menten constant (Km, mm), and effi-
ciency (kcat/Km, sÀ1 mmÀ1)] were estimated in bisubstrate kinetics at 258C, with use of Equations (1) and (2). Tran-
sient kinetic constants [including reduction constant (kred, sÀ1) and alcohol dissociation constant (Kd, mm)] were
estimated at 128C, with use of Equation (3) (meansÆS.D.s).
ble by use of different oxidoreductases.
constants, estimated under anaerobic conditions by stopped-
flow spectrophotometry, are also provided in Table 1.
In this study, a-monodeuterated—R and S enantiomers—
and a-dideuterated p-methoxybenzyl alcohol derivatives were
synthesized and evaluated as AAO substrates. Substrate and
solvent kinetic isotope effects (KIEs), modeling of p-methoxy-
benzyl alcohol docking at the active site of the crystal struc-
ture, and QM/MM calculations were used to gain information
on the mechanism of primary aromatic alcohol oxidation by
AAO, revealing that hydride abstraction stereoselectively af-
fects only one of the two a-hydrogens. The obtained conclu-
sions were extended to chiral aromatic alcohols, the stereo-
selective oxidation of which was enhanced by site-directed
mutagenesis, resulting in an AAO variant with an enlarged
active site.
The KIE values on the apparent steady state kinetic con-
stants for the three a-deuterated alcohols were estimated at
five O2 concentrations (0.051, 0.128, 0.273, 0.566, and
D
1.279 mm; Table S1). The kcat(app) values for the (R)-[a-2H]-p-me-
thoxybenzyl and [a-2H2]-p-methoxybenzyl alcohols increased
significantly with the concentration of O2, whereas their D(kcat-
(app)/Km(Al)(app)) values were significant but similar for the different
O2 concentrations, and the KIE values for (S)-[a-2H]-p-methoxy-
benzyl alcohol were always near unity (Figure 1). From the
above results, the steady state KIE values (Table 2) were calcu-
lated as the ratios between the values of the kinetic constants
for the protiated and the three a-deuterated alcohols, in the
bisubstrate kinetics.
The differences in the observed rates of AAO reduction by
the protiated and deuterated alcohol substrates, as well as
their concentration dependence, could be also observed by
anaerobic stopped-flow spectrophotometry (Figure 2). The
Results
AAO oxidation of a-deuterated p-methoxybenzyl alcohol
a-Monodeuterated and a,a -dideuterated p-methoxybenzyl al-
cohol derivatives were synthesized (by use of different deuter-
ated reducing agents) to confirm that primary alcohol oxida-
tion by AAO involves Ca hydride transfer and to investigate
the possible stereoselectivity of this reaction. The R and S mon-
odeuterated isomers were obtained with 96% enantiomeric
excesses (ees) and showed identical physical and spectroscopic
properties with the exception of the signs of optical rotation
1
(the H NMR spectra of the mono- and dideuterated alcohols
are shown in Figure S1 in the Supporting Information).
The steady state constants for the normal (a-protiated) and
different a-deuterated—(R)-[a-2H], (S)-[a-2H], and [a-2H2]—p-
methoxybenzyl alcohols were calculated by simultaneously
varying the concentrations of alcohol and O2 (in bisubstrate ki-
netics) to obtain maximum values by extrapolating to satura-
tion conditions. Michaelis–Menten constants (Km) and efficien-
cies (kcat/Km) for both alcohol (Al) and oxygen (Ox) are provided
in Table 1, together with the global catalytic constants (kcat).
The AAO transient state reduction (kred) and dissociation (Kd)
Figure 1. Influence of O2 concentration on steady state KIE values. The KIEs
on apparent kinetic constants (kcat and kcat/Km) for a-monodeuterated [(R)-2H
and (S)-2H], and a-dideuterated (2H2) p-methoxybenzyl alcohol oxidation by
AAO were estimated at five O2 concentrations (assays at 258C, and data
fitted to Equation (4)).
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AAO Stereoselectivity and Reaction Mechanisms
alcohol catalytic efficiency—D(kcat/Km(Al))—were slightly lower
Table 2. Substrate (p-methoxybenzyl alcohol) and solvent KIE values on
AAO steady state constants.[a]
D
than the kcat values, revealing that substrate binding is only
slightly affected by the isotopic substitutions, in agreement
with the low values of Kd KIE. The above primary values for the
KIEs on AAO turnover indicate that the breakdown of the alco-
hol (R)-Ca-1H/2H bond is limiting the rate of flavin reduction
and, therefore, of the overall catalysis. They also show that the
pro-R hydrogen (i.e., the hydrogen, the substitution of which
would provide the R enantiomer) is the one involved in the
hydride transfer reaction to the flavin.
kcat
Km(Al)
kcat/Km(Al)
Km(Ox)
kcat/Km(Ox)
(R)-[a-2H]
(S)-[a-2H]
[a-2H2]
5.2Æ0.1
1.4Æ0
1.4Æ0
3.6Æ0.1
1.2Æ0.1
3.9Æ0.1
1.6Æ0.1
3.4Æ0.1
1.2Æ0.1
5.0Æ0.2
1.4Æ0.1
1.5Æ0.1
1.2Æ0.1
1.6Æ0.1
1.1Æ0.1
1.1Æ0
7.9Æ0.1
1.5Æ0
2.0Æ0.1
1.3Æ0.1
2H2O
[a] The KIE values are the ratios between maximum activities (from bisub-
strate kinetics) on a-protiated/a-deuterated alcohols and in H2O/2H2O
buffer (meansÆS.D.s).
The small but significant kcat and kred KIEs for (S)-[a-2H]-p-me-
thoxybenzyl alcohol, together with the KIEs found for [a-2H2]-
p-methoxybenzyl alcohol, which are higher than found for the
R isomer, indicate a-secondary KIEs. The multiple KIEs for pri-
mary and secondary KIEs were especially evident under transi-
strongest rate decreases, with respect to the protiated p-me-
thoxybenzyl alcohol, were observed for the R enantiomer and
the dideuterated alcohol, but some decrease was also ob-
served for the S enantiomer. From these data, the values of
D
ent state conditions, under which the kred value for [a-2H2]-p-
methoxybenzyl alcohol was close to the product of those for
the KIEs on the AAO reduction kinetic constants (Dkred and Kd)
the R and S isomers.
D
were obtained (Table 3).
The AAO turnover (kcat) and reduction (kred) rates of the deu-
terated substrates showed significant KIEs for (R)-[a-2H]-p-me-
thoxybenzyl alcohol, close to those obtained for the a-dideu-
terated alcohol, whereas the KIEs for the (S)-[a-2H]-p-methoxy-
benzyl alcohol were very much lower. The KIE values for the
Solvent KIEs in alcohol oxidation by AAO
Oxidation of p-methoxybenzyl alcohol by AAO was also stud-
ied in deuterated buffer to confirm the timing of kinetic steps
involving solvent-exchangeable protons (including the alcohol
hydroxy proton), in the context of the hydride transfer de-
scribed above. The maximum KIE values for steady state reac-
tions in deuterated buffer, estimated as the ratios between the
2
maximum constants from bisubstrate kinetics in H2O and H2O,
are included in Table 2, and the transient state solvent KIE
values showing the effect of deuterated buffer on the enzyme
reduction constants are provided in Table 3.
Although the KIE values for solvent were much lower than
for substrate, low but consistent solvent KIEs were observed
for AAO kcat and kred, and a slightly higher value was obtained
for the alcohol catalytic efficiency, whereas the solvent KIE on
Kd was close to unity. The solvent effect is confirmed by a mul-
D,D2O
tiple transient state KIE (
k
, 13.5Æ0.9, from comparison of
red
AAO reduction by a-protiated alcohol in H2O and by a-dideu-
terated alcohol in 2H2O) being similar to the product of the
substrate and solvent kred KIE values. Multiple KIEs were not in-
vestigated in bisubstrate steady state reactions, but could be
Figure 2. Dependence of rates of AAO reduction on concentrations of proti-
ated and deuterated substrates. The effect of a-protiated (1H), a-monodeu-
terated [(R)-2H and (S)-2H], and a-dideuterated (2H2) p-methoxybenzyl alcohol
concentration on the transient state observed rates for AAO reduction were
estimated (assays at 128C by anaerobic stopped-flow spectrophotometry
and data were fitted to Equation (3)).
2
observed for apparent kinetic constants in air-saturated H2O
reactions, with kcat(app) (7.0Æ0.4) and D(kcat(app)/Km(Al)(app)) (6.1Æ
D
0.6) values being equal to or higher than the products of the
substrate and solvent KIEs for kcat(app) (5.4Æ0.1 and 1.3Æ0.1,
respectively) and kcat(app)/Km(Al)(app) (3.7Æ0.3 and 1.2Æ0.1, respec-
tively) under these reaction conditions.
2
Because H2O increases the viscosity of the medium (affect-
Table 3. Substrate (p-methoxybenzyl alcohol) and solvent values of KIEs
on AAO transient state constants.[a]
ing diffusion-limited steps) p-methoxybenzyl alcohol oxidation
was evaluated at a 30% glycerol concentration (resulting in
medium viscosity similar to 2H2O reactions). No significant
effect on bisubstrate kcat (0.99Æ0.01), kcat/Km(Al) (1.03Æ0.04), or
kcat/Km(Ox) (1.01Æ0.04) was observed, confirming that the ob-
served KIEs are due to the effect of solvent-exchangeable pro-
tons on the AAO reaction, and not to the increase in viscosity.
kred
Kd
kred
Kd
(R)-[a-2H]
[a-2H2]
6.5Æ0.1
9.3Æ0.1
1.5Æ0.4
1.1Æ0.4
(S)-[a-2H]
2H2O
1.5Æ0.1
1.4Æ0.1
1.2Æ0.3
1.1Æ0.3
[a] Transient state KIE values were calculated by fitting data to Equa-
tion (5) (meansÆS.D.s).
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429

A. T. Martꢁnez et al.
QM/MM calculations for docked p-methoxybenzyl alcohol
Reactions with secondary aromatic alcohols
The AAO crystal structure [Protein Data Bank (PDB), entry
3FIM] reveals a buried active site with direct connection to the
solvent blocked by the side chains of Tyr92, Phe397, and
Phe501. In a recent study,[9] PELE (protein energy landscape ex-
ploration), a state-of-the-art protein–ligand induced fit model-
ing software package, was used to elucidate the access of p-
methoxybenzyl alcohol to the AAO active site. The final posi-
tion of the p-methoxybenzyl alcohol is illustrated in Figure 3A
Figure 4A shows a flavin si-side view of p-methoxybenzyl alco-
hol docked at the AAO active site (also including Phe501,
His502, and His546). Although the Phe501 side chain points to-
wards its benzylic position, the p-methoxybenzyl alcohol can
Figure 4. Docking of primary and of secondary aromatic alcohols at the AAO
active site (view from flavin si-side). In contrast with the situation observed
in A) for p-methoxybenzyl alcohol, when (S)-1-(p-methoxyphenyl)ethanol is
positioned in the same catalytically relevant position, in B) steric hindrance
prevents accommodation of the a-methyl group due to collision (red arrow)
with the Phe501 side chain. Based on PDB ID: 3FIM. As CPK colored sticks.
Figure 3. Docking of p-methoxybenzyl alcohol at the AAO active site (lateral
view of flavin). A) At the position provided by PELE, the pro-R and hydroxy
hydrogens are at transfer distance from the flavin N5 and the unprotonated
Ne of His502, respectively (green lines). Moreover, the protonated Nd of
His546 forms a hydrogen bond to the hydroxy group (green line). B) Howev-
er, when a pro-S hydride transfer position was tried, the hydroxy group was
too far (red lines) from His502 and His546, and a new H-bond appears be-
tween the histidines (green line). Based on PDB ID: 3FIM. As Corey–Pauling–
Koltun (CPK) colored sticks.
easily adopt the catalytically relevant position described above
(with the hydroxy group and the pro-R Ca hydrogens at trans-
fer distances from the flavin N5 and the His502 Ne, respective-
ly). However, if the pro-S hydrogen is simply substituted by a
methyl group—in 1-(p-methoxyphenyl)ethanol—steric hin-
drance (red arrow in Figure 4B) is produced when the sub-
strate is forced to adopt a position compatible with catalysis.
The above predictions were confirmed by experimental data
showing a value for AAO apparent efficiency in oxidizing 1-(p-
methoxyphenyl)ethanol, estimated from H2O2 release (at 258C
under air saturation), of only 3.59ꢄ10À3 sÀ1 mmÀ1 (in compari-
son with 4070 sÀ1 mmÀ1 for p-methoxybenzyl alcohol under
the same conditions) with a kcat(app) value of only 0.179 sÀ1 (cf.
120 sÀ1 for p-methoxybenzyl alcohol). However, when an
F501A variant was obtained, and its activities on secondary al-
cohols (relative to p-methoxybenzyl alcohol activity) were com-
pared with those of native (wild-type) AAO (Table 4) it could
be observed that removal of the Phe501 side chain (hindering
AAO accommodation of secondary alcohols) resulted in a 60-
fold higher relative activity with 1-(p-methoxyphenyl)ethanol
and a 19-fold higher activity with (S)-1-(p-fluorophenyl)ethanol.
Interestingly, during the slow oxidation (of the order of
0.1 sÀ1 mÀ1) of 1-(p-fluorophenyl)ethanol by native AAO and,
especially, by the F501A variant, a net preference for the S
enantiomer was observed (Figure 5A and 5C) as revealed by
the formation of p-fluoroacetophenone shown in the differ-
ence spectra (Figure 5B and 5D).
and has similar distances (ꢀ2.5 ꢃ) between its hydroxy hydro-
gen and His502 Ne and between its pro-R Ca-hydrogen and
the flavin N5. The protonated Nd of His546 is also close to the
hydroxy group, and an edge-to-plane stacking interaction be-
tween the p-methoxybenzyl alcohol and Tyr92 aromatic rings
is produced as previously described[9] (a stereoview of p-me-
thoxybenzyl alcohol docking is provided in Figure S2).
With the aim of confirming the AAO preference for hydride
abstraction from the pro-R position, we performed a QM/MM
energy scan for the rotation of the dihedral between the
phenyl ring and the C1-Ca-OH plane, as shown on comparison
of Figure 3A and 3B. The energy scan is based on several ge-
ometry optimizations with constrained values for the dihedral
angles. The three main players in the hydrogen bond pat-
tern—the substrate, His502, and His546—were included in the
quantum region. The scan, involving six optimized intermedi-
ate points along the dihedral rotation, resulted in a 6 kcal
molÀ1 penalty for the pro-S orientation, with a 10 kcalmolÀ1 ro-
tation energy barrier. In this case (pro-S oxidation) larger dis-
tances would exist between the substrate hydroxy group and
the His502 and His546 side chains, which would form a new H-
bond, preventing their contribution to catalysis (Figure 3B).
In the case of racemic 1-(p-methoxyphenyl)ethanol, the ste-
reoselectivity of the reaction was confirmed by chiral HPLC,
which showed only one major peak after the AAO treatment
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AAO Stereoselectivity and Reaction Mechanisms
Table 4. AAO and F501A oxidation of secondary 1-(p-methoxyphenyl)e-
thanol and R and S enantiomers of 1-(p-fluorophenyl)ethanol relative to
primary p-methoxybenzyl alcohol activity (ꢄ10À6) and stereoselectivity
ratio.[a]
Native AAO
F501A variant
1-(p-methoxyphenyl)ethanol (racemic)
(R)-1-(p-fluorophenyl)ethanol
(S)-1-(p-fluorophenyl)ethanol
stereoselectivity S/R ratio
53
3130
0.263
17.37
66
0.043
0.914
21
[a] Linear oxidation of secondary alcohols (1 mm) to the corresponding
ketones was followed spectrophotometrically during long-term incuba-
tion in air-saturated (0.273 mm O2 concentration) phosphate (pH 6, 0.1m)
at 258C, and kobs values are relative to those obtained for p-methoxyben-
zyl alcohol oxidation by native AAO (120 sÀ1 mmÀ1) and F501A (3.8ꢄ
10À3 sÀ1 mmÀ1) under the same conditions.
(Figure 6) with an ee >98%. The absolute configuration of the
remaining enantiomer was ascertained by measuring the sign
of the optical rotation, which proved to be positive, corre-
sponding to the R isomer,[20,21] thus confirming that the
enzyme had selectively oxidized the S isomer. This highly ste-
reoselective oxidation implies abstraction of the R hydrogen,
as described above for the primary alcohols.
Figure 6. Chiral HPLC analysis confirming deracemization of 1-(p-methoxy-
phenyl)ethanol by AAO. A) Control chromatogram of the racemic mixture,
showing two peaks corresponding to the S and R enantiomers. B) Chromato-
gram after its treatment with AAO, resulting in enzymatic deracemization
yielding the R enantiomer (as shown by its optical rotation) with high ee
(>98%). The analyses were performed with a Chiralcel IB column, and peaks
were monitored at 225 nm. The UV spectrum corresponding to the 1-(p-me-
thoxyphenyl)ethanol peaks is included in the inset.
More importantly, the AAO preference for (S)-1-(p-fluorophe-
nyl)ethanol was increased threefold when the bulky side chain
of Phe501 was removed in the F501A variant, which shows a
stereoselectivity S/R ratio of 66:1 for this secondary alcohol
(Table 4). This is consistent with the higher relative rates of oxi-
dation of 1-(p-methoxyphenyl)ethanol and (S)-1-(p-fluorophe-
nyl)ethanol by the F501A variant
mentioned above (relative to
native AAO) due to better ac-
commodation of secondary al-
cohols at the active site of the
engineered variant.
Discussion
Oxidation mechanism by AAO,
a GMC oxidoreductase
The primary substrate KIEs on
AAO maximum steady state
constants (from bisubstrate ki-
netics) and transient state con-
stants for a-dideuterated p-me-
thoxybenzyl alcohol (ꢀ8–9 kcat/
kred KIE) indicate that this fla-
vooxidase abstracts one of the
Ca hydrogens (together with
the two electrons) during sub-
strate oxidation. Hydride trans-
Figure 5. Incubation of alcohol S and R enantiomers with the F501A variant. Initial (red) and final (black) UV spec-
tra were obtained during 20 h incubation of A) (S)-(p-fluorophenyl)ethanol and C) (R)-(p-fluorophenyl)ethanol
(both 1 mm) at 258C in phosphate (pH 6, 0.1m) containing F501A (1.7 mm). B) Difference spectra showed the p-
fluoroacetophenone maximum only for the S enantiomer. The UV spectrum of authentic p-fluoroacetophenone is
also shown (inset).
fer, to cofactor flavin N5, is
nowadays the consensus mech-
anism for substrate oxidation in
the GMC oxidoreductase super-
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431

A. T. Martꢁnez et al.
family,[10] and the substrate KIE values obtained here are of the
same order as those reported for the model GMC choline ox-
idase.[22,23] However, the KIE on the apparent efficiency of AAO
was independent of oxygen concentration, suggesting irrever-
sible hydride transfer in AAO, in contrast with what has been
the AAO hydride abstraction from primary aryl alcohols, which
are its natural substrates, is produced selectively from the pro-
R position. Stereoselective (or stereospecific) substrate oxida-
tion has been reported in some other oxidases, including the
copper-radical enzyme galactose oxidase[14] and the flavoen-
zymes d-amino acid oxidase[33] and vanillyl-alcohol oxidase.[11]
Moreover, glucose oxidase is selective, oxidizing the b-anome-
ric form of d-glucose.[34,35] However, as far as we know, this is
the first time that stereoselective primary alcohol oxidation
(with only one of the two a-hydrogens involved in hydride ab-
straction) has been reported for a member of the GMC super-
family of oxidoreductases.
reported for choline oxidase, with which the D(kcat(app)/Km(app)
)
values increase with oxygen concentration.[24]
Moreover, the low but consistent solvent KIE values (ꢀ1.5)
found on both AAO steady state (from bisubstrate kinetics)
and transient state constants are indicative of hydride transfer
to flavin being concerted with transfer of a solvent-exchangea-
ble proton (in a partially limiting reaction). The same tenden-
cies had been reported previously when the effects of sub-
strate and solvent deuteration on apparent kinetic constants
were measured from air reactions (instead of from bisubstrate
kinetic).[25] The multiple KIE found (from comparison of reac-
tions of a-protiated alcohol in H2O and reactions of a-dideuter-
In addition to the above primary KIE, a significant a-secon-
dary substrate KIE (ꢀ1.4) was also observed in the AAO reac-
tions, being indicative of hydrogen tunneling, as described for
tyrosine hydroxylase.[36] In recent years, quantum tunneling for
hydride transfer has been reported in many other oxidoreduc-
tases,[37,38] including related flavoproteins such as choline ox-
idase.[23,39,40] Secondary KIEs are often a consequence of a
change in the hybridization state of the donor in proceeding
from the reactants to the transition state. The multiple KIE
values obtained suggest that this rehybridization is occurring
at the same transition state as hydride transfer.
2
ated alcohol in H2O) confirms that the observed solvent effect
is due to the abstraction of the substrate hydroxy proton by a
catalytic base being concerted with hydride abstraction by the
flavin. The very strong decreases in both kcat and kred in the
H502A variant (2890- and 1830-fold, respectively),[9] together
with its position at the active site, strongly suggest that His502
is the catalytic base. A concerted transfer mechanism has also
been reported for AAO oxidation of some aromatic aldehydes
through their gem-diol species.[5]
The AAO stereoselectivity in hydride abstraction from the
pro-R Ca position in primary aromatic alcohols revealed by
deuterium labeling was also demonstrated by the use of sec-
ondary (chiral) aromatic alcohols as substrates, in spite of the
low activities of the enzyme with these compounds. This was
first revealed by the over 20-times faster oxidation of (S)-1-(p-
fluorophenyl)ethanol, implying hydride abstraction from the R
position, in relation to its R enantiomer. The selective removal
of the S enantiomer of racemic 1-(p-methoxyphenyl)ethanol by
AAO (resulting in an ee >98% in the remaining enantiomer)
was then confirmed by chiral HPLC. AAO could therefore be
used for deracemization of secondary alcohol mixtures for iso-
lation of chiral aromatic alcohols. These include products of in-
dustrial interest for which microbial deracemization has already
been considered.[41] The use of AAO for enzymatic deracemiza-
tion would not require the introduction of stereoselectivity but
the extension of its activity to secondary alcohols, as discussed
below.
In this respect, AAO differs from the related choline ox-
idase[23] and other members of the GMC oxidoreductase super-
family, in which nonconcerted (stepwise) hydride and proton
transfer (resulting in a stable alkoxide intermediate) has been
proposed as a general mechanism.[10] More recently, a noncon-
certed transfer mechanism to flavin and His548 (homologous
to AAO His502) has been reported in pyranose 2-oxidase.[26,27]
In AAO, recent QM/MM calculations found that proton transfer
precedes hydride transfer in alcohol oxidation, but no stable
intermediate is produced, so the two reactions are therefore
considered asynchronous concerted transfers.[9] AAO is not an
exception among flavoenzymes, because a highly concerted
transfer mechanism takes place in d-amino acid oxidase, the
paradigm of flavin enzymes.[28] Moreover, in choline oxidase
and some flavoproteins exhibiting similar catalysis (such as
flavocytochrome b2), changes from nonconcerted to concerted
mechanisms have been obtained by mutagenesis of residues
involved in abstraction of the hydroxy proton, as revealed by
low solvent and multiple KIEs,[29–31] similar to those reported
here for native AAO. Finally, small but significant solvent KIEs
have also been detected during the low-efficiency oxidation of
benzylic alcohols by galactose oxidase,[14,32] the stereoselectiv-
ity of which is discussed below.
In stereoselective galactose oxidase, which oxidizes benzyl
alcohol, although with much lower efficiency (0.36 sÀ1 mmÀ1)
than
it
does
1-O-methyl-a-d-galactopyranoside
(29.50 sÀ1 mmÀ1),[14] directed evolution from an improved var-
iant[42] has been performed to extend the activity to secondary
aryl alcohols.[43] The unique mutation (K330M) introduced
during evolution provides a more hydrophobic active site that
might favor 1-phenylethanol binding. In AAO, PELE docking of
p-methoxybenzyl alcohol at the crystal structure[44] found that
the benzylic position is at the bottom of the active site cavity
(with the primary hydroxy and pro-R hydrogens orientated as
discussed above) and the rest of the cavity is occupied by the
alcohol aromatic ring. Therefore, to accommodate secondary
alcohols, the bottom part of the cavity should be enlarged. To
conclude this study, engineering of the AAO active site was
addressed by rational design, resulting in the F501A variant, in
Stereoselectivity on primary (and secondary) aryl alcohols
When the two enantiomers of monodeuterated p-methoxy-
benzyl alcohol—the (S)-[a-2H] and (R)-[a-2H] forms—were as-
sayed as AAO substrates, primary KIE values of around 6 were
obtained for the R enantiomer under both steady state and
transient state conditions. This revealed for the first time that
432
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ChemBioChem 2012, 13, 427 – 435

AAO Stereoselectivity and Reaction Mechanisms
which the bulky aromatic side chain of Phe501 (located in
front of the pro-S hydrogen) was removed. As recently
shown,[45] this mutation reduces the efficiency of the enzyme
for oxidation of primary alcohols, but its relative activity with
secondary alcohols is significantly improved. More importantly,
when this variant was assayed on the R and S enantiomers of
1-(p-fluorophenyl)ethanol, its stereoselectivity was highly in-
creased with respect to native AAO.
nism. Moreover, our experimental results indicate, in agree-
ment with theoretical analysis, that substrate activation by the
catalytic base is concerted with hydride transfer, instead of the
stepwise process suggested for GMC oxidoreductases. Model-
ing of the active site geometry revealed the atomic mechanism
of stereoselectivity, involving alcohol hydrogen bonds to
His502 and His546. The catalytically relevant modeled position
also indicated the potential of the F501A variant for oxidation
of secondary alcohols. The variant was produced and showed
improved stereoselective activity with 1-phenylethanol deriva-
tives. These results provide relevant information on the catalyt-
ic mechanisms in GMC oxidoreductases and at the same time
show the potential for use of AAO or its engineered variants as
industrial biocatalysts in the production of chiral aromatic alco-
hols of interest for the fine chemicals or pharmaceutical sec-
tors.
Structural bases for AAO catalysis and stereoselectivity
With use of the AAO crystal structure[44] as the starting point,
the PELE software successfully simulated the migration of p-
methoxybenzyl alcohol to the buried active site of AAO,[9]
where it adopted a catalytically relevant position. Interestingly,
at this position its pro-R a-hydrogen is at 2.4 ꢃ from the fla-
vin N5, enabling hydride transfer consistently with the sub-
strate KIE results discussed above. Simultaneously, the hydroxy
hydrogen is 2.5 ꢃ from the His502 Ne, enabling its contribution
as a base in accepting the proton for alcohol activation. An ad-
ditional hydrogen bond to the substrate from His546, an im-
portant residue for catalysis, the removal of which reduced
(ꢀ40-fold) AAO activity and alcohol binding,[9] is also observed.
In contrast, if we try to place the p-methoxybenzyl alcohol
with the pro-S a-hydrogen in a suitable position for hydride
transfer to the flavin N5, the result is energetically unfavorable,
as revealed by QM/MM calculations. The pro-S structure pres-
ents an overall rearrangement of the hydrogen bond pattern.
The hydroxy group is too far from His502 and His546 to form
strong hydrogen bonds and efficient catalysis. As a result there
is a new intra-protein hydrogen bond between the two histi-
dines. Therefore, the KIEs for the two a-monodeuterated p-me-
thoxybenzyl enantiomers, as well as the small but stereoselec-
tive activity of AAO on chiral secondary aryl alcohols, are in
agreement with the predicted position of the alcohol at the
active site.
Experimental Section
Chemicals: p-Methoxybenzyl alcohol, p-fluorobenzyl alcohol, 1-(p-
methoxyphenyl)ethanol (racemic), (R)-1-(p-fluorophenyl)ethanol,
(S)-1-(p-fluorophenyl)ethanol, p-methoxybenzaldehyde, p-fluoroa-
cetophenone, methyl p-methoxybenzoate, and the deuterated re-
ducing agents used below were purchased from Sigma–Aldrich.
Synthesis of deuterated substrates: Dideuterated [a-2H2]-p-me-
thoxybenzyl alcohol was prepared by reduction of methyl p-me-
thoxybenzoate with LiAl2H4.[46] (R)-[a-2H]-p-Methoxybenzyl alcohol
was prepared from p-methoxybenzaldehyde,[47] with use of (S)-
alpine borane in the enantioselective reduction of the intermediate
deuterated aldehyde. The S enantiomer and the racemic mixture
were prepared in the same way but with use of (R)- and racemic
alpine borane, respectively. 1H NMR (400 MHz) and DEPT 13C NMR
(100 MHz) spectra in C2HCl3 were used to confirm the products ob-
tained.
Native enzyme and mutated variant: Native recombinant AAO
from P. eryngii was obtained by E. coli expression of the mature
AAO cDNA (GenBank AF064069) followed by in vitro activation for
cofactor incorporation and correct folding.[48] The F501A variant
was prepared by PCR with use of the QuikChange site-directed
mutagenesis kit (Stratagene; the oligonucleotides used are de-
scribed in the Supporting Information). The mutation was con-
firmed by sequencing (GS-FLX sequencer from Roche) and the var-
iant was produced and refolded as for the native enzyme. Enzyme
concentrations were determined with a Cary-100-Bio spectropho-
We postulate that the stereoselectivity in AAO is related to
the active site architecture and reaction mechanism revealed
by the KIE studies, which imply the asynchronous but concert-
ed transfer of hydroxy proton (to His502) and Ca hydride (to
flavin N5),[9] in contrast to the nonconcerted (stepwise) mecha-
nism suggested as a consensus in GMC oxidoreductases.[10] In
the concerted transfer catalyzed by AAO, once the alcohol sub-
strate is accommodated with the hydroxy group pointing to-
wards His502, as shown by PELE docking, only the pro-R hy-
drogen is facing the flavin N5, resulting in the high hydride ab-
straction selectivity, whereas in stepwise transfer mechanisms
some conformational changes are possible.
tometer and use of the molar absorbances of native AAO (e463
=
11050mÀ1 cmÀ1) and its F501A variant (e463 =10389mÀ1 cmÀ1) esti-
mated by heat denaturation.
Steady-state and transient-state kinetics: Oxidation of p-methox-
ybenzyl alcohol to p-methoxybenzaldehyde (e285 =16950mÀ1 cmÀ1
)
was followed spectrophotometrically (Cary-100-Bio). Maximum
steady state constants were estimated in bisubstrate kinetics by
varying both the alcohol and O2 concentrations and extrapolating
to saturation conditions. Transient-state kinetic measurements of
enzyme reduction constants were performed by stopped-flow
spectrophotometry (Applied Photophysics SX18.MV equipment)
under anaerobic conditions. The experimental details and equa-
tions used for steady state and transient state data analyses are
provided in the Supporting Information.
Conclusions
Using the synthesized deuterated enantiomers of the natural
alcohol substrate of AAO, we have found, for the first time in a
member of the GMC superfamily, highly stereoselective ab-
straction of one of the two primary alcohol hydrogens. The sig-
nificant KIE previously observed for the a-dideuterated sub-
strate was indicative of a hydride transfer oxidation mecha-
ChemBioChem 2012, 13, 427 – 435
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433

A. T. Martꢁnez et al.
KIEs in p-methoxybenzyl alcohol oxidation: The effect of sub-
strate a-deuteration—R, S, and dideuterated forms—on AAO reac-
tions was estimated (in the pH-independent portion of the pH ac-
tivity profile) under both steady state and transient state condi-
tions, the former in bisubstrate kinetics to obtain maximum values.
Solvent KIE values were also estimated in both steady state and
M.M.), CTQ2010-19606 (to P.M.), and CTQ2010-18123 (to V.G.),
and by PEROXICATS (KBBE-2010-4-265397; to A.T.M.) and PELE
(ERC-2009-Adg 25027; to V.G.) European projects. The authors
thank Jesffls Jimꢃnez-Barbero (CIB, Madrid) for his useful com-
ments and suggestions, and the Barcelona Supercomputing
Center for computational resources. A.H.-O. acknowledges a con-
tract from the Comunidad de Madrid.
2
transient state reactions with use of H2O buffer, and possible vis-
cosity effects were considered (the experimental details and equa-
tions used for data analysis are provided in the Supporting Infor-
mation).
Keywords: enantioselectivity
·
enzyme
catalysis
·
Secondary alcohol oxidation: Oxidation of 1-(p-methoxyphenyl)-
ethanol by native AAO and by its F501A variant was measured
from the H2O2 generated, by means of an HRP-coupled assay with
AmplexRed (Invitrogen) at 258C in air-saturated sodium phosphate
(pH 6, 0.1m). Assays were initiated by addition of AAO, and forma-
tion of the resofurin product was monitored (e585 52000mÀ1 cmÀ1).
flavooxidases · isotope effects · QM/MM
[1] F. J. Ruiz-DueÇas, ꢅ. T. Martꢁnez, Microb. Biotechnol. 2009, 2, 164–177.
[2] ꢅ. T. Martꢁnez, F. J. Ruiz-DueÇas, M. J. Martꢁnez, J. C. del Rꢁo, A. Gutiꢆrrez,
Curr. Opin. Biotechnol. 2009, 20, 348–357.
[3] A. J. Ragauskas, C. K. Williams, B. H. Davison, G. Britovsek, J. Cairney,
C. A. Eckert, W. J. Frederick, J. P. Hallett, D. J. Leak, C. L. Liotta, J. R. Mie-
lenz, R. Murphy, R. Templer, T. Tschaplinski, Science 2006, 311, 484–489.
[4] F. Guillꢆn, A. T. Martꢁnez, M. J. Martꢁnez, Eur. J. Biochem. 1992, 209, 603–
611.
[5] P. Ferreira, A. Hernꢀndez-Ortega, B. Herguedas, J. Rencoret, A. Gutiꢆrrez,
M. J. Martꢁnez, J. Jimꢆnez-Barbero, M. Medina, A. T. Martꢁnez, Biochem. J.
2010, 425, 585–593.
[6] F. Guillꢆn, C. S. Evans, Appl. Environ. Microbiol. 1994, 60, 2811–2817.
[7] A. Gutiꢆrrez, L. Caramelo, A. Prieto, M. J. Martꢁnez, A. T. Martꢁnez, Appl.
Environ. Microbiol. 1994, 60, 1783–1788.
[8] M. Medina, P. Ferreira, F. Guillꢆn, M. J. Martꢁnez, W. J. H. van Berkel, ꢅ. T.
Martꢁnez, Biochem. J. 2005, 389, 731–738.
Oxidation of (R)-1-(p-fluorophenyl)ethanol and (S)-1-(p-fluoropheny-
l)ethanol to p-fluoroacetophenone, the molar absorbance of which
was determined here (e247 11616mÀ1 cmÀ1), was evaluated in air-sa-
turated phosphate buffer (pH 6, 0.1m) at 258C, after long-term in-
cubation (up to 24 h), due to the very low AAO activity with this al-
cohol. For chiral HPLC (see below), long-term oxidation of racemic
1-(p-methoxyphenyl)ethanol (20 mm, 5–10 mg) with AAO (20 mm)
was also assayed. After incubation in phosphate (pH 6, 50 mm) at
258C, the enzyme was removed by ultrafiltration and the sample
was freeze-dried for analysis. Activities relative to p-methoxybenzyl
alcohol (under the same conditions) were estimated for both sec-
ondary alcohols for purposes of comparison.
[9] A. Hernꢀndez-Ortega, K. Borrelli, P. Ferreira, M. Medina, A. T. Martꢁnez, V.
Guallar, Biochem. J. 2011, 436, 341–350.
[10] G. Gadda, Biochemistry 2008, 47, 13745–13753.
[11] R. H. H. van den Heuvel, M. W. Fraaije, C. Laane, W. J. H. van Berkel, J.
Bacteriol. 1998, 180, 5646–5651.
[12] H. L. Holland, H. K. Weber, Curr. Opin. Biotechnol. 2000, 11, 547–553.
[13] M. J. Knapp, K. Rickert, J. P. Klinman, J. Am. Chem. Soc. 2002, 124, 3865–
3874.
[14] S. G. Minasian, M. M. Whittaker, J. W. Whittaker, Biochemistry 2004, 43,
13683–13693.
[15] M. A. Gilabert, L. G. Fenoll, F. Garcia-Molina, P. A. Garcia-Ruiz, J. Tudela, F.
Garcia-Canovas, J. N. Rodrꢁguez-Lꢇpez, Biol. Chem. 2004, 385, 1177–
1184.
Chiral HPLC and optical rotation: The enantioselectivity of race-
mic 1-(p-methoxyphenyl)ethanol oxidation was quantified by HPLC
(Waters Alliance), after salt removal from the sample, with use of a
Chiralcel IB column (4.6ꢄ250 mm, 5 mm; Daicel Chemical Indus-
tries, Ltd.), elution with n-hexane/propan-2-ol (98:2, v/v) at
1.0 mLminÀ1, and a diode-array detector (peak monitoring at
225 nm). Optical rotation was measured in chloroform with a Jasco
polarimeter.
QM/MM: QM/MM calculations were performed with the Qsite pro-
gram.[49] The DFT method with the M06 functional,[50] the ultrafine
pseudospectral grid, and the 6–31G* basis set were applied for the
QM/MM geometry optimization. The quantum region included the
substrate, His502, and His546. The classical region used a conju-
gate gradient minimizer (with an rmsg of 0.01) and the OPLS-AA
force field. Because of the large influence of the classical point
charges on the wave function, the non-bonding cutoff was set to
100 ꢃ and updated every 10 steps. In the scan optimization, all
atoms within 20 ꢃ of the substrate were allowed to move. The di-
hedral scan was built with six intermediate minimizations along
the C1–Ca dihedral (maintaining the aromatic ring fixed). The ini-
tial structure (pro-R) was taken from our previous simulation,[9]
based on migration of p-methoxybenzyl alcohol into the AAO crys-
tal structure (3FIM).[44] In this previous study we performed an ex-
haustive analysis of the histidine protonation steps and an initial
equilibration of the system and the explicit solvent. A PDB file with
the initial coordinates for the simulation is available in the Sup-
porting Information.
[16] S. ꢅguila, R. Vꢀzquez-Duhalt, R. Tinoco, M. Rivera, G. Pecchi, J. B. Alder-
ete, Green Chem. 2008, 10, 647–653.
[17] B. Yuan, A. Page, C. P. Worrall, F. Escalettes, S. C. Willies, J. J. W. McDouall,
N. J. Turner, J. Clayden, Angew. Chem. 2010, 122, 7164–7167; Angew.
Chem. Int. Ed. 2010, 49, 7010–7013.
[18] T. Matsuda, R. Yamanaka, K. Nakamura, Tetrahedron: Asymmetry 2009,
20, 513–557.
[19] J. S. Carey, D. Laffan, C. Thomson, M. T. Williams, Org. Biomol. Chem.
2006, 4, 2337–2347.
[20] A. S. Y. Yim, M. Wills, Tetrahedron 2005, 61, 7994–8004.
[21] B. A. Barros-Filho, M. D. F. de Oliveira, T. L. G. Lemos, M. C. de Mattos, G.
de Gonzalo, V. Gotor-Fernꢀndez, V. Gotor, Tetrahedron: Asymmetry 2009,
20, 1057–1061.
[22] G. Gadda, Biochim. Biophys. Acta Proteins Proteomics 2003, 1650, 4–9.
[23] F. Fan, G. Gadda, J. Am. Chem. Soc. 2005, 127, 2067–2074.
[24] F. Fan, G. Gadda, J. Am. Chem. Soc. 2005, 127, 17954–17961.
[25] P. Ferreira, A. Hernꢀndez-Ortega, B. Herguedas, A. T. Martꢁnez, M.
Medina, J. Biol. Chem. 2009, 284, 24840–24847.
[26] J. Sucharitakul, T. Wongnate, P. Chaiyen, Biochemistry 2010, 49, 3753–
3765.
[27] T. Wongnate, J. Sucharitakul, P. Chaiyen, ChemBioChem 2011, 12, 2577–
2586.
[28] L. Pollegioni, W. Blodig, S. Ghisla, J. Biol. Chem. 1997, 272, 4924–4934.
[29] P. Sobrado, P. F. Fitzpatrick, Biochemistry 2003, 42, 15208–15214.
[30] M. Ghanem, G. Gadda, Biochemistry 2005, 44, 893–904.
[31] K. Rungsrisuriyachai, G. Gadda, Biochemistry 2010, 49, 2483–2490.
[32] M. M. Whittaker, D. P. Ballou, J. W. Whittaker, Biochemistry 1998, 37,
8426–8436.
Acknowledgements
This work was supported by Spanish projects BIO2008-01533,
BIO2011-26694 (to A.T.M. and co-workers), BIO2010-1493 (to
434
www.chembiochem.org
ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2012, 13, 427 – 435

AAO Stereoselectivity and Reaction Mechanisms
[33] A. Mattevi, M. A. Vanoni, F. Todone, M. Rizzi, A. Teplyakov, A. Coda, M.
Bolognesi, B. Curti, Proc. Natl. Acad. Sci. USA 1996, 93, 7496–7501.
[34] D. Keilin, E. F. Hartree, Biochem. J. 1952, 50, 331–341.
[35] V. Leskovac, S. Trivic, G. Wohlfahrt, J. Kandrac, D. Pericin, Int. J. Biochem.
Cell Biol. 2005, 37, 731–750.
[43] F. Escalettes, N. J. Turner, ChemBioChem 2008, 9, 857–860.
[44] I. S. Fernꢀndez, F. J. Ruiz-DueÇas, E. Santillana, P. Ferreira, M. J. Martꢁnez,
A. T. Martꢁnez, A. Romero, Acta Crystallogr. D 2009, 65, 1196–1205.
[45] A. Hernꢀndez-Ortega, F. Lucas, P. Ferreira, M. Medina, V. Guallar, A. T.
Martꢁnez, J. Biol. Chem. 2011, 286, 41105–41114.
[36] P. A. Frantom, R. Pongdee, G. A. Sulikowski, P. F. Fitzpatrick, J. Am. Chem.
Soc. 2002, 124, 4202–4203.
[46] M. Fetizon, Y. Henry, N. Moreau, G. Moreau, M. Golfier, T. Prange, Tetra-
hedron 1973, 29, 1011–1014.
[37] J. Pu, J. Gao, D. G. Truhlar, Chem. Rev. 2006, 106, 3140–3169.
[38] Z. D. Nagel, J. P. Klinman, Nat. Chem. Biol. 2009, 5, 543–550.
[39] M. J. Sutcliffe, L. Masgrau, A. Roujeinikova, L. O. Johannissen, P. Hothi, J.
Basran, K. E. Ranaghan, A. J. Mulholland, D. Leys, N. S. Scrutton, Phil.
Trans. R. Soc. Lond. B 2006, 361, 1375–1386.
[47] J. R. Walker, R. W. Curley, Tetrahedron 2001, 57, 6695–6701.
[48] F. J. Ruiz-DueÇas, P. Ferreira, M. J. Martꢁnez, A. T. Martꢁnez, Protein Expres-
sion Purif. 2006, 45, 191–199.
[49] Schrçdinger Inc., QSite 5.6, LCC, New York, 2010.
[50] Y. Zhao, D. G. Truhlar, Theor. Chem. Acc. 2008, 120, 215–241.
[40] I. Lans, J. R. Peregrina, M. Medina, M. Garcia-Viloca, A. Gonzalez-Lafont,
J. M. Lluch, J. Phys. Chem. B 2010, 114, 3368–3379.
[41] G. R. Allan, A. J. Carnell, J. Org. Chem. 2001, 66, 6495–6497.
[42] L. H. Sun, I. P. Petrounia, M. Yagasaki, G. Bandara, F. H. Arnold, Protein
Eng. 2001, 14, 699–704.
Received: November 11, 2011
Published online on January 23, 2012
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ꢂ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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435