A conserved active-site threonine is important for both sugar and flavin oxidations of pyranose 2-oxidase

Article abstract of DOI:10.1074/jbc.M109.073247

Pyranose 2-oxidase (P2O) catalyzes the oxidation by O₂ of D-glucose and several aldopyranoses to yield the 2-ketoaldoses and H ₂O₂. Based on crystal structures, in one rotamer conformation, the threonine hydroxyl of Thr¹⁶⁹ forms H-bonds to the flavin-N5/O4 locus, whereas, in a different rotamer, it may interact with either sugar or other parts of the P2O·sugar complex. Transient kinetics of wild-type (WT) and Thr¹⁶⁹ → S/N/G/A replacement variants show that D-Glc binds to T169S, T169N, and WT with the same K_d (45-47mM), and the hydride transfer rate constants (kred) are similar (15.3-9.7 s ^-1 at 4 °C ). k_red of T169G with D-glucose (0.7 s ^-1, 4 °C) is significantly less than that of WT but not as severely affected as in T169A (k_red of 0.03 s^-1 at 25 °C). Transient kinetics of WT and mutants using D-galactose show that P2O binds D-galactose with a one-step binding process, different from binding of D-glucose. In T169S, T169N, and T169G, the overall turnover with D-Gal is faster than that of WT due to an increase of k_red. In the crystal structure of T169S, Ser¹⁶⁹ Oγassumes a position identical to that of Oγ1 in Thr¹⁶⁹; in T169G, solvent molecules may be able to rescue H-bonding. Our data suggest that a competent reductive half-reaction requires a side chain at position 169 that is able to form an H-bond within the ES complex. During the oxidative half-reaction, all mutants failed to stabilize a C4a-hydroperoxyflavin intermediate, thus suggesting that the precise position and geometry of the Thr¹⁶⁹ side chain are required for intermediate stabilization.

Full text of DOI:10.1074/jbc.M109.073247

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 13, pp. 9697–9705, March 26, 2010
2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
©
A Conserved Active-site Threonine Is Important for Both
□
S
*
Sugar and Flavin Oxidations of Pyranose 2-Oxidase
Received for publication, October 6, 2009, and in revised form, December 29, 2009 Published, JBC Papers in Press, January 20, 2010, DOI 10.1074/jbc.M109.073247
¶2
‡
1
§
‡
¶
Warintra Pitsawong , Jeerus Sucharitakul , Methinee Prongjit , Tien-Chye Tan , Oliver Spadiut ,
ʈ3
¶2
‡4
Dietmar Haltrich , Christina Divne , and Pimchai Chaiyen
‡
From the Department of Biochemistry and Center of Excellence in Protein Structure and Function, Faculty of Science,
Mahidol University, Bangkok 10400, Thailand, the Department of Biochemistry, Faculty of Dentistry, Chulalongkorn University,
Henri-Dunant Road, Patumwan, Bangkok 10300, Thailand, the Division of Glycoscience, Kungliga Tekniska H o¨ gskolan
Biotechnology, Royal Institute of Technology, Albanova University Center, SE-106 91 Stockholm, Sweden, and the Department of
§
¶
ʈ
Food Science and Technology, Bodenkultur-University of Natural Resource and Applied Life Sciences, A-1190 Vienna, Austria
Pyranose 2-oxidase (P2O) catalyzes the oxidation by O of and geometry of the Thr1 side chain are required for interme-
69
2
D-glucose and several aldopyranoses to yield the 2-ketoaldoses diate stabilization.
and H O . Based on crystal structures, in one rotamer confor-
2
2
1
69
mation, the threonine hydroxyl of Thr forms H-bonds to the
flavin-N5/O4 locus, whereas, in a different rotamer, it may
interact with either sugar or other parts of the P2Oꢀsugar com-
5
Pyranose 2-oxidase (P2O, pyranose:oxygen 2-oxidoreduc-
tase, EC 1.1.3.10) from Trametes multicolor is a homotet-
1
69
plex. Transient kinetics of wild-type (WT) and Thr 3 S/N/
rameric enzyme with each subunit carrying one FAD covalently
⑀
2
167
G/A replacement variants show that D-Glc binds to T169S,
linked to N (N3) of His via the FAD 8␣-methyl group (1).
P2O catalyzes the oxidation by molecular oxygen of D-glucose
(D-Glc) and several aldopyranoses at the C2 position to yield the
corresponding 2-ketoaldoses and hydrogen peroxide (2). The
overall catalytic reaction can be divided into two half-reactions
T169N, and WT with the same K (45–47 mM), and the hydride
d
؊
1
transfer rate constants (k ) are similar (15.3–9.7 s at 4 °C ).
red
؊1
k
of T169G with D-glucose (0.7 s , 4 °C) is significantly less
red
than that of WT but not as severely affected as in T169A (k of
red
؊
1
0
.03 s at 25 °C). Transient kinetics of WT and mutants using
(
Scheme 1) obeying a Ping-Pong type mechanism at pH 7 (3): a
D-galactose show that P2O binds D-galactose with a one-step
binding process, different from binding of D-glucose. In T169S,
T169N, and T169G, the overall turnover with D-Gal is faster
reductive half-reaction in which the protein-bound flavin
receives a hydride equivalent from a sugar substrate to produce
Ϫ
the reduced FAD (FADH ) and the 2-keto-sugar, and an oxi-
than that of WT due to an increase of k . In the crystal struc-
red
dative half-reaction in which two electrons are transferred from
the reduced flavin to O to form H O (2, 3).
169
ture of T169S, Ser O␥ assumes a position identical to that of
2
2
2
169
O␥1 in Thr ; in T169G, solvent molecules may be able to res-
cue H-bonding. Our data suggest that a competent reductive
half-reaction requires a side chain at position 169 that is able to
form an H-bond within the ES complex. During the oxidative
half-reaction, all mutants failed to stabilize a C4a-hydroperoxy-
flavin intermediate, thus suggesting that the precise position
We reported the first detection of a C4a-hydroperoxyfla-
vin intermediate for a flavoprotein oxidase (4). This flavin
intermediate has not been previously observed for flavopro-
tein oxidases but has been limited to reactions of flavoprotein
monooxygenases (5–7). Besides P2O, an intermediate in fla-
voprotein oxidases has been detected only in the crystalline
forms of choline oxidase (1.86 Å) (8) and nitroalkane oxidase (in
the presence of cyanide) (9), and in the mutant form, C42S, of
an NADH oxidase (10). In the case of glucose oxidase from
*
This work was supported in part by the Thailand Research Fund through
Grants BRG5180002 (to P. C.), MRG4980117 (to J. S.), and PHD/0151/2547
of the Royal Golden Jubilee Ph.D. program (to M. P.) and by the Faculty of Aspergillus niger, the generation of a flavin semiquinone-super-
Science, MahidolUniversity(toP. C.)andfromtheFacultyofDentistryChu-
lalongkorn University (to J. S.).
The atomic coordinates and structure factors (codes 3K4B and 3K4C) have been
oxide radical pair using pulse radiolysis resulted in formation of
a putative C4a-hydroperoxyflavin intermediate (11). Studies on
deposited in the Protein Data Bank, Research Collaboratory for Structural the P2O reductive half-reaction indicate that P2O binds D-Glc
Bioinformatics, Rutgers University, NewBrunswick, NJ(http://www.rcsb.org/).
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental text, Tables S1 and S2, and Figs. S1–S3.
according to a two-step binding mechanism: first an initial
□
complex is formed, followed by an isomerization step to form
1
2
Supported by the Development and Promotion of Science and Technology an active Michaelis ES complex (P2Oꢀglucose). Interestingly,
Talent Project, Thailand.
this complex shows higher absorbance at 395 nm than does the
Supported by the Swedish Research Council Formas, the Swedish Research
oxidized enzyme, which is unique among flavoprotein oxidases
Council Vetenskapsrådet, the Carl Tryggers Foundation, and the Swedish
Foundation for Strategic Research through the Swedish Center for Biomi- (3).
metic Fiber Engineering (through Biomime).
P2O belongs to the large family of glucose-methanol-choline
3
4
Supported by a grant from the Austrian Research Foundation (Fonds zur
F o¨ rderung der wissenschaftlichen Forschung Translational Project
L213-B11).
548
oxidoreductases (12, 13) with the catalytic side chains His
–
To whom correspondence should be addressed: Dept. of Biochemistry and
Center of Excellence in Protein Structure and Function, Faculty of Science,
Mahidol University, Rama 6 Road, Bangkok 10400, Thailand. Tel.: 66-2-201-
5
The abbreviations used are: P2O, pyranose 2-oxidase; ABTS, 2,2Ј-azino-
bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt; ␭, wave-
length; WT, wild type; Mes, 4-morpholineethanesulfonic acid.
5
596; Fax: 66-2-354-7174; E-mail: scpcy@mahidol.ac.th.
MARCH 26, 2010•VOLUME 285•NUMBER 13
JOURNAL OF BIOLOGICAL CHEMISTRY 9697

169
Roles of Thr in Catalysis by Pyranose 2-Oxidase
EXPERIMENTAL PROCEDURES
Reagents—D-Glc (99.5% purity), D-Gal (99% purity), and
horseradish peroxidase were purchased from Sigma-Aldrich.
ABTS (2,2Ј-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)
diammonium salt) was purchased from Roche Applied Science
SCHEME 1
5
93
Asn positioned at the re-face of the flavin isoalloxazine ring
13). The active site is gated by a dynamic active-site loop (res-
(Germany). Concentrations of the following compounds were
(
determined using the known absorption coefficients at pH 7.0:
idues 451–461), which has been shown to harbor several resi-
dues important for substrate binding and catalysis (13–15).
Comparison of P2O structures in sugar-free and sugar-bound
forms reveals the presence of at least two well defined active-
site loop conformations, each of which is relevant to one of the
half-reactions (14). The structure of a less active P2O mutant
5
Ϫ1
Ϫ1
⑀
ϭ 10 cm for horseradish peroxidase; ⑀ ϭ 1.3 ϫ
M
4
0
03
458
4
Ϫ1
Ϫ1
1
M
cm for the value of FAD per each subunit of T169S;
4 Ϫ1 Ϫ1 4 Ϫ1
⑀
ϭ 1.2 ϫ 10
M
cm for T169N; ⑀ ϭ 1.1 ϫ 10
M
4
58
458
M
Ϫ1
4
Ϫ1
Ϫ1
cm for T169G; and ⑀ ϭ 1.18 ϫ 10
cm for T169A.
4
53
All mutants were expressed in Escherichia coli without His tag,
6
and purified according to the protocol described earlier (14).
(
H167A), in complex with a slow substrate (2-deoxy-2-fluoro-
Site-directed Mutagenesis—Site-directed mutagenesis at
D-glucose (14)) or the reaction product (2-keto-D-glucose (16)),
shows the active-site loop in an open conformation. The open
loop state is able to accommodate spatially the sugar substrate,
and thus this conformation is relevant for the reductive half-
reaction (3, 14). The wild-type structure of P2O in complex
with the small competitive inhibitor acetate revealed the loop in
a different well defined conformational state (13). This state is
characterized as fully closed, and the active site is solvent-inac-
cessible and provides a more hydrophobic substrate environ-
ment than does the open state. Thus, this structural state pro-
vides a suitable environment for the reaction of the reduced
enzyme and oxygen, and can spatially accommodate the C4a-
hydroperoxy-FAD intermediate during the oxidative half-reac-
tion (4, 13, 14).
1
69
position Thr of P2O was performed using the QuikChangeꢁ
II site-directed mutagenesis kit. All primers were from Sigma-
Proligo (Singapore). The plasmid pET11a was used as a tem-
plate for the mutagenic PCR (GeneAmp PCR system, Applied
TM
Biosystems, model 2004 or MyCycler , Thermal Cycler, Bio-
Rad). All plasmids were confirmed using T7 forward and reverse
primers, as well as a primer to an internal region of the p2o gene.
The DNA sequencing was performed by MACROGEN (Korea).
The nucleotide sequences of the synthetic oligonucleotides are
listed in supplemental Table S1.
Reduction Potential Measurement—Measurements of re-
oЈ
duction potential values (E ) of the mutant enzymes were car-
m
ried out according to the protocol described in a previous study
(
19), using xanthine/xanthine oxidase as a reduction system.
We have discussed previously that the conserved residue
169
Reactions for all mutants were carried out in reaction mixtures
Thr occupies an important position in the P2O active site,
positioned immediately “above” the flavin, on the flavin si-side,
where it forms a hydrogen bond to the flavin N5 and O4 atoms
when the loop is in the closed conformation (13, 14). Flavin-N5-
protein interactions are recurrent features in structures of
many flavoprotein oxidases (17), and little is known about the
functional effects of this H-bonding interaction. When the
containing 500 ␮M xanthine, 3 nM xanthine oxidase (side arm),
oЈ
standard dye (26.8 ␮M indigo carmine (E ϭ Ϫ116 mV), 18 ␮M
m
oЈ
oЈ
cresyl violet (E ϭ Ϫ166 mV), or 20 ␮M methylene blue (E ϭ
m
m
1
69
ϩ10 mV)), and Thr
mutants (26.8 ␮M T169S, 26.8 ␮M
T169N, 18 ␮M T169A, or 20 ␮M T169G) in 50 mM sodium
phosphate (pH 7.0). Details of the procedure are provided in
69 supplemental Procedures.
1
active-site loop assumes the open conformation in P2O, Thr
Steady-state Kinetics—Initial velocities at various concentra-
discards its flavin interaction by means of a rotamer change
169
tions of oxygen with D-Glc or D-Gal were measured at 4 °C and
(
14). The position of the Thr side chain relative to the sugar
2
5 °C using a stopped-flow spectrophotometer (3). Steady-state
substrate and FAD also provides a simple, straightforward
explanation for the poor performance of D-galactose (D-Gal)
as a substrate. The oxidation of D-Gal by P2O is industrially
useful, because it produces the intermediate for further syn-
thesis of D-tagatose, a low caloric and non-glycemic sweet-
ener (18). In wild-type P2O (WT), the axial O4 hydroxyl
group of D-Gal is probably unfavorable due to steric clashes
kinetics of the P2O variants was studied by coupling to the
reaction of horseradish peroxidase and ABTS. Progress of the
Ϫ1
Ϫ1
reaction was followed at 420 nm (⑀
ϭ 42.3 mM cm
)
420
where the oxidized ABTS absorbs (3). The assay reaction typi-
cally contained 20–40 nM of a mutant enzyme, 0.6 ␮M peroxi-
dase, 2 mM ABTS, varying concentrations of D-Glc or D-Gal,
and varying oxygen concentrations in 50 mM sodium phosphate
1
69
with the Thr
side chain (14). The kinetics of the binding
and reaction mechanism of P2O with D-Gal have not been (pH 7.0).
rationalized in detail, because transient kinetics of the reac-
tion have not been investigated.
Rapid Reaction Experiments—Reductive and oxidative half-
169
reactions of the Thr variants were studied using a stopped-
flow spectrophotometer (Hi-Tech Scientific). Experimental
1
69
In this report, steady-state and transient kinetics of Thr
variants, T169S, T169N, T169G, and T169A, with D-Glc and protocols are similar to those in the previous report (3, 4) and
D-Gal were investigated. Rapid kinetics of WT with D-Gal are described in detail in supplemental Procedures. The reduction
also reported. These results provide mechanistic insights of the enzyme-bound FAD was monitored at 395 and 458 nm.
1
69
regarding the role of Thr in the reductive and oxidative half- For studying the oxidative half-reaction, solutions containing
reactions. In addition, three-dimensional structures of T169G the reduced enzyme were prepared as described previously (3,
and T169S were solved and used in conjunction with kinetic 4) and then mixed with buffers equilibrated with oxygen
169
data for interpretation of the roles of Thr in catalysis of P2O. ([O ] ϭ 0.13, 0.31, 0.61, and 0.96 mM) using the stopped-flow
2
9
698 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 13•MARCH 26, 2010

169
Roles of Thr in Catalysis by Pyranose 2-Oxidase
TABLE 1
169
Steady-state kinetic parameters for the wild-type and Thr mutants of P2O
Ping-Pong
T169S
Ternary
T169A
Parameters
WT
T169N
T169G
Ϫ1
D-Glucose (4 °C)
k
(s
)
9.7 Ϯ 0.15
1.13 Ϯ 0.07
0.09 Ϯ 0.008
8.6
13.7 Ϯ 0.2
5.6 Ϯ 0.05
4.6 Ϯ 0.1
4.8 Ϯ 0.1
1.2
0.7 Ϯ 0.01
0.9 Ϯ 0.02
4.5 Ϯ 0.5
0.8
0.06 Ϯ 0.005
cat
Glc
K
K
k
(mM)
1.7 Ϯ 0.05
0.99 Ϯ 0.03
8.1
30 Ϯ 1
m
O2
(mM)
0.2 Ϯ 0.03
0.002
m
Glc
Ϫ1 Ϫ1
/K (mM
s
)
cat
m
O2
k
/K (mMϪ1sϪ1)
110.0
13.8
1.2
0.2
0.4
cat
m
Ϫ1
D-Galactose
k
(s
)
1.2 Ϯ 0.05
15.0 Ϯ 0.3
0.03 Ϯ 0.002
0.08
2.5 Ϯ 0.02
16.2 Ϯ 0.5
0.07 Ϯ 0.01
0.15
5.3 Ϯ 0.1
16 Ϯ 1
1.32 Ϯ 0.1
0.3
1.5 Ϯ 0.03
3.1 Ϯ 0.08
3.7 Ϯ 0.08
0.5
0.03 Ϯ 0.002
50 Ϯ 2
cat
Gal
(25 °C)
K
K
k
(mM)
m
O2
(mM)
0.4 Ϯ 0.03
0.005
m
Gal
Ϫ1 Ϫ1
/K (mM
s
)
cat
m
O2
k
/K (mMϪ1sϪ1)
44.2
32.0
3.4
0.4
0.07
cat
m
oЈ
Ϫ105a
Reduction potential
Redox potential value is from a previous report (14).
E (mV)
Ϫ106
Ϫ95
Ϫ1
Ϫ197
m
a
spectrophotometer. The reactions were performed in 50 mM previously (21). For all proteins, crystals grew from micro-
sodium phosphate (pH 7.0), and monitored at 5 nm intervals in seeded hanging drops containing a 1:1 mixture of 0.1 M Mes (pH
the ␭ range between 300 and 550 nm.
5.2), 50 mM MgCl , 10% (w/v) monomethyl ether polyethylene
2
Data Analysis: Transient Kinetics—Analyses of observed rate glycol 2000 and protein at a concentration of 20 mg/ml in 20
constants (k ) from kinetic traces were conducted by fitting mM Mes (pH 5.2). Crystals were stabilized in mother liquor with
obs
data to exponential equations using the software Kinetic Studio 28 (w/v) % polyethylene glycol 2000, followed by vitrification in
(
Hi-Tech Scientific, Salisbury, UK) or Program A (developed by liquid nitrogen. Data were collected at 100 K using synchrotron
C. J. Chiu, R. Chung, J. Diverno, and D. P. Ballou at the Univer- radiation at beam line I911-2, MAX-lab, Lund, Sweden, and
sity of Michigan, Ann Arbor, MI). Rate constants were deter- processed and scaled using the XDS package (22). Details of
mined from plots of k versus sugar or oxygen concentrations data processing are provided in supplemental Procedures. Data
obs
using Marquardt-Levenberg nonlinear fitting algorithms collection and refinement statistics are given in supplementary
included in KaleidaGraph (Synergy Software) according to Table S2.
Equations 1 and 2 (3). Rate constants are defined as in Table 2.
RESULTS
k2͓G͔
Reduction Potentials of Thr1 Variants—The absorbances at
69
kobs͑first_phase͒ ϭ
ϩ kϪ2
(Eq. 1)
Kd ϩ ͓G͔
4
58 nm, an isosbestic point for indigo carmine, and at 610 nm
k k
2
where P2O has no absorbance, were used for calculating the
3
͓G͔
amount of the enzyme (E) and dye (D), respectively (19). Plots of
͑
k ϩ k ͒
2 Ϫ2
kobs͑second_phase͒ ϭ
ϩ kϪ3 (Eq. 2)
log(D /D ) versus log(E /E ) gave a slope of ϳ1.2
k
red ox
red ox
Ϫ2
Kdͩ ͪϩ ͓G͔
(supplemental Fig. S1), indicating that both T169S and T169N
k ϩ k
2
Ϫ2
were reduced by xanthine/xanthine oxidase via a two-electron
Data Analysis: Steady-state Kinetics—Initial rates of steady- process (indigo carmine as the reference dye). No absorption
state measurements were calculated using Program A. The ini- was detected at the long wavelength or 370 nm regions, which
tial rates were analyzed for steady-state kinetic parameters of a suggests that no semiquinone was stabilized during the reduc-
oЈ
two-substrate enzyme reaction according to a double-recipro- tion of FAD. The midpoint potentials (E
) calculated from
m
cal plot of a Ping-Pong (Equation 3) or a ternary complex mech- these plots are Ϫ106 mV for T169S and Ϫ95 mV for T169N,
anism (Equation 4) (20). In addition, data were analyzed which are similar to that of the wild-type enzyme (WT), Ϫ105
according to direct plots (Equations 5 and 6) using the EnzFitter mV (14). T169A and cresyl violet were also reduced by xan-
program (Biosoft, Cambridge, UK).
thine/xanthine oxidase via a two-electron process (absorbance
at 414 and 585 nm was used for calculating concentrations of
e
v
␾G
␾
O2
the oxidized enzyme and dye, respectively). The slope of the
ϭ ␾ ϩ
0
ϩ
(Eq. 3)
(Eq. 4)
(Eq. 5)
(Eq. 6)
oЈ
͓G͔
͓O2͔
plot was ϳ0.92 (data not shown), and E was calculated to be
m
Ϫ197 mV, which is significantly lower than WT. For T169G
e
v
␾G
␾
O2
␾
G:O2
(
methylene blue as the standard dye), calculations using
ϭ ␾ ϩ
0
ϩ
ϩ
͓G͔ ͓O2͔ ͓G͔͓O2͔
oЈ
absorbance at 458 and 664 nm gave a slope of 0.86, and E of
m
Ϫ1.2 mV. For each mutant, at least three standard dyes were
used to ensure that there was no interference from dyes to the
measurement. All data are summarized in Table 1.
VAB
v ϭ
v ϭ
K B ϩ K A ϩ AB
a
b
Steady-state Kinetics—Steady-state kinetic parameters for all
P2O variants were determined from initial rates of the assay
reactions at various concentrations of oxygen and D-Glc or
VAB
K K ϩ K A ϩ AB
ia
b
b
Glc
Gal
Crystallographic Analysis—Expression and purification of D-Gal. K denotes K of D-Glc while K represents K of
m
m
m
m
the Thr1 mutants used for crystallization have been reported D-Gal. Double-reciprocal plots for T169N (Fig. 1, A and C),
69
MARCH 26, 2010•VOLUME 285•NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 9699

169
Roles of Thr in Catalysis by Pyranose 2-Oxidase
respectively, again confirming that alterations at the amino acid
position 169 significantly affect the oxidative half-reaction.
169
Reduction of the Oxidized Thr Mutants by D-Glucose—To
1
69
investigate the role of Thr in P2O catalysis and to identify the
specific reaction steps affected by the mutations, transient
kinetics were investigated. For analysis of the reductive half-
reaction with D-Glc as a substrate, anaerobic enzyme solutions
were mixed with buffers containing various concentrations of
D-Glc, and reduction of the flavin was monitored at 458 and 395
nm. Absorbance at 458 nm gives the maximum difference
between the oxidized and the reduced enzyme, and the signal at
3
95 nm indicates formation of an active P2Oꢀsugar complex, as
previously observed in the wild-type reaction (3).
Only the kinetic traces of the T169S and T169N reduction
showed an obvious increase in absorbance at 395 nm. The first
phase corresponds to a small increase in absorbance at 395 nm,
concomitant with a lag at 458 nm (Fig. 2, A and B). This phase
was interpreted as the formation of an active P2Oꢀsugar com-
plex required for flavin reduction (3). The macroscopic equilib-
1
69
FIGURE 1. Steady-state kinetics of Thr mutants with D-Glc or D-Gal. The
assay reactions were performed at 4 °C (for D-Glc) and 25 °C (for D-Gal) using
the stopped-flow spectrophotometer. Double reciprocal plots of initial rates rium dissociation constants (K ) associated with the binding of
d
of T169N (A) with final D-Glc concentrations of 0.1, 0.2, 0.4, 1, 2, 4, 8, 16, 25, and
D-Glc to the oxidized state of T169S and T169N (45 and 47 mM,
5
1
0 mM, and T169A (B) with final D-Glc concentrations of 8, 12.5, 25, 50, 100,
50, and 200 mM. The upper to lower lines represent, respectively, the plots of
respectively (Table 2)) are identical to that of WT (45 mM) (3),
the final oxygen concentrations of 0.13, 0.26, 0.44, 0.74, and 1.09 mM. Double indicating the same binding affinity and similar nature of the
reciprocalplotsofinitialratesofT169N(C)withfinalD-Galconcentrationsof2,
, 8, 16, 32, 64, and 150 mM, and T169A (D) with final D-Gal concentrations of 8,
6, 32, 64, 150, 300, and 400 mM are shown. The upper to lower lines represent,
P2OꢀGlc complex in the mutants and WT.
4
1
Analysis of the flavin reduction phase (the decrease in
respectively, the final oxygen concentrations of 0.13, 0.26, 0.44, and 0.74 mM. absorbance at 458 nm in the second phase) is in agreement with
The reactions of T169S and T169G with D-Glc or D-Gal showed parallel-line
the effects of the mutations on the first phase (Fig. 2 and Table
patterns similar to that of T169N (supplemental information).
2
). In the reactions of T169S and T169N, flavin was reduced by
T169S, and T169G (supplemental Fig. S2) all display parallel- D-Glc with a rate constant k (4 °C) of 13.8 Ϯ 0.4 and 9.7 Ϯ 0.1
red
Ϫ1
line patterns consistent with a Ping-Pong mechanism (Equa-
s
, respectively, which is similar to k of the wild type (15.3 Ϯ
red
Ϫ1
tion 3) similar to that of WT (3), whereas T169A shows an 0.4 s ) (3). However, we noted that the biphasic reductive half-
intersecting-line pattern for reactions both with D-Glc (Fig. 1B) reaction of T169N with D-Glc (Fig. 2B) could only be observed
and D-Gal (Fig. 1D), consistent with a ternary-complex mecha- after storing the purified enzyme on ice for 3 weeks prior to the
nism (Equation 4). All steady-state kinetic parameters are sum- experiment. When freshly prepared T169N was used in the
marized in Table 1.
experiment, the reductive half-reaction resulted in three
Glc
When D-Glc was used as a substrate, the value of kcat/K for phases; the first phase was substrate binding with the absorb-
m
Ϫ1 Ϫ1
T169S was similar to that of WT (8.1 versus 8.6 mM
s
), ance increase at 395 nm (K ϭ 47 mM), and the second phase
d
Ϫ1
whereas those of T169N and T169G were ϳ7- and 11-fold was flavin reduction (9.7 Ϯ 0.1 s ) as described above, while
lower than that of WT, respectively (Table 1). This shows that the third phase corresponded to slow flavin reduction and
the reductive half-reaction is less affected in T169S, compared accounted for ϳ20–30% of the total amplitude at 458 nm (Fig.
O2
with T169N and T169G. When compared with WT, k /K
2B). The first two phases are identical to those of the stored
cat
m
was reduced by 8-, 92-, and 549-fold for T169S, T169N, and enzyme. The observed rate constants of the third phase were
169
T169G, respectively, indicating that Thr
is significantly hyperbolically dependent on the concentrations of D-Glc with a
Ϫ1
important for the oxidative half-reaction. For T169A, both k
/
limiting rate constant of 0.002 s . Following storage on ice for
cat
Glc
O2
K
and k /K decreased considerably, indicating that, in at least 3 weeks, the third phase diminished, and most of the
m
cat
m
addition to altering the kinetic reaction pattern, replacing the flavin reduction was now according to the fast second phase
side-chain hydroxyl group by a methyl group severely impairs (Fig. 2B).
the overall catalytic competence. It is interesting to note that
Kinetic traces of T169A and T169G do not show significant
with D-Glc as a substrate, T169S shows the highest k value increase in absorbance at 395 nm (Fig. 2C), implying that the
cat
Ϫ1
(
13.7 s ), also exceeding that of the WT.
nature of the complex formed is different in these mutants. In
Gal
With D-Gal as a substrate, the k /K
values of T169S, addition, the reductive half-reaction in T169G and T169A
cat
m
T169N, and T169G were all higher (2-, 4-, and 6-fold, respec- showed significantly slower flavin reduction (k of 0.7 Ϯ 0.01
red
Ϫ1
tively) than that of wild-type P2O, which can be mainly attrib- (at 4 °C) and 0.03 Ϯ 0.007 (at 25 °C) s , respectively) when
Ϫ1
Ϫ1
uted to the increase in k of these mutants (Table 1). This compared with WT (15.3 Ϯ 0.4 s (at 4 °C) and 48 Ϯ 2 s (at
cat
implies that by replacing the Thr side chain with Ser, Asn, and 25 °C)) (Table 2 and Fig. 2C).
169
Gly, the overall turnover of the reaction with D-Gal has
Reduction of the Oxidized Thr Mutants by D-Galactose—A
O2
improved. When compared with WT, the k /K values were similar set of experiments were conducted using D-Gal as a
cat
m
1
.4-, 13-, and 111-fold lower for T169S, T169N, and T169G, substrate. However, due to the slow reaction rate, flavin reduc-
9
700 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 13•MARCH 26, 2010

169
Roles of Thr in Catalysis by Pyranose 2-Oxidase
tion of D-Glc (Fig. 2) (3). Interestingly, in contrast to the reac-
tion with D-Glc (Fig. 2) (3), k of the first substrate-binding
obs
phase is linearly dependent on [D-Gal] (insets in Fig. 3), consist-
ent with a simple one-step binding model (Fig. 4B). The one-
step binding model is also supported by agreement between
Gal
(
K
) obtained from steady-state kinetics at 4 °C (3.83 mM, data
m
not shown), and the value calculated based on the model in Fig.
4
B and kinetic parameters in Table 2 (3.88 mM). Macroscopic
K values for T169S (5.9 Ϯ 0.3 mM), T169N (6.6 Ϯ 0.3 mM), and
d
T169G (4.2 Ϯ 0.2 mM) are all higher than that of WT (3.7 Ϯ 0.2
mM), indicating that D-Gal binds to the mutants with lesser
affinity than to WT. The second phase, flavin reduction, in
Ϫ1
Ϫ1
T169S (0.5 Ϯ 0.04 s ), T169N (0.9 Ϯ 0.01 s ), and T169G
Ϫ1
Ϫ1
(
2.7 Ϯ 0.02 s ) is faster than that of WT (0.3 Ϯ 0.03 s ) (Table
2
). Thus, the rapid kinetic results suggest that, by replacing the
169
Thr side chain with Ser, Asn, or Gly, only the rate constant of
the flavin reduction step is enhanced without a concomitant
increase in the binding affinity of D-Gal.
169
Oxidation of the Reduced Thr Mutants by Oxygen—The
1
69
oxidative half-reactions of the Thr variants were investigated
using stopped-flow experiments. Kinetic traces at 395 and 458
nm for all mutants show one single phase (Fig. 5), which con-
trasts the reaction kinetics of WT where biphasic kinetics was
observed (3, 4). In the reaction of WT, the biphasic kinetics is
due to formation and decay of a C4a-hydroperoxyflavin inter-
mediate (3, 4). All mutants show similar kinetics at all ␭, indi-
cating that only the oxidized flavin was formed, and no tran-
sient C4a-hydroperoxyflavin intermediate was stabilized. The
k
values for flavin oxidation analyzed from the traces at 395
obs
and 458 nm were linearly dependent on [O ] and intercepts of
2
all plots approach zero, suggesting that the reverse rate is neg-
ligible. Biomolecular rate constants for the enzyme oxidation
are summarized in Table 3.
Crystal Structure Analysis—We analyzed two of the four
replacements at position 169, Thr 3 Ser and Thr 3 Gly, by
solving the crystal structures (Fig. 6). Data collection and
refinement statistics are given in supplementary Table S2. The
overall structures of the P2O monomer and homotetramer are
very similar to those reported earlier for wild-type and mutant
T. multicolor P2O (13, 14, 23, 24). As expected, based on our
previous structures, the only region in P2O that displays signif-
icant structural differences besides the point of mutation is the
1
69
FIGURE 2. Reduction of the oxidized Thr mutants with D-Glc. Stopped- active-site loop (residues 451–461). Hence, only the structure
flow traces at 458 nm are shown in dotted lines, whereas those at 395 nm are
of the active site will be discussed below. The two major con-
in solid lines. A, a solution of T169S (23 ␮M) was mixed with various concen-
trationsofD-Glc(0.4, 0.8, 1.6, 3.2, 6.4, 15, 30, and60mM). Allconcentrationsare formational states previously observed for P2O, the open
givenasaftermixing. Theinsetashowsaplotofkobs ofthefirstphase(absorb-
ance increase at 395 nm) versus D-Glc concentrations, whereas the same plot
of the second phase (large decrease in A458 nm) is shown in inset b. B, after
(2IGO, Fig. 7A (14)) and the closed state (1TT0, Fig. 7B (13))
serve as reference structures for structural comparisons. These
storing on ice for at least 3 weeks, the third slow phase of the flavin reduction two states have been discussed above.
(
lowest dotted line) diminished, and kinetic traces of the T169N reaction
showed biphasic kinetics. C, stopped-flow experiments similar to A were per-
The active-site loop in the T169S structure (Fig. 6A) folds
formed with T169G. Traces monitored at 395 nm did not show significant into a slightly different conformation from the “open” state seen
absorbance increase as in the reaction of T169S in A.
in other unliganded structures or the 2-deoxy-2-fluoro-D-glu-
cose complex (14). Because the structure is unliganded, it is
tion of T169A was monitored in an anaerobic cuvette. As uncertain whether this loop conformer is relevant for the sugar-
observed with D-Glc, only kinetic traces of WT, T169S, and binding state (i.e. during the reductive half-reaction), or the
T169N showed obvious increases in absorbance at 395 nm (Fig. oxidative half-reaction. The conformation is best characterized
3
and supplemental Fig. S3). Overall, the reductive half-reac- as “semi-open” (Figs. 6A and 7C), and the Ser O␥ atom assumes
169
tion with D-Gal showed two observed phases similar to the reac- a position identical to that of O␥1 in Thr of the sugar-bound
MARCH 26, 2010•VOLUME 285•NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 9701

169
Roles of Thr in Catalysis by Pyranose 2-Oxidase
TABLE 2
169
Summary of rate constants for the flavin reduction of Thr variants at pH 7
Substrate
D-Glc, 4 °C
Parameter
WT
T169S
T169N
T169G
T169A
Ϫ1
a
k
(s
)
15.3 Ϯ 0.4
13.8 Ϯ 0.4
9.7 Ϯ 0.1 (fast) 0.002 (slow)
0.7 Ϯ 0.01
ND
red
a
b
K (mM)
45
45
47
ND
ND
d
Ϫ1
c
D-Glc, 25 °C
D-Gal, 4 °C
D-Gal, 25 °C
k
(s
)
48 Ϯ 2
ND
ND
ND
ND
0.03 Ϯ 0.007
ND
red
d
red
d
K (mM)
ND
ND
ND
Ϫ1
k
(s
)
0.3 Ϯ 0.03
3.7 Ϯ 0.2
1.2 Ϯ 0.04
ND
0.5 Ϯ 0.04
5.9 Ϯ 0.3
ND
0.9 Ϯ 0.01 (fast) 0.006 (slow)
2.7 Ϯ 0.02
4.2 Ϯ 0.2
ND
ND
K (mM)
6.6 Ϯ 0.3
ND
ND
Ϫ1
c
k
(s
)
0.005 Ϯ 0.0008
red
K (mM)
ND
ND
ND
ND
d
a
b
c
d
Rate constant and K are from the previous report (3).
d
ND, not determined. The K value could not be determined because the absorbance increase at 395 nm of the first phase was not significant.
These numbers contain large errors, because the highest concentrations of D-Glc and D-Gal used were not in a range that gives the saturating rate.
DISCUSSION
Based on our kinetic data presented here and its strategic
1
69
position in the P2O active site (14, 13), it is clear that Thr is
an intriguing and important amino acid in catalysis by P2O.
Our findings indicate that a side chain at position 169 capable of
forming H-bonds near the productive P2Oꢀsugar complex is
needed for efficient flavin reduction and identify mutants that
can help improving application of P2O in D-tagatose synthesis.
Stabilization of C4a-hydroperoxyflavin in P2O also requires the
1
69
presence of threonine at position 169. Thr has been observed
to assume two rotamer conformations in P2O: a rotamer with
the O␥ pointing away from the flavin and toward amino acids in
the dynamic active-site loop, as seen when the loop is open to
allow sugar binding (Fig. 7A), and another rotamer where O␥ is
FIGURE 3. Reduction of the oxidized WT with D-Gal. Stopped-flow traces at pointing toward the flavin and offers H-bond(s) to the N5/O4
4
58 nm are shown as dotted lines while at those at 395 nm are in solid lines. A
locus when the substrate loop is closed (Fig. 7B) (14). Based on
solution of the wild-type P2O (23 ␮M) was mixed with various concentrations
of D-Gal (0.4, 0.8, 1.6, 3.2, 6.4, 12.5, and 50 mM). All concentrations are given as
1
69
available crystal structures, the O␥ atom of Thr
in P2O
in electron
2
66
after mixing. Inset a shows a plot of kobs of the first phase (absorbance assumes a position equivalent to that of Thr
increase at 395 nm) versus D-Gal concentrations, whereas kobs of the second
transfer flavoprotein from Methylophilus methylotrophus (25);
phase (large decrease in A458 nm) was plotted against D-Gal concentration in
inset b. Data of the mutant are in supplemental Fig. S3.
2
54
Ser in electron transfer flavoprotein from the methylotro-
101
phic bacterium W3A1 (26); Ser in choline oxidase (27);
170
1
06
state observed for the H167A variant in complex with 2-deoxy- Ser
in alditol oxidase (28); Asp
in vanillyl alcohol oxi-
120
2
-fluoro-D-glucose (2IGO (14)) (Fig. 7, A and C).
dase (29); the backbone amide group of Gly in Streptomy-
In the case of the glycine replacement, we crystallized the ces or Brevibacterium cholesterol oxidase (30). The observed
double mutant, H167A/T169G, where also the flavinylation distances between atoms of the proteins and the flavin-
167
ligand (His ) had been mutated, as in the previously deter- N5/O4 locus indicate that they are mostly within H-bonding
mined sugar-bound state (2IGO (14)). It has been shown previ- distance. Hydrogen-bonding interactions in the vicinity of
ously that the H167A replacement does not introduce any sig- the flavin N5/O4 locus are recurrent features in flavoen-
nificant change in the positioning of flavin or protein atoms in zymes (17). Despite the commonality of these interactions,
167
⑀2
the active site beyond the absence of His
N -flavin C8␣ little is known about the precise functional roles and mech-
bond. The active-site loop in H167A/T169G is in a fully closed anistic implications. In electron transfer flavoprotein from
conformation (Fig. 6B), identical to that observed for P2O with methylotrophic bacterium W3A1, the H-bond to N5 has been
oЈ
bound acetate (1TT0 (13)) (Fig. 7, B and D), the only notable suggested to increase E of the enzyme (26). Clearly, more
m
difference is at the C-terminal end of the active-site loop, in detailed studies on other flavoproteins are needed to determine
1
69
the region 457–460 that is further away from the active site. whether the mechanistic implications of Thr in P2O, espe-
These differences may rather be due to the binding of a poly- cially in flavin reduction, can be generalized to other
ethylene glycol molecule in 1TT0 in this region, which is not flavoenzymes.
present in the unliganded H167A/T169G structure shown
Our results suggest that a protein side chain capable of form-
here. In H167A/T169G, two water molecules are positioned in ing H-bonds near the productive P2Oꢀsugar complex is re-
front of the isoalloxazine ring (Fig. 6B): one ϳ3.1 Å below the quired for efficient flavin reduction. Results with D-Glc as a
1
69
flavin N5 atom and another water 2.8 Å in front of the flavin O4 substrate indicate that the replacement of Thr
with Asn
oxygen. Neither of these assumes the exact position of the miss- and Ser has a negligible effect on the reductive half-reaction.
ing Thr side-chain hydroxyl oxygen, but the water interacting The K values for the binding of D-Glc in WT, T169S, and
d
with O4 is relatively close to that position. The other water T169N are almost identical. In addition, T169S and T169N all
molecule, slightly “below” the flavin N5 in Fig. 6B, would not be form an active P2OꢀGlc complex characterized by an absorb-
oЈ
present when sugar is bound.
ance increase at 395 nm (3). The E values (Table 1) and the
m
9
702 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 13•MARCH 26, 2010

169
Roles of Thr in Catalysis by Pyranose 2-Oxidase
This suggests that the binding
mode of D-Glc in T169G is different
from that in WT, T169S, and
T169N, thus resulting in lower effi-
ciency of the hydride transfer. In
T169A, the flavin reduction by
D-Glc is significantly impaired
(
Table 2). The small absorbance
increase at 395 nm accompanying
the binding of D-Glc to T169G and
T169A also supports the idea that
substrate binding is different than in
1
69
FIGURE4. KineticmechanismsofWTandThr variants. A, describeskineticmechanismsofreactionsofWT WT, T169N, and T169S. We may
1
69
and Thr variants with D-Glc. The reverse rate constant (kϪ3) is only present in the reaction of T169A, explain-
ing a convergent-line pattern found in this mutant. B, D-Gal binds to WT and Thr
one-step binding prior to the flavin reduction step.
conclude that efficient hydride
transfer from the sugar C2 to flavin
N5 during the reductive half-reac-
1
69
variants with simple
tion requires an enzyme conformational state that is compati-
ble with Thr, Ser, and Asn at the position 169.
Stopped-flow experiments of WT-P2O with D-Gal show
that, unlike the binding of D-Glc, P2O binds D-Gal in one step.
1
69
Replacement of Thr
by Ser, Asn, and Gly increased flavin
reduction rates when compared with that of WT without
increasing the binding affinity. This result can be interpreted as
D-Gal binds to the mutants with the geometry allowing the
reactants to carry out the hydride-transfer reaction more opti-
mal than in the WT. This is likely due to the relief of steric
hindrance close to the axial C4 hydroxyl group of D-Gal, as
predicted by our previous structural analysis (14). In T169A,
flavin reduction by D-Gal is very slow, resulting in an almost
inactive enzyme. As for the reaction with D-Glc discussed
1
69
above, these data suggest that, by substituting Thr with Ala,
FIGURE 5. Oxidation of the reduced T169S at various concentrations of
oxygen. A solution of reduced T169S (23 ␮M) was mixed with solutions con- the reductive half-reaction is significantly impaired.
taining various concentrations of oxygen: 0.13, 0.31, 0.61, and 0.96 mM. All
concentrationswereasaftermixing, andthereactionsweremonitoredat395
nm (dotted lines) and 458 nm (solid lines). The trace with empty circles repre-
The crystal structures of T169S and T169G mutants also
support the idea that the H-bonding ability of a side chain at
sents the reaction of WT with 0.13 mM oxygen monitored at 395 nm, showing position 169 is likely to be required for efficient flavin reduc-
formation and decay of C4a-hydroperoxyflavin (4). The inset shows kobs as a
tion. The structure of T169S shows that the O␥ of Ser assumes
function of [O ]. A bimolecular rate constant without a reverse rate was cal-
2
169
4
Ϫ1 Ϫ1
the same position as O␥ of Thr in the sugar-bound state of
culated to be 1.6 Ϯ 0.08 ϫ 10
M
s
.
1
69
H167A P2O where the Thr side chain is pointing away from
the flavin N5/O4 locus (Figs. 6A and 7C). Thus, by allowing
identical positioning of the Thr/Ser O␥, the reductive half-re-
action remains unperturbed. In T169G, which lacks the possi-
bility of forming a H-bond, two water molecules are found near
the flavin N5/O4, offering the possibility of H-bonds. In T169G,
the rate of flavin reduction by D-Glc decreased, but the rate with
D-Gal increased when compared with that of the WT (Table 2).
This mutant is thus quite competent in the reductive half-reac-
tion, maybe due to the possibility of solvent-mediated H-bonds
TABLE 3
Summary of bimolecular rate constants of the oxidative
half-reaction of Thr
1
69
variants at pH 7
Variants
Bimolecular rate constant
Ϫ1 Ϫ1
M
s
4
4
4
T169S
T169N
T169A
T169G
1.6 Ϯ 0.08 ϫ 10
0.1 Ϯ 0.01 ϫ 10
1.3 Ϯ 0.07 ϫ 10
80 Ϯ 4
rates of flavin reduction (k , Table 2) of WT, T169S, and to the productive P2OꢀGal complex. Although the structure
red
T169N are also in the same range, indicating that the oxidative of T169N is not available, the Asn side chain should also be able
power of these mutants is preserved. Interestingly, although the to H-bond to important groups in the productive ES complex.
oЈ
E of T169G was significantly higher than the other mutant Hence, by maintaining the H-bonding ability at position 169 (as
m
enzymes (Ϫ1 mV versus Ϫ105 mV or lower), the flavin in T169S, T169N, and T169G), efficient flavin reduction can be
reduction by D-Glc was affected inversely to the thermody- maintained.
1
69
namic changes, i.e. Ͼ10-fold decrease in k (Table 2). It has
Thr
is important for formation of a C4a-hydroperoxy-
red
been shown for the flavoenzyme 2-methyl-3-hydroxypyri- FAD intermediate. Our previous study with the WT has shown
oЈ
dine-5-carboxylic monooxygenase that, despite similar E
that P2O is thus far unique among flavoprotein oxidases for its
m
values, flavin reduction rates can differ up to 1600-fold due ability to stabilize the C4a-hydroperoxy-FAD during the oxida-
to changes in the precise geometry of the reactants (31, 32). tive half-reaction (4). It was suggested that the cavity at the
MARCH 26, 2010•VOLUME 285•NUMBER 13
JOURNAL OF BIOLOGICAL CHEMISTRY 9703

169
Roles of Thr in Catalysis by Pyranose 2-Oxidase
active site of this enzyme can provide the required space for restricted to allow formation of the intermediate (4, 28, 33). We
accommodating a peroxide group at the flavin C4a position (4). could not detect the C4a-hydroperoxy-FAD intermediate in
1
69
In other flavoprotein oxidases, the active sites may be too any of the Thr variants. All mutants reacted with oxygen in a
second-order fashion without a reverse rate constant, similar to
other flavoprotein oxidases (34, 35). The closed loop conforma-
tion observed in the wild-type P2O-acetate complex (1TT0
1
69
(
13)) shows Thr
O␥1 interacting with the flavin N5/O4
locus, offering the possibility of H-bonds to N5 and O4 (Fig.
1
69
7
B). In the structure of T169S, although the position of Ser
169
O␥ (Fig. 6) is different from the position of Thr O␥1 in the
closed loop structure of WT (1TT0 (13)), a similar H-bond as in
WT may be possible if loop movement occurs during the oxi-
dative half-reaction. However, the presence of a group capable
of making a H-bond to the flavin N5/O4 is unlikely to be the
main factor for stabilizing the intermediate, because in T169S,
no intermediate was detected. A possible explanation may be
1
69
that, in the closed conformational state, the position of Thr
provides an occluded reaction chamber that helps to control
precisely the reactivity of the reduced flavin and oxygen to
ensure the formation of the flavin-C4a intermediate prior to the
subsequent H O elimination.
2
2
The steady-state kinetic data are in agreement with the
kinetic model interpreted from stopped-flow data and suggest
that T169S and T169N can be considered to be good biocata-
lysts. With D-Glc as a substrate, k of T169S is higher than the
cat
k
of WT and other mutants. In all mutants, the effect on k
cat
cat
followed the trend seen for k (T169S Ͼ T169N Ͼ T169G Ͼ
red
T169A). In WT, the overall turnover with D-Glc is governed by
two steps: flavin reduction (k ) and C4a-hydroperoxyflavin
red
FIGURE 6. Active-site structure in T169S and H167A/T169G. Position 169
with Ser in T169S (A); and Gly in H167A/T169G (B).
decay (3). Because in all of the mutants the oxidative half-reac-
tion is second-order without an
intermediate (Fig. 5), the reaction is
limited only by flavin reduction
(
k
). Therefore, k of T169S with
red
cat
D-Glc is higher than the value of WT
Table 1). These data also imply
(
that, if the reaction is equilibrated
with high concentration of oxygen,
T169S can be employed as a better
catalyst than WT when using D-Glc
as a substrate. When using D-Gal as
a substrate, k
of T169S and
cat
T169N are 2- and 4-fold higher than
the value of WT, due to the increase
in k
(rate-limiting step) of the
red
mutants (Fig. 3 and Tables 1 and 2).
These two mutants would also be
better biocatalysts than the WT in
D-Gal oxidation for producing
D-tagatose. Effects of the mutations
based on steady-state parameters
reported in Table 1 are different
Glc,Gal
from
k
and
K
values
cat
m
reported before (15, 21) where the
values were from steady-state assay
under air saturation. Deviations of
the values reported in previous
studies (15, 21) from the actual
FIGURE 7. Comparison of active-site structures. The active site in H167A in complex with 2-deoxy-2-fluoro-
D-glucose (A); wild-type with bound acetate (B); superposition of T169S (green) and H167A2FG (yellow) (C); and
superposition of H167A/T169G (green) with wild-type P2O (yellow) (D). No water molecules have been drawn.
1
69
H-bonds from Thr in the wild type to the flavin N5/O4 atoms are indicated as dashed lines.
9
704 JOURNAL OF BIOLOGICAL CHEMISTRY
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169
Roles of Thr in Catalysis by Pyranose 2-Oxidase
O2
kinetic parameters in Table 1 are due to high values of K
,
3. Prongjit, M., Sucharitakul, J., Wongnate, T., Haltrich, D., and Chaiyen, P.
m
(
2009) Biochemistry 48, 4170–4180
especially for the mutants T169N and T169G; the concentra-
O2
4. Sucharitakul, J., Prongjit, M., Haltrich, D., and Chaiyen, P. (2008) Bio-
chemistry 47, 8485–8490
tion of oxygen under air-saturation (0.26 mM) is below the K
m
values of all the mutants (Table 1).
5
. Palfey, B. A., and Massey, V. (1998) in Comprehensive Biological Catalysis,
Vol. III (Michael, S., ed) pp. 83–153, San Diego, Academic Press
All variants except T169A showed parallel-line patterns
when D-Glc or D-Gal were used as substrates. This is similar to
the reaction of WT with D-Glc as a substrate (3). The kinetic
mechanism of WT at pH 7.0 is Ping-Pong where the 2-keto-D-
sugar product is released prior to the oxygen reaction. The par-
allel-line pattern can also be explained by a model in which the
sugar product remains bound during the reaction of molecular
6. Ballou, D. P., Entsch, B., and Cole, L. J. (2005) Biochem. Biophys. Res.
Commun. 338, 590–598
7
8
9
. van Berkel, W. J., Kamerbeek, N. M., and Fraaije, M. W. (2006) J. Biotech-
nol. 124, 670–689
. Orville, A. M., Lountos, G. T., Finnegan, S., Gadda, G., and Prabhakar, R.
(
2009) Biochemistry 48, 720–728
. H e´ roux, A., Bozinovski, D. M., Valley, M. P., Fitzpatrick, P. F., and Orville,
oxygen, but the reduction step is essentially irreversible (k ϾϾ
3
A. M. (2009) Biochemistry 48, 3407–3416
k
) (3). Interestingly, the steady-state kinetics of T169A with 10. Mallett, T. C., and Claiborne, A. (1998) Biochemistry 37, 8790–8802
Ϫ3
1
1
1
1. Ghisla, S., and Massey, V. (1989) Eur. J. Biochem. 181, 1–17
D-Glc or D-Gal as substrates show an intersecting-line pattern
instead of a parallel-line pattern, suggesting that, for T169A,
2. Albrecht, M., and Lengauer, T. (2003) Bioinformatics 19, 1216–1220
3. Hallberg, B. M., Leitner, C., Haltrich, D., and Divne, C. (2004) J. Mol. Biol.
regardless of the sugar used, the reaction of O with the reduced
2
341, 781–796
enzyme may occur while the 2-keto-D-sugar remains bound to
the enzyme. These data also imply that the reverse rate constant
1
4. Kujawa, M., Ebner, H., Leitner, C., Hallberg, B. M., Prongjit, M., Suchari-
takul, J., Ludwig, R., Rudsander, U., Peterbauer, C., Chaiyen, P., Haltrich,
D., and Divne, C. (2006) J. Biol. Chem. 281, 35104–35115
(
k
in Fig. 4A) of T169A is significant, in agreement with its
Ϫ3
oЈ
low E . The ternary-complex pattern indicates that a 2-keto- 15. Salaheddin, C., Spadiut, O., Ludwig, R., Tan, T. C., Divne, C., Haltrich, D.,
m
and Peterbauer, C. (2009) Biotechnol. J. 4, 535–543
sugar product resulting from the reductive half-reaction fails to
1
6. Bannwarth, M., Heckmann-Pohl, D., Bastian, S., Giffhorn, F., and Schulz
G. E. (2006) Biochemistry 45, 6587–6595
dissociate and remains bound to the active site during the reac-
tion with O in the oxidative half-reaction. One possible expla-
2
1
1
7. Fraaije, M. W., and Mattevi, A. (2000) Trends Biochem. Sci. 25, 126–132
8. Haltrich, D., Leitner, C., Neuhauser, W., Nidetzky, B., Kulbe, K. D., and
Volc, J. (1998) Ann. N.Y. Acad. Sci. 864, 295–299
nation is that space allocation at the re-side of the isoalloxazine
ring is increased in T169A compared with the WT and other
mutants investigated. A shift in the reaction mechanism from 19. Massey, V. (1991) in: Flavins and Flavoproteins (Curti, B., Ronchi, S., and
Zanetti, G., eds) pp. 59–66, Walter de Gruyter, Berlin
the typical Ping-Pong type to a ternary-complex mechanism
2
2
0. Dalziel, K. (1957) Acta Chem. Scand. 11, 1706–1723
was also observed in the reaction of P2O at pH 8 or above (36).
In conclusion, our results suggest that the residue at position
1. Spadiut, O., Leitner, C., Tan, T. C., Ludwig, R., Divne, C., and Haltrich, D.
(
2008) Biocatal. Biotran. 26, 120–127
1
69 is important for both half-reactions in P2O catalysis. Effi-
2
2
2. Kabsch, W. (1993) J. Appl. Crystallogr. 26, 795–800
cient hydride transfer from the sugar C2 to flavin N5 during the
reductive half-reaction requires a residue at this position that
3. Spadiut, O., Leitner, C., Salaheddin, C., Varga, B., Vertessy, B. G., Tan,
T. C., Divne, C., and Haltrich, D. (2009) FEBS J. 276, 776–792
can maintain a H-bonding within the ES complex, i.e. Thr 24. Spadiut, O., Radakovits, K., Pisanelli, I., Salaheddin, C., Yamabhai, M.,
Tan, T. C., Divne, C., and Haltrich, D. (2009) Biotechnol. J. 4, 525–534
(
WT), Ser, Asn, and Gly (H-bond possibly maintained by sol-
2
5. Roberts, D. L., Frerman, F. E., and Kim, J. J. (1996) Proc. Natl. Acad. Sci.
U.S.A. 93, 14355–14360
vent). When compared with the WT, the overall turnover of
T169S and T169N with D-Gal was faster due to higher rate
constants of the flavin reduction. In addition, the side chain of
2
2
6. Yang, K. Y., and Swenson, R. P. (2007) Biochemistry 46, 2289–2297
7. Quaye, O., Lountos, G. T., Fan, F., Orville, A. M., and Gadda, G. (2008)
Biochemistry 47, 243–256
169
Thr is crucial for stabilizing the C4a-hydroperoxy-FAD dur-
ing the oxidative half-reaction and cannot be substituted by 28. Forneris, F., Heuts, D. P., Delvecchio, M., Rovida, S., Fraaije, M. W., and
Mattevi, A. (2008) Biochemistry 47, 978–985
other residues.
2
3
3
9. van den Heuvel, R. H., Fraaije, M. W., Mattevi, A., and van Berkel, W. J.
(
2000) J. Biol. Chem. 275, 14799–14808
0. Yue, Q. K., Kass, I. J., Sampson, N. S., and Vrielink, A. (1999) Biochemistry
8, 4277–4286
1. Chaiyen, P., Brissette, P., Ballou, D. P., and Massey, V. (1997) Biochemistry
Acknowledgments—We thank Janewit Wongratana for constructing
plasmids for expression of T169S, T169N, and T169A and the beam-
line staff scientists at MAX-lab (Lund, Sweden) for support during
data collection. We thank Bruce Palfey for critical reading of the
manuscript.
3
36, 2612–2621
3
3
2. Chaiyen, P. (2010) Arch. Biochem. Biophys. 493, 62–70
3. Alfieri, A., Fersini, F., Ruangchan, N., Prongjit, M., Chaiyen, P., and Mat-
tevi, A. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 1177–1182
4. Mattevi, A. (2006) Trends Biochem. Sci. 31, 276–283
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MARCH 26, 2010•VOLUME 285•NUMBER 13
JOURNAL OF BIOLOGICAL CHEMISTRY 9705

Enzymology:
A Conserved Active-site Threonine Is
Important for Both Sugar and Flavin
Oxidations of Pyranose 2-Oxidase
Warintra Pitsawong, Jeerus Sucharitakul,
Methinee Prongjit, Tien-Chye Tan, Oliver
Spadiut, Dietmar Haltrich, Christina Divne
and Pimchai Chaiyen
J. Biol. Chem. 2010, 285:9697-9705.
doi: 10.1074/jbc.M109.073247 originally published online January 20, 2010
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