Báo cáo khoa học: Importance of the gating segment in the substrate-recognition loop of pyranose 2-oxidase pptx

Importance of the gating segment in the
substrate-recognition loop of pyranose 2-oxidase
Oliver Spadiut
1,
*, Tien-Chye Tan
1,2,
*, Ines Pisanelli
3
, Dietmar Haltrich
3
and Christina Divne
1,2
1 KTH Biotechnology, Royal Institute of Technology, Stockholm, Sweden
2 Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden
3 Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU – University of Natural Resources and Applied Life
Sciences, Vienna, Austria
Keywords
active-site loop; alanine-scanning
mutagenesis; crystal structure; pyranose
2-oxidase; site-saturation mutagenesis
Correspondence
C. Divne, KTH Biotechnology, Royal Institute
of Technology, Albanova,
Roslagstullsbacken 21, SE-106 91
Stockholm, Sweden
Fax: +46 8 5537 8468
Tel: +46 8 5537 8296
E-mail:
or D. Haltrich; Food Biotechnology
Laboratory, Department of Food Science
and Technology, BOKU – University of

Natural Resources and Applied Life
Sciences, A-1190 Vienna, Austria
Fax: +43 1 47654 6251
Tel: +43 1 47654 6140
E-mail:
*These authors contributed equally to this
work
Database
The atomic coordinates and structure
factors for the models are available in the
Protein Data Bank database under the
accession numbers 3K4J (H450Q),
3K4K (F454N), 3K4L (F454N
2FG
),
3K4M (Y456W
2FG
) and 3K4N
(F454A ⁄ S455A ⁄ Y456A)
(Received 22 February 2010, revised 2 May
2010, accepted 6 May 2010)
doi:10.1111/j.1742-4658.2010.07705.x
Pyranose 2-oxidase from Trametes multicolor is a 270 kDa homotetrameric
enzyme that participates in lignocellulose degradation by wood-rotting
fungi and oxidizes a variety of aldopyranoses present in lignocellulose to
2-ketoaldoses. The active site in pyranose 2-oxidase is gated by a highly
conserved, conformationally degenerate loop (residues 450–461), with a
conformer ensemble that can accommodate efficient binding of both elec-
tron-donor substrate (sugar) and electron-acceptor substrate (oxygen or
quinone compounds) relevant to the sequential reductive and oxidative

half-reactions, respectively. To investigate the importance of individual resi-
dues in this loop, a systematic mutagenesis approach was used, including
alanine-scanning, site-saturation and deletion mutagenesis, and selected
variants were characterized by biochemical and crystal-structure analyses.
We show that the gating segment (
454
FSY
456
) of this loop is particularly
important for substrate specificity, discrimination of sugar substrates, turn-
over half-life and resistance to thermal unfolding, and that three conserved
residues (Asp
452
, Phe
454
and Tyr
456
) are essentially intolerant to substitu-
tion. We furthermore propose that the gating segment is of specific impor-
tance for the oxidative half-reaction of pyranose 2-oxidase when oxygen is
the electron acceptor. Although the position and orientation of the slow
substrate 2-deoxy-2-fluoro-glucose when bound in the active site of pyra-
nose 2-oxidase variants is identical to that observed earlier, the substrate-
recognition loop in F454N and Y456W displays a high degree of confor-
mational disorder. The present study also lends support to the hypothesis
that 1,4-benzoquinone is a physiologically relevant alternative electron
acceptor in the oxidative half-reaction.
Abbreviations
ABTS, 2,2¢-azinobis(3-ethylbenz-thiazolinesulfonic acid; 2FG, 2-deoxy-2-fluoro-
D-glucose; BQ, 1,4-benzoquinone; Fc

+
, ferrocenium ion; P2O,
pyranose 2-oxidase; PDB, Protein Data Bank; TLS, translation, libration, screw-rotation.
2892 FEBS Journal 277 (2010) 2892–2909 ª 2010 The Authors Journal compilation ª 2010 FEBS
Introduction
Pyranose 2-oxidase (P2O; pyranose:oxygen 2-oxidore-
ductase; EC 1.1.3.10) from Trametes multicolor (syno-
nym Trametes ochracea) is a 270 kDa, 8a-(N3)-histidyl
flavinylated, homotetrameric flavoprotein oxidase
found in the fungal hyphal periplasmic space [1–5].
P2O has been suggested to perform a dual function
during lignin degradation by wood-rotting fungi, by
providing H
2
O
2
for ligninolytic enzymes [6,7] and
reducing quinones as part of the extracellular quinone
redox cycling machinery [8]. In some white-rot basid-
iomycetes, P2O participates in a secondary meta-
bolic pathway in which d-glucose is first converted
to 2-keto-d-glucose (2-arabino-hexos-2-ulose; d-gluco-
sone; (Scheme 1) and then further oxidized by
aldos-2-ulose dehydratase to the b-pyrone antibiotic
cortalcerone [9,10].
During the reductive half-reaction, P2O catalyses
the oxidation at the C2 of several aldopyranoses
(Scheme 1) present in lignocellulose to the correspond-
ing 2-ketoaldoses, accompanied by electron transfer to
FAD, yielding the reduced flavin, FADH

2
[11]. In the
oxidative half-reaction, the cofactor is re-oxidized by
an electron acceptor (e.g. O
2
or quinone compounds),
producing either H
2
O
2
or reduced quinone [1,8]. The
reaction mechanism is of the Ping Pong Bi Bi type [12],
which is common in flavoprotein oxidoreductases
[13,14]. In analogy with the hydride-transfer mecha-
nism proposed for flavoproteins in general [15,16], and
in particular for the reductive half-reaction of Phanero-
chaete chrysosporium cellobiose dehydrogenase [17,18],
which is the closest relative of P2O among glucose-
methanol-choline family members, His
548
in P2O is
suitably positioned to act as a general base to deproto-
nate the equatorial substrate 2-OH group, with support
from an O2–Asn
593
N
d2
hydrogen bond, accompanied
by transfer of the axial 2-hydrogen atom as a hydride
from C2 to the flavin N5 atom [5,19]. The two half-

reactions catalysed by P2O (i.e. oxidation of electron
donor and reduction of electron acceptor) have differ-
ent prerequisites with respect to the local chemical and
structural environment. Our crystal structures of a
closed state of wild-type P2O with the competitive
inhibitor acetate bound (Fig. 1A) [5], and an open state
with a slow sugar substrate bound (Fig. 1B) [19], have
shown that a conserved substrate-recognition loop
450
HRDAFSYGAVQQ
461
is highly dynamic and offers
a conformational gating mechanism to P2O.
In T. multicolor P2O, b-d-glucose is oxidized regi-
oselectively at C2 without traces of 3-keto product.
For the related Peniophora gigantea P2O, however,
oxidation at C3 can take place as a side reaction
when the glucose 2-OH group is either absent (e.g.
2-deoxy-d-glucose) or modified (e.g. 2-keto-d-glucose)
[11]. This was also observed for the T. multicolor P2O
Scheme 1. Substrates (electron acceptors
and donors) discussed in the text.
O. Spadiut et al. Substrate-recognition loop mutations in P2O
FEBS Journal 277 (2010) 2892–2909 ª 2010 The Authors Journal compilation ª 2010 FEBS 2893
mutant H167A with the slow substrate 2-deoxy-2-flu-
oro-d-glucose (2FG) (Scheme 1, Fig. 1B) [19]. In the
H167A variant, the flavinylation ligand His
167
has
been mutated to an alanine and the FAD is noncova-

lently bound. It was also shown that the flavin reduc-
tion was significantly slower than in the wild-type,
which enabled structure determination of an ordered
complex with 2FG [19]. The ability of the enzyme to
oxidize some substrates at both C2 and C3 requires
that the sugar can bind in two productive binding
modes. On the basis of the in silico modelling of glu-
cose, we have suggested that these two binding modes
are related by a 180° rotation about an axis running
through two points in the pyranose ring (one point
between C5 and O5, and the other between C2 and
C3), producing almost isosteric substrate-binding
modes [19]. Although d-galactose (Scheme 1) is a
relatively poor substrate for wild-type P2O (6%
relative activity compared to d-glucose) [8], it is struc-
turally very similar to d-glucose, differing only by the
axial C4 hydroxyl group (equatorial in glucose), and
thus, the C4 position must be important for the
substrate selectivity mechanism in P2O. We have
shown that d-galactose cannot be well accommodated
in the P2O active site as a result of possible steric
hindrance between the axial O4 and the side chain of
Thr
169
[20,21]. Engineering of P2O for improved
d-galactose turnover is also highly relevant for indus-
trial purposes because C2 oxidation of d-galactose
yields 2-keto-d-galactose, which can be further
reduced at C1 to give the low caloric sweetener
d-tagatose [22].

In the present study, we report the results obtained
from a systematic mutagenesis approach that aimed to
investigate the importance of key amino acids in the
substrate-recognition loop of T. multicolor P2O by
means of alanine-scanning, site-saturation and deletion
mutagenesis, as well as the characterization of mutants
by biochemical and crystal-structure analysis. We dis-
cuss the catalytic competence and stability of the
mutants and their preference for electron donors and
electron acceptors in light of the steady-state kinetics,
stability and structural data presented. The finding of
the present study demonstrate that the gating segment
of the substrate-recognition loop is of particular
importance for P2O function and substrate specificity,
as well as for catalytic and thermal stability at elevated
temperatures.
AB
Fig. 1. Overall P2O subunit structure of the closed and open state. Subunit structure of (A) wild-type P2O in complex with acetate (PDB
code 1TT0) with the substrate-recognition loop closed [5] and (B) the P2O variant H167A in complex with 2FG (PDB code 2IGO) where the
loop is completely open [19]. The H167A mutant has the flavinylation ligand His
167
mutated to alanine, and the FAD is noncovalently bound.
The Rossmann domain is coloured pink, and the substrate-binding domain is shown in light blue. Loops are shown in beige and the FAD
cofactor is shown in yellow. The ‘head domain’, which is not present in related glucose-methanol-choline members, is shown in light green.
The substrate-recognition loop (residues 450–461) is highlighted as a purple coil, with the side chains that form the gating segment (i.e.
Phe
454
, Ser
455
and Tyr

456
) shown as stick representations. The ligands (coloured bright green), acetate in the closed form and 2FG in the
open state, are bound at the re face of the isoalloxazine ring.
Substrate-recognition loop mutations in P2O O. Spadiut et al.
2894 FEBS Journal 277 (2010) 2892–2909 ª 2010 The Authors Journal compilation ª 2010 FEBS
Results
Site-saturation, alanine-scanning and deletion
mutagenesis
Site-saturation mutagenesis was employed to generate
a library of T. multicolor P2O variants targeting the
substrate-recognition loop, combined with high-
throughput screening of mutants in a 96-well plate
format. A procedure for generating and screening P2O
variants targeting position 450 has been described
previously [23]. We used this approach to generate
enzyme variants by targeting the positions 452, 454
and 456. The P2O mutant library covered > 95% of
all possible combinations of variants at the selected
positions (His
450
, Asp
452
, Phe
454
and Tyr
456
), and high-
throughput screening included 360 colonies for each
position, which were tested for activity towards two
electron-donor substrates: d-glucose and d-galactose.

The statistics show that a large number of mutations
at these positions result in inactive P2O variants: 60%,
44%, 56% and 48% inactive variants for His
450
,
Asp
452
, Phe
454
and Tyr
456
, respectively (site-saturation
mutagenesis at position 450 has been reported sepa-
rately) [23]. This demonstrates the importance of the
targeted loop residues for enzymatic activity and ⁄ or
proper folding and stability. In addition, alanine-scan-
ning and site-directed mutagenesis were performed.
A set of mutants that displayed interesting characteris-
tics with respect to d-glucose or d-galactose turnover
were selected and subjected to more detailed analysis.
These included the single-substitution variants H450Q,
F454P, F454N and Y456W; two multiple-alanine
mutants targeting the gating segment (F454A ⁄ Y456A
and F454A ⁄ S455A ⁄ Y456A); and one deletion mutant
lacking the FSY segment ( D454–456). Typical yields of
mutant P2Os were in the range 15–30 mgÆL
)1
culture
medium, although the D454–456 and alanine mutants
were expressed at significant lower levels (0.2–

0.8 mgÆL
)1
) and overexpression was accompanied by
an increase in inclusion-body formation.
Kinetic characterization of mutants
Apparent kinetic constants were determined for the
loop variants as a preliminary means to assess the effect
of these mutations on the P2O-catalysed reaction;
accordingly, one of the substrates of P2O (either the
electron acceptor or donor) was fixed, with the other
one being varied, and the obtained data were fit to the
Michaelis–Menten equation for a single substrate. For
all variants, the apparent k
app
cat
values with d-glucose
and O
2
(fixed at a concentration of 256 lm, air satura-
tion) as substrates, k
cat[Glc ⁄ O2]
, decreased dramatically
compared to the wild-type enzyme, and K
m[Glc]
values
increased by a factor in the range 1.9–3.3 (Table 1),
resulting in decreased catalytic efficiency constants
(k
cat[Glc ⁄ O2]
⁄ K

m[Glc]
). The only exceptions are Y456W, a
conservative mutation with one bulky hydrophobic
amino acid replacing another one, which shows a simi-
lar k
cat[Glc ⁄ O2]
, albeit with a doubled K
m[Glc]
and an
associated three-fold decrease in k
cat[Glc ⁄ O2]
⁄ K
m[Glc]
,as
well as H450Q, where k
cat[Glc ⁄ O2]
was decreased by a
factor of 2. Consistently, turnover numbers for the elec-
tron donor ⁄ acceptor substrate pair d-galactose ⁄ O
2
for
the variants H450Q and Y456W were comparable to
that of the wild-type. Other variants, in particular the
Ala mutants and D454-456, showed three- to 12-fold
lower k
cat[Gal ⁄ O2]
, and up to six-fold elevated K
m[Gal]
values (Table 1).
Mutations in the gating segment affected sugar

substrate specificity significantly. The wild-type enzyme
displays a clear preference for d-glucose over d-galac-
tose, as indicated by a selectivity ratio of  160, with
Table 1. Apparent steady-state kinetic constants of T. multicolor P2O wild-type and mutants with D-glucose (0.1–50 mM)orD-galactose
(0.1–200 m
M) as electron donor and O
2
(air) under saturation as electron acceptor.
Variant
Glc ⁄ O
2
Gal ⁄ O
2
K
m
[Glc]
(m
M)
k
cat
[Glc ⁄ O
2
]
(s
)1
)
k
cat
⁄ K
m

[Glc]
(
M
)1
Æs
)1
)
K
m
[Gal]
(m
M)
k
cat
[Gal ⁄ O
2
]
(s
)1
)
k
cat
[Gal ⁄ O
2
] ⁄ K
m
[Gal]
(
M
)1

Æs
)1
)
Wild-type 0.76 ± 0.05 33 ± 0 43 · 10
3
6.1 ± 0.3 1.7 ± 0 0.27 · 10
3
H450Q 2.5 ± 0.3 17 ± 1 7.0 · 10
3
34 ± 7 2.0 ± 0.1 0.059 · 10
3
F454P 1.8 ± 0.3 0.99 ± 0.04 0.54 · 10
3
13 ± 1 0.30 ± 0.01 0.024 · 10
3
F454N 1.5 ± 0.1 12 ± 0 8.2 · 10
3
26 ± 2 1.2 ± 0 0.046 · 10
3
Y456W 1.7 ± 0.3 26 ± 1 15 · 10
3
29 ± 2 1.5 ± 0 0.053 · 10
3
F454A ⁄ Y456A 1.5 ± 0.2 7.1 ± 0.2 4.7 · 10
3
10 ± 1 0.64 ± 0.02 0.062 · 10
3
F454A ⁄ S455A ⁄ Y456A 2.1 ± 0.3 0.20 ± 0.01 0.094 · 10
3
13 ± 3 0.14 ± 0.01 0.011 · 10

3
D454–456 1.4 ± 0 3.1 ± 0 2.2 · 10
3
7.7 ± 0.9 0.29 ± 0.01 0.038 · 10
3
O. Spadiut et al. Substrate-recognition loop mutations in P2O
FEBS Journal 277 (2010) 2892–2909 ª 2010 The Authors Journal compilation ª 2010 FEBS 2895
the ratio of the specificity constants [24] for the
two substrates being [(k
cat[Glc ⁄ O2]
⁄ K
m[Glc]
) ⁄ (k
cat[Gal ⁄ O2]
⁄
K
m[Gal]
)]. Most of the mutations in the gating segment
reduced the selectivity ratio. The lowest ratio [(k
cat[Glc ⁄
O2]
⁄ K
m[Glc]
) ⁄ (k
cat[Gal ⁄ O2]
⁄ K
m[Gal]
)] of  8.4 was observed
for the triple-alanine mutant, which, however, still
retains some preference for d-glucose over d-galactose.

By contrast, the replacements F454N or Y456W show
increased [(k
cat[Glc ⁄ O2]
⁄ K
m[Glc]
) ⁄ (k
cat[Gal ⁄ O2]
⁄ K
m[Gal]
)] values
of  180 and 290, respectively. This effect on substrate
selectivity is even more pronounced when considering
the disaccharide melibiose (D-Gal-a(1 fi 6)-D-Glc;
Scheme 1). Melibiose is a rather poor substrate for
P2O, mainly because of its very high K
app
m
value
(K
m[Mel]
= 1530 mm and k
cat
= 7.6 for wild-type;
Table 2), and the selectivity ratio [(k
cat[Glc ⁄ O2]
⁄ K
m[Glc]
) ⁄
(k
cat[Mel ⁄ O2]

⁄ K
m[Mel]
)] for wild-type is  8700, indicat-
ing very strong discrimination of P2O in favour of the
monosaccharide substrate d-glucose over the disaccha-
ride melibiose, with oxygen as acceptor. Again, muta-
tions in the gating segment of the substrate loop
reduced the substrate selectivity for all of the variants
to values in the range 12–1630. The lowest [(k
cat[Glc ⁄
O2]
⁄ K
m[Glc]
) ⁄ (k
cat[Mel ⁄ O2]
⁄ K
m[Mel]
)] ratios of 93 and 12
were found for the F454P and triple-Ala mutant,
respectively.
Furthermore, apparent kinetic constants were deter-
mined for the reduction of the two-electron acceptor
1,4-benzoquinone (BQ) to hydroquinone (Scheme 1,
Table 3), and for the reduction of the 1-electron accep-
tor ferrocenium (Fc
+
) to ferrocene (Scheme 1,
Table 4), with either d-glucose or d-galactose at satu-
rating concentrations. Variant Y456W is characterized
by improved BQ and Fc

+
binding, as indicated by
lower K
app
m
values, and increased k
app
cat
values, resulting
in an  2.5-fold higher k
cat[BQ ⁄ Glc]
⁄ K
m[BQ]
and k
cat[BQ ⁄
Gal]
⁄ K
m[BQ]
relative to wild-type. The triple-Ala and
loop-deletion mutants also show considerably lower
K
m[BQ]
with Glc as electron donor, although with an
associated decrease in k
cat[BQ ⁄ Glc]
values. Similar results
were obtained for the reduction of BQ with d-galac-
tose, and for Fc
+
with d-glucose or d-galactose as sat-

urating electron-donor substrates. In addition, H450Q,
F454N and Y456W show improved kinetic properties
Table 2. Apparent steady-state kinetic constants of T. multicolor P2O wild-type and mutants with melibiose (5.0–500 mM) as electron donor
and O
2
(air) under saturation as electron acceptor.
Variant
Mel ⁄ O
2
Substrate selectivity
K
m
[Mel] (mM) k
cat
[Mel ⁄ O
2
](s
)1
)
k
cat
[Mel ⁄ O
2
] ⁄ K
m
[Mel]
(
M
)1
Æs

)1
)
(k
cat
[Glc ⁄ O
2
] ⁄ K
m
[Glc]) ⁄
(k
cat
[Mel ⁄ O
2
] ⁄ K
m
[Mel])
Wild-type 1500 ± 300 7.6 ± 1.3 5.0 8700
H450Q 390 ± 50 3.3 ± 0.2 8.4 830
F454P 50 ± 4 0.29 ± 0.01 5.8 93
F454N 240 ± 40 2.7 ± 0.2 11.3 720
Y456W 260 ± 10 4.4 ± 0.1 16.6 920
F454A ⁄ Y456A 350 ± 110 1.3 ± 0.2 3.7 1300
F454A ⁄ S455A ⁄ Y456A 23 ± 4 0.19 ± 0.01 8.0 12
D454–456 210 ± 60 0.28 ± 0.04 1.3 1600
Table 3. Apparent steady-state kinetic constants of T. multicolor P2O wild-type and mutants with BQ (0.01–1.5 mM) as electron acceptor
and
D-glucose or D-galactose at saturation (100 mM each) as electron donor.
Variant
BQ ⁄ Glc BQ ⁄ Gal
K

m
[BQ]
(l
M)
k
cat
[BQ ⁄ Glc]
(s
)1
)
k
cat
[BQ ⁄ Glc] ⁄ K
m
[BQ]
(
M
)1
Æs
)1
)
K
m
[BQ]
(l
M)
k
cat
[BQ ⁄ Gal]
(s

)1
)
k
cat
[BQ ⁄ Gal] ⁄ K
m
[BQ]
(
M
)1
Æs
)1
)
Wild-type 140 ± 20 160 ± 10 1.2 · 10
6
27 ± 4 3.8 ± 0.1 0.14 · 10
6
H450Q 240 ± 80 220 ± 30 0.94 · 10
6
13 ± 2 3.0 ± 0.1 0.23 · 10
6
F454P 72 ± 29 30 ± 4 0.42 · 10
6
7.1 ± 1.0 2.0 ± 0.1 0.28 · 10
6
F454N 52 ± 13 130 ± 10 2.6 · 10
6
5.2 ± 0.8 2.7 ± 0.1 0.52 · 10
6
Y456W 72 ± 15 220 ± 10 3.0 · 10

6
10 ± 1 3.3 ± 0.1 0.33 · 10
6
F454A ⁄ Y456A 29 ± 4 61 ± 2 2.1 · 10
6
78 ± 21 2.9 ± 0.3 0.037 · 10
6
F454A ⁄ S455A ⁄ Y456A 29 ± 11 15 ± 1 0.52 · 10
6
8.9 ± 1.0 1.2 ± 0 0.13 · 10
6
D454–456 41 ± 4 25 ± 1 0.60 · 10
6
37 ± 19 1.2 ± 0.2 0.032 · 10
6
Substrate-recognition loop mutations in P2O O. Spadiut et al.
2896 FEBS Journal 277 (2010) 2892–2909 ª 2010 The Authors Journal compilation ª 2010 FEBS
for Fc
+
(lower K
m
and higher k
cat
values) both with
glucose and galactose as saturating substrate compared
to wild-type. Both BQ and Fc
+
are considerably larger
molecules compared to O
2

, and, most likely, shorten-
ing the loop or introducing smaller side chains pro-
motes the reaction with the larger electron-acceptor
substrates.
Heat inactivation, pH optima, UV-visible spectra
ThermoFAD analysis
The half-life of P2O activity (i.e. the time during which
the enzyme remains active) was measured for wild-type
and mutants at constant pH (6.5) at 60 or 70 °C. The
inactivation constant, k
in
, and the half-life of activity,
s
1 ⁄ 2
, were determined (Table 5). On the basis of
[ln(residual activity) versus time] plots, all mutants
show first-order inactivation kinetics (Fig. 2). H450Q
shows pronounced destabilization (Fig. 2A), whereas
the other variants show similar or improved stability
(Fig. 2A,B). The substitutions Phe
454
fi Asn, and
Tyr
456
fi Trp result in a 29- and 34-fold increase in
s
1 ⁄ 2
values at 60 °C, respectively. Interestingly, the ala-
nine-substituted variants are also more stable at 60 °C,
with a 12-fold and 23-fold increase in s

1 ⁄ 2
for
F454A ⁄ Y456A and F454A ⁄ S455A ⁄ Y456A, respec-
tively. Some stabilization is also seen for D454–456
(four- to five-fold increase) at 60 °C. A similar trend
of increased heat inactivation half-life was observed
for all variants at 70 °C (Fig. 2C).
All variants show pH optima at pH 6.5 (data not
shown), suggesting that the altered kinetics is not inti-
mately correlated with changes in pH profile. All
enzymes also display typical flavoprotein UV-visible
spectra with absorption maxima k
max
at 345 and
456 nm (data not shown), and reduction of the
enzymes with d-glucose and sodium dithionite in the
absence of oxygen resulted in the disappearance of
the absorption peak at 456 nm that was expected for
the fully reduced state. FAD was not released upon
trichloroacetic acid treatment, demonstrating that,
despite extensive mutagenesis in the vicinity of the
FAD-binding pocket, the mutants remain properly fla-
vinylated (not shown). The thermal stability was inves-
tigated using the ThermoFAD technique to derive
thermal unfolding transition values (T
m
). The T
m
val-
ues are summarized in Table 6. Of the variants ana-

lyzed, all but two mutants show slightly decreased T
m
values (1–3 °C). Y456W and F454A⁄ Y456A show
improved T
m
values by 5.5 and 1.5 °C, respectively.
Overall monomer structure of loop variants
The mutants analyzed structurally include the unbound
forms of H450Q, F454N and F454A ⁄ S455A ⁄ Y456A,
and F454N or Y456W with bound 2FG. Data for the
triple-Ala mutant were obtained to medium-low resolu-
tion (2.75 A
˚
), and the model is included mainly to evalu-
ate the backbone conformation of the substrate loop.
Table 4. Apparent steady-state kinetic constants of T. multicolor P2O wild-type and mutants with Fc
+
(0.005–1.5 mM) as electron acceptor
and
D-glucose or D-galactose under saturation (100 mM each) as electron donor.
Variant
Fc
+
⁄ Glc Fc
+
⁄ Gal
K
m
[Fc
+

]
(l
M)
k
cat
[Fc
+
⁄ Glc]
(s
)1
)
k
cat
[Fc
+
⁄ Glc] ⁄ K
m
[Fc
+
]
(
M
)1
Æs
)1
)
K
m
[Fc
+

]
(l
M)
k
cat
[Fc
+
⁄ Gal]
(s
)1
)
k
cat
[Fc
+
⁄ Gal] ⁄ K
m
[Fc
+
]
(
M
)1
Æs
)1
)
Wild-type 400 ± 110 210 ± 30 0.51 · 10
6
100 ± 70 6.6 ± 1.8 0.063 · 10
6

H450Q 380 ± 110 470 ± 70 1.25 · 10
6
49 ± 15 17 ± 2 0.34 · 10
6
F454P 140 ± 60 67 ± 12 0.48 · 10
6
23 ± 14 4.5 ± 0.7 0.20 · 10
6
F454N 350 ± 210 420 ± 110 1.21 · 10
6
25 ± 10 7.3 ± 1.1 0.29 · 10
6
Y456W 240 ± 40 400 ± 30 1.64 · 10
6
41 ± 9 7.3 ± 0.4 0.18 · 10
6
F454A ⁄ Y456A 150 ± 30 74 ± 5 0.50 · 10
6
16 ± 7 2.9 ± 0.3 0.18 · 10
6
F454A ⁄ S455A ⁄ Y456A 280 ± 70 110 ± 10 0.37 · 10
6
15 ± 9 5.4 ± 0.7 0.36 · 10
6
D454–456 300 ± 60 15 ± 1 0.050 · 10
6
11 ± 1 1.3 ± 0.1 0.12 · 10
6
Table 5. Heat inactivation half-life of T. multicolor P2O wild-type
and mutants at 60 and 70 °C. k

in
, inactivation constant; s
1 ⁄ 2
, half-
life; ND, not determined.
Variant
60 °C70°C
k
in
(min
)1
)
s
1 ⁄ 2 (60 °C)
(min)
k
in
(min
)1
)
s
1 ⁄ 2 (70 °C)
(min)
Wild-type )58 · 10
)3
12 )9.9 0.07
H450Q )86 · 10
)3
8.1 ND ND
F454P )67 · 10

)3
10 ND ND
F454N )2.0 · 10
)3
350 )1.8 0.39
Y456W )1.7 · 10
)3
410 )1.1 0.63
F454A ⁄ Y456A )4.8 · 10
)3
140 )1.1 0.64
F454A ⁄ S455A ⁄
Y456A
)2.5 · 10
)3
280 )1.2 0.60
D454–456 )13 · 10
)3
55 ND ND
O. Spadiut et al. Substrate-recognition loop mutations in P2O
FEBS Journal 277 (2010) 2892–2909 ª 2010 The Authors Journal compilation ª 2010 FEBS 2897
All structures show satisfactory model statistics
(Table 7), and are very similar overall to the previously
determined crystal structures of T. multicolor P2O
[5,19,25,26], demonstrating that the mutations do
not result in significant structural changes beyond the
targeted region. In the unbound form of variants
H450Q, F454N and F454A ⁄ S455A ⁄ Y456A, the sub-
strate loop is in the open conformation. Therefore,
structural comparisons are made with H167A rather

than the wild-type because H167A has the substrate-
recognition loop in the fully open conformation [19].
This is in agreement with our earlier observation that
the substrate loop tends to be in the open state either
when the active site is unoccupied, or when sugar (elec-
tron donor) is bound. However, in the former case, we
typically observe varying degrees of disorder of the loop,
whereas the loop becomes well defined when sugar
substrate is bound [19,25,26]. The same is observed in
the present study where the unbound variants display
varying degrees of fluctuation of the open state, which is
manifested as weak but interpretable electron density
indicative of multiple conformers. Furthermore, the two
2FG-bound F454N and Y456W models show the open
state of the substrate-recognition loop.
Structure of the Y456W-2FG complex
Despite the larger tryptophan side chain, the substrate
loop in Y456W
2FG
(Fig. 3A) assumes the same open
state as observed in H167A
2FG
[Protein Data Bank
(PDB) code 2IGO] [19]. However, the electron density
is weak for the Trp
456
indole ring, as well as for the sub-
strate loop, suggesting that the larger side chain induces
local disorder and suboptimal side-chain packing.
Despite this local disorder, the 2FG molecule is orderly

bound in an orientation identical to that in H167A
2FG
,
corresponding to the C3-oxidation binding mode
(Fig. 3B) (i.e. oriented for oxidation at the substrate C3
atom). The only notable difference is that the side chain
of Asp
452
assumes a different conformation, offering
the possibility of a hydrogen bond between its Od2
carboxylic oxygen and the glucosyl O1 of 2FG, thus
replacing the 2FG O1-Gln
448
Ne2 interaction observed
in H167A
2FG
[19]. The Tyr
456
side chain of the open
state in H167A
2FG
is located some 13 A
˚
from the
A
B
C
Fig. 2. Inactivation kinetics of P2O wild-type and mutants. Inactiva-
tion at 60 °C (pH 6.5). (A)
, wild-type; , H450Q; , F454P; ,

F454N;
, Y456W. (B) , wild-type; , F454A ⁄ Y456A; , F454A ⁄
S455A ⁄ Y456A;
, D454–456. (C) Same as in (A) and (B), but at
70 °C.
Table 6. Melting temperature T
m
of T. multicolor P2O wild-type
and mutants. ND, not determined.
Variant T
m
(°C)
Wild-type 63.5
H450Q 60.5
F454P 62.0
F454N 62.5
Y456W 69.0
F454A ⁄ Y456A 65.0
F454A ⁄ S455A ⁄ Y456A 62.5
D454–456 ND
Substrate-recognition loop mutations in P2O O. Spadiut et al.
2898 FEBS Journal 277 (2010) 2892–2909 ª 2010 The Authors Journal compilation ª 2010 FEBS
Table 7. Data collection and crystallographic refinement statistics. Statistics for the high-resolution shell are given in parentheses.
H450Q F454N F454N
2FG
Y456W
2FG
F454A ⁄ S455A ⁄ Y456A
Data collection
Cell constants: a, b, c (A

˚
); b (°) 101.91, 101.91, 120.47 102.06, 102.06, 119.69 101.57, 101.57, 250.05 168.19, 103.14, 168.71; 106.467 101.58, 101.58, 250.00
Space group P4
2
2
1
2 P4
2
2
1
2 P4
3
2
1
2 P2
1
P4
3
2
1
2
Number of molecules ⁄ a.s.u. 1 1 2 8 2
Beamline, k (A
˚
) I911-2, 1.0379 I911-2, 1.0379 I911-2, 1.0379 I911-2, 1.0379 I911-2, 1.0379
Resolution range (A
˚
) 29–2.0 (2.10–2.00) 30–1.60 (1.70–1.60) 30–1.75 (1.80–1.75) 30–2.20 (2.30–2.20) 30–2.75 (2.80–2.75)
Unique reflections 43 397 (5 784) 83 548 (13 637) 132 005 (10 540) 273 077 (32 973) 34 840 (1776)
Multiplicity 14.5 (14.5) 12.9 (9.9) 7.3 (7.3) 2.7 (2.4) 6.8 (6.3)

Completeness (%) 99.8 (99.9) 99.9 (99.6) 99.9 (99.9) 97.2 (94.6) 99.7 (99.8)
<I ⁄ rI> 17.3 (5.2) 18.1 (3.1) 15.4 (2.5) 7.6 (1.5) 11.8 (2.6)
R
sym
a
(%) 18.7 (85.9) 10.0 (86.0) 10.2 (88.3) 12.9 (82.7) 19.2 (88.3)
Refinement
Resolution range (A
˚
) 30–2.0 (2.11–2.00) 30–1.60 (1.69–1.60) 30–1.75 (1.84–1.75) 30–2.20 (2.32–2.20) 30–2.75 (2.90–2.75)
Completeness % 99.9 (100) 99.9 (99.5) 99.9 (99.9) 97.4 (94.9) 99.8 (99.8)
R
factor
b
⁄ work reflns 15.8 ⁄ 41 420 18.6 ⁄ 81 587 17.6 ⁄ 130 031 19.4 ⁄ 270 272 19.6 ⁄ 32 909
R
free
⁄ free reflns 20.0 ⁄ 1990 21.3 ⁄ 1961 20.9 ⁄ 1974 25.8 ⁄ 2798 27.4 ⁄ 1931
Nonhydrogen atoms 4891 5021 9836 38 783 9264
Mean B (A
˚
2
) protein all ⁄ mc ⁄ sc 13.3 ⁄ 12.3 ⁄ 14.4 15.1 ⁄ 14.0 ⁄ 16.2 16.0 ⁄ 14.9 ⁄ 17.1 22.9 ⁄ 22.3 ⁄ 23.5 25.9 ⁄ 25.8 ⁄ 26.0
Mean B (A
˚
2
) solvent ⁄ number of molecules 22.4 ⁄ 309 27.1 ⁄ 437 25.2 ⁄ 726 23.9 ⁄ 1930 24.0 ⁄ 150
rmsd bond lengths (A
˚
), angles (°) 0.022, 1.86 0.020, 1.84 0.021, 2.00 0.021, 1.98 0.018, 1.81

Ramachandran
c
favoured ⁄ allowed (%) 98.1 ⁄ 100 97.7 ⁄ 100 97.6 ⁄ 100 96.2 ⁄ 100 93.3 ⁄ 99.5
PDB accession code 3K4J 3K4K 3K4L 3K4M 3K4N
a
R
sym
=[R
hkl
R
i
|I ) <I>| ⁄ R
hkl
R
i
|I |] · 100%.
b
R
factor
= R
hkl
||F
o
| ) |F
c
|| ⁄ R
hkl
|F
o
|.

c
Ramachandran analysis according to MOLPROBITY [24a].
O. Spadiut et al. Substrate-recognition loop mutations in P2O
FEBS Journal 277 (2010) 2892–2909 ª 2010 The Authors Journal compilation ª 2010 FEBS 2899
2FG molecule, where it forms hydrogen bonds to
Gln
365
Ne2 and one water molecule. In the open state
of Y456W
2FG
, the Trp
456
side chains adopts the same
position and conformation as the tyrosine, and also
makes the same edge-to-face ring stacking interaction
with Phe
454
as observed in H167A
2FG
. The tryptophan
side chain is unable to form the tyrosine Og hydrogen
bonds, although a new hydrogen bond is possible
between Trp
456
Ne1 and Asp
101
Od2 (Fig. 3C). The
observation that the loop is highly ordered in the
H167A
2FG

complex was attributed to two principal
factors: first, that the H167A variant is redox impaired
(i.e. removal of the covalent His
167
-FAD bond reduces
the oxidative power of FAD) and, second, that 2FG
is a very slow substrate for P2O [i.e. reduction by 2FG
of wild-type P2O (k
obs
= 0.0064 min
)1
) and of H167A
(k
obs
= 0.000027 min
)1
)] [19]. Because H167A
2FG
and
Y456W
2FG
bind the same slow substrate, the difference
is therefore mainly attributed to the introduction of the
larger tryptophan side chain at position 456, which may
alter the conformational ensemble accessible to the
loop, and to the intact His
167
-FAD bond, which retains
the oxidative power of FAD in Y456W to allow higher
2FG turnover rates.

Structure of the F454N-2FG complex
In the F454N
2FG
complex, more pronounced changes
occur in the substrate-recognition loop (Fig. 4). The
electron density is of very high quality for the overall
protein, including the 2FG molecule bound in the
same C3-oxidation mode as in H167A
2FG
[19] and
A
B
C
Fig. 3. Active-site structure of the Y456W mutant with bound 2FG.
(A) Active-site loop conformation in Y456W
2FG
(yellow) superim-
posed onto H167A
2FG
(green) [19]. The comparison of the mutant
is made with H167A rather than the wild-type because the sub-
strate-recognition loop is open in H167A and the same ligand is
bound. The 2FG molecule is bound for oxidation at C3. (B) Close-up
of region around 2FG. Superposition of models as in (A). For clarity,
no water molecules are shown. (C) Details of the interactions made
by Trp
456
(Y456W) and Tyr
456
(H167A). The overall loop conforma-

tion, the bound ligand, the flavin cofactor and most active-site resi-
dues are strikingly similar in the two complexes. Small backbone
changes at position 452 induce a different conformation of the Asp
side chain, and the interactions made by Tyr
456
are abolished by
the mutation. The Trp side chain is instead stabilized by a hydrogen
bond to Asp
101
.
Fig. 4. Active-site structure of the F454N mutant with bound 2FG.
Loop conformation in F454N
2FG
(yellow) superimposed onto
H167A
2FG
(green) [19]. The 2FG molecule is bound for oxidation at
C3. The Phe fi Asn replacement at position 454 induces significant
changes in the 452–454 backbone and side chains without affecting
the position or orientation of the bound 2FG molecule.
Substrate-recognition loop mutations in P2O O. Spadiut et al.
2900 FEBS Journal 277 (2010) 2892–2909 ª 2010 The Authors Journal compilation ª 2010 FEBS
Y456W
2FG
(present study). Nonetheless, the substrate
loop in F454N
2FG
is disordered beyond the mutated
residue 454, lacking interpretable electron density for
the segment 455–460. Thus, the Phe

454
fi Asn replace-
ment appears to have a large influence on the local
conformation of the loop compared to other variants
for which structural data have been analyzed. We have
reported previously that, in the open loop conforma-
tion in the C3 oxidation mode (H167A
2FG
), residues
454 and 456 do not form specific interactions with the
sugar substrate but, rather, they are folded away from
the active site [19]. It is therefore particularly interest-
ing to note that Asn
454
assumes a position not previ-
ously observed in 2FG complexes of P2O, and that its
side-chain amide group approaches the exocylic O6
hydroxyl group of the substrate, but does not come
close enough to form a hydrogen bond (Fig. 4). In a
structure superposition, the distance between the Ca
position of Asn
454
in F454N
2FG
and that of Phe
454
in
the closed wild-type acetate complex (WT
ACT
; PDB

code 1TT0) [5] is  1.5 A
˚
. The lack of density beyond
position 454 does not allow further analysis of this
loop conformer, indicating that, even in the presence
of orderly bound substrate analogue, the loop under-
goes significant dynamic fluctuations. Despite the unu-
sual position of Asn
454
, Asp
452
is positioned as in the
Y456W mutant, forming a potentially tight interaction
with 2FG O1 (distance 2FG O1–Asp
452
Od2, 2.6 A
˚
).
Structure of H450Q
In the WT
ACT
complex, Arg
451
, the residue immedi-
ately following the mutated histidine in H450Q has
two well defined alternative side-chain conformations,
each of which appears appropriately stabilized. In the
first conformation, two interactions are possible:
Arg
451

Ng1–Asp
470
Od2 and Arg
451
Ng2–water. In the
second conformation, the possible interactions are:
Arg
451
Ng1–Ser
465
O and Arg
451
Ng2–water. Thus, it
appears that the arginine can alternate between these
two alternative conformations when the loop is in the
fully closed state. In the open loop state of H167A
2FG
,
the latter conformation is prevalent (with a Ser
465
interaction). H450Q assumes the same open loop con-
formation as H167A
2FG
; however, in H450Q, we
observe backbone displacements of 1 A
˚
at positions
451 and 452, and the Arg
451
side chain assumes a dif-

ferent conformation than those observed in the fully
closed and open states, participating in a different set
of side-chain interactions (Fig. 5): Arg
451
Ng1–Asp101
Od1, Arg
451
Ng1–Tyr
456
Og and Arg
451
Ng1–water.
The changes introduced in the region 450–452 are
also manifested as a somewhat weak density for the
side-chain carboxylate group of Asp
452
, most likely
because the carboxylate group lacks interaction possi-
bilities in H450Q. In both the WT
ACT
and H167A
2FG
complexes, Asp
452
can participate in side-chain interac-
tions with nearby residues and solvent (WT
ACT
: Asp
452
Od2–Lys

91
Nf, Asp
452
Od2–Ala
453
N, Asp
452
Od1–two
water molecules; H167A
2FG
: Asp
452
Od1–Asp
470
Od2,
Asp
452
Od1–Arg
472
Ng1).
Comparison of the active-site volumes
To determine whether there are changes in the volume
of the active site as a result of mutations that may
explain the ability to accommodate substrates other
than glucose, the cavity volumes were calculated for
the mutant models, and compared with those of the
closed state in WT
ACT
(PDB code 1TT0) [5] and
the open state in H167A

2FG
(PDB code 2IGO) [19].
The substrate-recognition loop in F454N with bound
2FG is heavily disordered in the region 455–460 and,
because these residues were not modelled, the volume
could not be calculated for this mutant. In all models,
the substrate-recognition loop is in the open state,
which means that the gorge leading to the active site is
open to the large internal void at the homotetramer
centre from which the active sites are accessible.
This complicates any attempt at computation of the
Fig. 5. Active-site structure in the H450Q mutant without ligand.
Changes of the 450–452 backbone region (in particular, Asp
452
and
Arg
451
) in H450Q (yellow) compared to H167A
2FG
(green) [19].
Hydrogen bonds formed by Arg
451
are coloured yellow in H450Q,
and green in H167A. The mutation at position 450 leads to back-
bone perturbation accompanied by compensatory stabilizing interac-
tions formed by Arg
451
. Although no sugar is bound in H450Q, the
overall structure of the substrate-recognition loop is similar to that
of the open sugar-binding state of H167A

2FG
.
O. Spadiut et al. Substrate-recognition loop mutations in P2O
FEBS Journal 277 (2010) 2892–2909 ª 2010 The Authors Journal compilation ª 2010 FEBS 2901
volume, and each subunit had to be closed off prior to
the calculation (see Materials and methods). The vol-
umes should therefore be regarded as approximations
because the delineation of active-site gorge and inter-
nal void is somewhat arbitrary. For the structures
where the complete substrate-recognition loop had
been modelled, the following active-site volumes were
obtained: H167A
2FG
(714 A
˚
3
) > F454N (668 A
˚
3
)>
H450Q (664 A
˚
3
) > triple-Ala (619 A
˚
3
) > Y456W
2FG
(585 A
˚

3
)>WT
ACT
(173 A
˚
3
).
When calculating the volume of the active site for
the unbound state of F454N, we noted an additional
opening formed between Asp
452
, Lys
91
and Thr
169
. The
opening creates a channel that appears to merge with
part of the larger solvent channels leading from the
surface into the internal void. The opening is created
by the different conformation of Asp
452
. An estimated
volume of the active site in F454N (668 A
˚
3
) was
obtained by rotating the Asp
452
backbone by  100°,
which forces the channel to close, although this was

made only to serve as a rough comparison with the
other models. Besides Asp
452
, the loop conformation is
very similar to H167A
2FG
, but the Asn
454
side chain
appears less favourably packed in F454N than the
wild-type Phe
454
.
The H450Q active site is only slightly smaller than
that in H167A
2FG
, where the net decrease is mainly the
result of a series of small adjustments of residues lining
the active site. Again, Asp
452
has a different conforma-
tion, although this can be rationalized by the nearby
H450Q replacement, which causes backbone shifts at
positions 451 and 452. These changes do not appear to
decrease the cavity volume but rather to increase it.
In the triple-Ala mutant, the drastic replacement of
the
454
FSY
456

segment by
454
AAA
456
did not result in
an increased active-site volume in the open state. The
unexpected result can be rationalized such that, despite
the Phe
454
fi Ala mutation increasing the volume, the
movement of Asp
452
and Ala
453
closer to the active site
leads to a net volume decrease. In addition, the Thr
169
side chains rotates 35° about v
1
, and Val
104
undergoes
a rotamer shift to further decrease the volume. In the
fully open state represented in H167A
2FG
, Tyr
456
is
folded away from the active site and, indeed, the
Tyr

456
fi Ala replacement is too far away to affect
the active-site volume. This is probably not true when
the loop is closed but, because none of the structures
shows a closed loop, the effects of mutation on the
closed state are unknown.
The Y456W
2FG
model confirms that Tyr
456
is too
remote to affect the volume in the open state. Again,
the difference in volume between this structure
(585 A
˚
3
) and that of the original open state H167A
2FG
(714 A
˚
3
) is the result of conformational changes at the
Asp
452
backbone, and a small rotation of the Leu
545
side chain in response to minor side-chain adjustments
of the nearby Glu
542
. All of these changes appear

remote and uncoupled from Tyr
456
.
Discussion
Loop dynamics is essential for P2O function
Although the mechanisms by which oxidoreductases
operate are a matter of wide biological interest, special
interest can be assigned to P2O not only as a part of
the fungal lignocellulose-degrading machinery, but also
because of its usefulness in biofuel cells, as well as its
unique potential as a biocatalyst in industrial sugar
biotransformation reactions of bulk carbohydrates to
produce added-value rare sugars, fine chemicals and
drugs. It is well established that active-site loops have
important roles in substrate binding and the specificity
and catalysis of enzymes [27–29], as well as in stabiliz-
ing tertiary and quaternary structure [30–32]. On the
basis of previous crystal-structure data, we have
observed that the loop (residues 450–461) covering the
active site in P2O displays substantial conformational
degeneracy, and proposed that this region is important
for substrate recognition, binding and regioselectivity
[5,19]. We hypothesize that the substrate-recognition
loop samples a temporal ensemble of conformations
during its cycle: in the absence of substrate; during
substrate recognition, substrate binding and product
release; and when toggling between the reductive
and oxidative half-reactions. The most well defined
conformers observed by crystal-structure analysis
have been the extremes of a closed and an open state

as related by a major conformational transition
(Fig. 1) [5,19]. In addition, a number of less occupied
conformers, including transient states along with
highly occupied conformers relevant to the gating
mechanism, are likely to be represented in the confor-
mational ensemble.
One segment of the substrate-recognition loop
undergoes a particularly large rearrangement during
the transition from the closed to the open state
(Fig. 1) [19]. This region, which we refer to as the
gating segment, involves only the tip of the loop
(
454
FSY
456
) but, nonetheless, appears to play a most
prominent role in determining the local environment
and physicochemical characteristics of the active site
[5,19]. The term conformational gating refers in
general to the opening of a loop to allow substrate
entry, followed by closure during binding and cataly-
sis, and subsequent opening to release product. The
Substrate-recognition loop mutations in P2O O. Spadiut et al.
2902 FEBS Journal 277 (2010) 2892–2909 ª 2010 The Authors Journal compilation ª 2010 FEBS
gating mechanism discussed here has a slightly differ-
ent meaning because the enzyme turns over two funda-
mentally different substrates sequentially. The gating
mechanism in P2O enables at least two distinct struc-
tural conformers for reaction with different substrates.
For aldopyranose substrates to be oxidized during the

reductive half-reaction, the loop is most likely required
to be in a more open state. This is evident when con-
sidering the loop in its fully closed state because, here,
the side chains of Phe
454
and Tyr
456
swing into the
active site, restricting access for the monosaccharide
substrates and their corresponding 2-keto products.
The closed loop conformer, as defined by the WT
ACT
complex [5], does not appear feasible during the reduc-
tive half-reaction because it is too restricted to accom-
modate sugar substrates [19], although it is probably
favourable during the oxidative half-reaction with O
2
as electron-acceptor substrate. In the closed state, the
active site is solvent-inaccessible and restricted, and
appears more to be hydrophobic [5,19]. These charac-
teristics likely favour the oxygen reaction [19] and
enable stabilization of a flavin C4a-hydroperoxo
adduct [33] during the oxidative half-reaction.
Electron-donor preference
Site-saturation mutagenesis at positions 452, 454 and
456 did not return variants with improved characteris-
tics with respect to substrate binding and catalytic per-
formance using the preferred substrate (d-glucose) as
an electron-donor substrate and molecular oxygen as
the electron-acceptor substrate. This indicates that

these positions are evolutionary intolerant to mutation,
at least in a context-independent perspective (i.e. when
not considering nearby compensatory substitutions),
and assigns particular functional importance to Asp
452
,
Phe
454
and Tyr
456
of the substrate-recognition loop.
Relative to the wild-type, Y456W shows increased
K
app
m
, but only slightly lower k
app
cat
values for all sub-
strates tested. In the open state of the loop in
H167A
2FG
, where 2FG is bound for oxidation at C3,
the aromatic side chain of Tyr
456
is some 13 A
˚
away
from the sugar [19]. In Y456W, the loop assumes the
same conformation as in H167A

2FG
(Fig. 3A), result-
ing in a minimum edge-to-edge distance of 14 A
˚
between the 2FG glucosyl and Trp
456
indole rings. On
the basis of the position of the side chain and its
remote location from the ligand in the Y456W
2FG
structure, the Trp
456
side chain would be unable to
form any interaction with either the mono- or disac-
charides in the open C3 oxidation state. Whether this
is true also for the open C2 oxidation state is not pos-
sible to assess in the absence of a structure represent-
ing this mode. However, considering the inherent
plasticity and conformational degeneracy of the P2O
substrate-recognition loop, additional conformers
should be possible, especially in light of the new and
unexpected loop conformation of F454N
2FG
(Fig. 4).
In previous modelling of d-glucose in position for oxi-
dation at C2, we speculated that the open state of the
loop, as represented in the C3 oxidation mode, would
be fully compatible also with the preferred binding
mode (C2 oxidation), and even support a mechanism
of regioselectivity through the side chains of Asp

452
and Arg
472
[19].
Substituting the aromatic side chain of Phe
454
by
Pro, Asn or Ala was expected to create a more spa-
cious active site or to facilitate access to the isoalloxa-
zine ring, allowing larger substrates to enter the active
site and bind in a productive way. Interestingly, how-
ever, the calculated active-site volume of F454N is
somewhat smaller than that of H167A. Furthermore,
the aromatic side chain of Phe
454
is crucial for the
gating mechanism in the closed state and efficient
overall turnover. Replacement of Phe
454
consistently
gives rise to variants with dramatically decreased k
app
cat
values for the substrate pair sugar ⁄ O
2
. The observa-
tion that Asn
454
is approaching the ligand without
offering specific side-chain interactions (Fig. 4) is

interesting, and also adds to the ensemble of possible
P2O substrate-recognition loop conformers observed
to date. Although most of the loop is disordered
(455–461) in this structure, we may conclude that the
new conformation represents an intermediary state
between the extremes of the closed and open loop
conformers seen in the WT
ACT
and H167A
2FG
com-
plexes, respectively.
In the related enzyme cholesterol oxidase, Sampson
et al. [34] deleted residues at the tip of the active-site
loop, yielding a correctly folded and active enzyme.
The rationale for the retained catalytic competence of
this mutant was that, although truncation had affected
the binding of substrate negatively, active-site accessi-
bility had increased, thus facilitating binding and dis-
sociation of substrate and product. Although this
explanation is probably valid for the cholesterol oxi-
dase deletion mutant, a similar correlation cannot be
conclusively shown for P2O. As discussed above, the
FSY segment undergoes a large conformational rear-
rangement during the transition from the open to the
closed state, providing a conformational gating mecha-
nism in which Phe
454
⁄ Tyr
456

appear to play key roles.
On the basis of the large changes in position and local
environment of Phe
454
and Tyr
456
in the two states, we
expect each of these residues to have distinct roles and
importance in the two loop states, and conceivably
O. Spadiut et al. Substrate-recognition loop mutations in P2O
FEBS Journal 277 (2010) 2892–2909 ª 2010 The Authors Journal compilation ª 2010 FEBS 2903
also in the two half-reactions, as well as in the sub-
strate selectivity mechanism. In the case of the alanine
and D454–456 variants, and most other mutants exam-
ined, we observe both increased K
app
m
and decreased
k
app
cat
values for d-glucose (and d-galactose) with O
2
as
acceptor substrate, indicating the significance of these
positions for substrate binding and catalysis.
By contrast to what was expected, the triple-Ala
mutant shows a decrease in active-site volume of the
open state. To fully assess the structural impact of the
mutations on active-site volume, however, we would

also need to obtain the mutant structure of the closed
state. It is possible that the volume decreases in the
open state, whereas it increases in the closed state.
Although the K
app
m
values for Glc and Gal are both
approximately two-fold higher for the triple-Ala
mutant compared to the wild-type, the k
app
cat
with
Glc ⁄ O
2
is decreased 165-fold for Glc and only two-fold
for Gal, which gives a greatly reduced preference for
Glc (nine-fold over Gal) compared to the wild-type.
Hence, the reason for the altered preference is proba-
bly not to be found in the binding, but rather is the
result of a differential destabilization of the transition
state for Glc in the variant. An already poor transi-
tion-state stabilization of Gal in the wild-type may
explain why the turnover of this substrate is not as
sensitive to the mutations. Despite being a crippled
enzyme, the triple-Ala variant displays behaviour that
is in the desired direction with respect to the simulta-
neous conversion of glucose and galactose with O
2
as
acceptor. However, our results also highlight the inher-

ent difficulty of structure-based rationalization of
mutational effects when only one of several active con-
formers of a thermodynamically accessible ensemble
can be analyzed.
The loop in P2O is somewhat different from most
other active-site loops because the majority of these
loops close to perform their function (i.e. to sequester
a reactive intermediate and restrict the access of sol-
vent) [34,35]. The loop in P2O has to open to allow
access of its electron-donor substrate and has to stay
open when the sugar substrate is bound during turn-
over, or at least more open than the closed conforma-
tion observed in WT
ACT
. This is reminiscent of
cholesterol oxidase where the open state of the loop
represents the ligand-bound form. Cholesterol oxidase
is active on lipophilic substrates, and the open form of
the loop interacts with the hydrophobic part of choles-
terol and is thus responsible for substrate specificity
[34]. Similarly, the loop of P2O in its open state, and
some of the residues in the gating segment appear to
play an essential role in sugar substrate recognition
and binding. This is evident when comparing the
sugar-substrate selectivity for the wild-type and the
variants studied. Wild-type P2O shows a clear catalytic
preference for d-glucose over d-galactose or melibiose,
whereas most of the variants considered in the present
study show a decreased selectivity ratio (i.e. the ratio
of the specificity constants k

app
cat
⁄ K
app
m
) and hence a
reduced discrimination between these substrates. Fur-
thermore, all of the variants showed reduced K
app
m
val-
ues for the disaccharide melibiose, indicating that
access to the active site might have been improved by
removal of bulkier side chains in the substrate-recogni-
tion loop (e.g. most pronounced in F454P,
F454A ⁄ S455A ⁄ Y456A), even though the total active-
site volume of these variants in the open-loop state
decreased compared to the 2FG-bound form of
H167A. The homotetramer P2O shows a peculiarly
restricted access to its four identical active sites
because these can only be accessed through a large
central cavity, which, in turn, is connected with the
surrounding solvent through narrow channels [5]. The
active-site loop and some of its bulkier hydrophobic
residues in the gating segment might contribute to this
restricted access. Such strict control of access ⁄ activity
could be important for the function of the enzyme
because highly reactive hydrogen peroxide is one of
the primary reaction products of P2O, and its uncon-
trolled formation in the fungal periplasm (i.e. the pro-

posed location of P2O) or in the direct vicinity of the
fungal cell through oxidation of various sugars, and
even oligosaccharides, might be detrimental for the
organism.
Electron-acceptor preference
Although it is well established that P2O can use molec-
ular oxygen during the oxidative half-reaction, the
identity of the preferred, biologically relevant electron-
acceptor substrate is ambiguous. Besides oxygen,
quinone compounds are physiologically and function-
ally suitable electron acceptors because they constitute
natural breakdown products of ligninolysis by wood-
degrading basidiomycetes [8] and, as a result of benzo-
quinone reduction comprising an important part of
extracellular quinone redox cycling in these organisms,
this could be a biologically important reaction. In
addition, various complexed metal ions play a role in
lignin degradation, and could be electron-acceptor
substrates for P2O during lignocellulose breakdown.
The assignment of a dual function to P2O during
the oxidative events of lignin degradation (i.e. as an
H
2
O
2
-generating and a quinone-reducing enzyme) also
suggests a possible function for the large internal
cavity of the homotetrameric assembly [5]. With the
Substrate-recognition loop mutations in P2O O. Spadiut et al.
2904 FEBS Journal 277 (2010) 2892–2909 ª 2010 The Authors Journal compilation ª 2010 FEBS

electron donor-acceptor substrate pair Glc ⁄ BQ, the
wild-type shows a five-fold increase in overall k
app
cat
compared to the Glc ⁄ O
2
pair, and a very low K
app
m
of
0.14 mm for BQ, whereas K
app
m
for O
2
was reported
to be 0.74 mm [5]. Although the artificial substrate
ferrocenium Fc
+
is certainly not a biologically relevant
substrate of P2O, it can serve as a model compound
for complexed metal ions. Again, the substrate pair
Glc ⁄ Fc
+
shows an overall higher k
app
cat
than the Glc ⁄ O
2
pair, and Fc

+
also shows a very low K
app
m
value,
despite being a very bulky substrate (Scheme 1). These
observations corroborate the hypothesis that quinone
compounds can be natural and physiologically relevant
electron acceptors for P2O, at least when functioning
as quinone-reducing enzyme.
Most of the mutations in the gating segment did not
affect considerably the catalytic efficiency k
app
cat
⁄ K
app
m
obtained for BQ (as well as for Fc
+
) and, indeed,
some of these values for selected variants were slightly
increased. This is in sharp contrast to the catalytic effi-
ciency determined for the variants for Glc and Gal
when oxygen was the saturating electron acceptor.
When determining apparent steady-state kinetic con-
stants in these two-substrate reactions, the results
reflect the overall reaction and hence both half-reac-
tions, and it is not possible to determine which of the
half-reactions is primarily affected by the mutation. In
a recent study on the kinetic mechanism of P2O by a

combination of pre-steady-state and steady-state kinet-
ics, it was shown that the overall turnover of the reac-
tion between sugar and oxygen is limited by the steps
of flavin reduction and decay of the C4a-hydroperoxy-
FAD, which is formed as a transient intermediate in
the oxidative half-reaction of P2O [12]. It has also
been proposed that movement of the active-site loop in
the presence of oxygen facilitates the release of the
oxidized sugar product in this reaction. P2O was the
first flavoprotein oxidase for which the C4a hydroper-
oxyflavin intermediate was observed under natural
turnover conditions. It was suggested that the struc-
tural features possibly facilitating the formation and
stabilization of this intermediate include an elongated,
mainly hydrophobic cavity, which is formed at the re
side of the isoalloxazine ring upon closure of the sub-
strate loop, and which is large enough to accommo-
date a peroxide group at the C4a position [33]. When
considering the importance of the active-site loop for
this reaction, it is feasible that mutations in the gating
segment are affecting mainly the oxidative half-reac-
tion when oxygen is the electron acceptor. However, to
obtain further insight regarding this, more detailed
studies are necessary, including pre-steady-state kinet-
ics of the two half reactions.
We do not propose that O
2
is a non-natural electron
acceptor, but rather that the enzyme has dual electron-
acceptor preference (i.e. that of an oxidase and of a

quinone reductase), depending on the metabolic state
of the fungus. The quinone compounds to which P2O
would be exposed (typically BQ and substituted BQ)
are similar in size to the aldopyranose substrates.
Therefore, we do not expect the loop to be in the
closed state during the oxidative half-reaction with qui-
nones as acceptors. The closed state is probably mainly
relevant for the oxygen reaction and flavin C4a hydro-
peroxide intermediate stabilization. To enable oxygen
reactivity of the reduced flavin (singlet state) activation
of O
2
is required to overcome the forbidden spin bar-
rier [36]. The conversion of triplet-state oxygen to the
singlet state requires a precisely controlled physico-
chemical and structural environment, which can indeed
be offered when the loop is in the fully closed confor-
mation. Quinone reduction, however, is expected to be
a more facile process involving the transfer of two
electrons from the two-electron donor (reduced flavin)
to the two-electron acceptor (1,4-benzoquinone). An
open loop conformation and the less strict require-
ments of a direct two-electron transfer reaction may
explain why the substrate-recognition loop appears
insensitive to mutagensis with quinones as electron
acceptors, but highly sensitive with O
2
. This sequence-
independence would also support the inherent promis-
cuity in the quinone specificity of P2O, which would

enable P2O to reduce a heterogeneous pool of quinone
substrates generated during ligninolysis. We do not
claim that the improved binding and higher turnover
rates of BQ comprise absolute proof that this is a bio-
logically more relevant acceptor than oxygen; rather,
these findings suggest that BQ is a physiologically rele-
vant substrate, and our results indeed support a role
of P2O as a quinone reductase. The alternative elec-
tron acceptor Fc
+
is an unnatural, nonphysiological
electron acceptor of P2O but, nonetheless, performs
better than both BQ and O
2
(Table 4). Although not
being relevant for the natural function of P2O, the
improved kinetics of wild-type and mutants with Fc
+
as electron acceptor serves to highlight the power of
mutagenesis in conjunction with electron-acceptor
screening to optimize conversion rates of carbohydrate
substrates for industrial applications of P2O.
Stability
Analysis of the correlation between heat inactivation
half-life (Table 5) and the resistance to thermal unfold-
ing (Table 6) shows that half-life and melting tempera-
ture are correlated for variants H450Q, F454P,
O. Spadiut et al. Substrate-recognition loop mutations in P2O
FEBS Journal 277 (2010) 2892–2909 ª 2010 The Authors Journal compilation ª 2010 FEBS 2905
Y456W and F454A⁄ Y456A. For H450Q and F454P,

reduced s
1 ⁄ 2
is accompanied by a decrease in T
m
; and,
for Y456W and F454A ⁄ Y456A, an increase in s
1 ⁄ 2
is
associated with higher T
m
. Most notably, Y456W
shows a 34-fold increase in half-life, and an increase in
T
m
of 5.5 °C. However, for F454N and the triple-Ala
mutant, large improvements in half-life at 60 °C are
observed (29- and 23-fold increase, respectively) with
only minor effects on the melting temperature (T
m
val-
ues reduced by 1 °C). On the basis of our earlier struc-
tures of the closed and open states of P2O [5,19], it is
noted that the residues in the beginning of the sub-
strate-recognition loop (450–452) provide important
interactions. His
450
modulates the electrostatic environ-
ment and is important for the correct spatial position-
ing of Asp
452

and Arg
451
. In addition, Arg
451
helps to
stabilize selected conformers of the substrate-recogni-
tion loop and Asp
452
was proposed to be directly
involved in substrate recognition and regioselectivity
[19]. H450Q shows the largest decrease in T
m
(3 °C) of
the mutants. Most likely, this is partly the result of
backbone displacements and conformational changes
occurring at positions 451 and 452 in direct response
to the His
450
fi Gln replacement (Fig. 5), which
impose a different spatial relationship between these
residues and the surrounding active-site residues. Vol-
ume calculations of the active sites when the substrate-
recognition loop is in the open state also show that
conformational perturbations leading to changes in the
volume of the active sites tend to be manifested mainly
in the 452 backbone region.
Conclusions
Following site-saturation mutagenesis of the positions
His
450

, Asp
452
, Phe
454
and Tyr
456
in the substrate-
recognition loop of P2O, where Phe
454
and Tyr
456
con-
stitute the gating segment, a high fraction of inactive
enzyme variants was obtained, indicating that this
region is highly sensitive to amino acid replacements,
as well as being functionally important for P2O. As
judged from apparent steady-state kinetics and struc-
tural data, this loop, and specifically the gating seg-
ment, appears to play a prominent role in the binding
and selectivity of sugar substrates. The loop contrib-
utes to the strong discrimination against various sugars
in favour of the preferred electron donor, d-glucose,
and also appears to be important for the oxidative
half-reaction when oxygen is the electron acceptor.
However, before any unequivocal conclusions can be
made, more detailed studies concerning the positions
of the gating segment identified in the present study
are required.
Materials and methods
Site-saturation, alanine-scanning and deletion

mutagenesis
The pET21d
+
⁄ p2o vector (pHL2) expressing a construct of
the T. multicolor p2o gene (GenBank AY291124) fused to a
C-terminal His
6
tag, controlled by the T7 promoter has
been described previously [19]. The plasmid pHL2 was used
as template for all mutagenic PCRs. Alanine mutants were
prepared by the sequence overlap extension method [37]
using the primers F454A⁄ Y456A
fwd
, F454A⁄ S455A ⁄
Y456A
fwd
and F454A ⁄ S455A ⁄ Y456A
rev
, and the flanking
primers T7
fwd
and T7
rev
. Site-directed mutants were pre-
pared by a two-step mutagenesis approach using PCR and
digestion with DpnI [38]. A deletion mutant (D454–456) was
generated by deleting three amino acids from the substrate-
recognition loop (i.e. amino acids Phe
454
, Ser

455
and Tyr
456
,
which constitute the gating segment). The amino acid posi-
tions 452, 454 and 456 were also targeted by site-saturation
mutagenesis. The procedure has been as described recently
for site-saturation mutagenesis and screening of P2O vari-
ants targeting His
450
[23]. P2O variants were screened in a
96-well plate-screening assay using d-glucose and d-galac-
tose as substrates [23,25]. The nucleotide sequences of the
synthetic oligonucleotides are provided in Table S1. A ran-
dom set of colonies was picked for variants showing high,
moderate or low level of catalytic activity with either of the
two sugar substrates (ten colonies each), and their genes
were sequenced to confirm mutagenesis. Variants showing
interesting characteristics with respect to substrate specific-
ity or catalytic stability (e.g. F454P, F454N and Y456W)
were selected for further analysis. The subsequent prepara-
tive-scale production of recombinant enzymes was per-
formed in Escherichia coli strain BL21 Star (DE3), and
purified using Ni
2+
-immobilized metal affinity chromatog-
raphy as described previously [23,25].
Activity assays, steady-state kinetics and heat
inactivation experiments
Enzyme activity was assayed at 30°C using the 2,2¢-azin-

obis(3-ethylbenz-thiazolinesulfonic acid) (ABTS) method
[39], as described previously [23,25]. Kinetic constants
were determined for the substrates d-glucose (0.1–50 mm),
d-galactose (0.1–200 mm) and melibiose (5.0–500 mm) using
the ABTS assay under air saturation, and the kinetic param-
eters calculated using the Henri–Michaelis–Menten equa-
tion. Additionally, the catalytic constants were measured for
the two-electron, proton acceptor, 1,4-benzoquinone and
the one-electron, nonproton, acceptor ferrocenium (ferroce-
nium hexafluorophosphate) at a saturating concentration of
100 mm of either d -glucose or d-galactose [25]. Heat inacti-
vation half-life of the T. multicolor P2O variants was
determined in 50 mm KH
2
PO
4
buffer (pH 6.5) at 60 °C and
Substrate-recognition loop mutations in P2O O. Spadiut et al.
2906 FEBS Journal 277 (2010) 2892–2909 ª 2010 The Authors Journal compilation ª 2010 FEBS
70 °C in accordance with a previously described protocol
[23]. At various time points, residual enzyme activity was
measured (standard ABTS assay with 20 mmd-glucose) in a
thermal cycler. The inactivation constant k
in
was calculated
by linear regression of ln(residual activity) versus time, and
the half-life of heat inactivation (s
1 ⁄ 2
) as defined by
s

1 ⁄ 2
=ln2⁄ k
in
[40]. The pH optima of the enzyme variants
were determined by measuring the activity at 30 °C using
the standard ABTS assay at different pH values [50 mm
citrate buffer (pH 2.5–6.0), 50 mm phosphate buffer (pH
6.0–8.0), 50 mm borate buffer (pH 8.0–10.0). FAD absorp-
tion spectra of all enzymes were recorded using a DU 7400
spectrophotometer (Beckmann Instruments, Vienna, Aus-
tria) with diode-array detection at room temperature.
Enzymes were reduced using 10 mmd-glucose or 5 mm
sodium dithionite in the absence of oxygen, and recording
of the spectra of the reduced states was repeated. To investi-
gate the effect of the mutations on the flavinylation state of
the P2O variants, trichloroacetic acid was added to enzyme
solutions to a final concentration of 5% (v ⁄ v) and the
enzymes denatured at 100 °C (10 min). Denatured protein
was partioned from the supernatant by centrifugation, and
the flavin absorption spectra of the supernatants were
recorded.
ThermoFAD analysis
The Thermofluor
Ò
-based ThermoFAD method [41] was used
to monitor protein unfolding for analysis of thermal stabil-
ity of wild-type and mutants. This method takes advantage
of the intrinsic fluorescence of the FAD cofactor, and does
not depend on fluorescent dyes. All enzymes were diluted in
KH

2
PO
4
-buffer (50 mm, pH 6.5) to a final concentration of
5mgÆmL
)1
and subsequently analyzed in six wells, each in
a thin-walled 96-well PCR plate in 50 lL aliquots per well.
The real-time PCR machine used was an i-Cycler (Bio-Rad,
Hercules, CA, USA) providing a MyiQ Optics Module, and
SYBR-Green filters (523–543 nm) were used to record the
signals. The samples were heated from 30 to 95 °C with
0.5 °C intervals for 20 s. After each step, the fluorescence
signal was measured.
Crystallization, data collection and X-ray
crystallographic refinement
Crystals of the mutant P2O variants were produced using
the hanging drop vapour diffusion method where the drops
were prepared by equal volumes of 20 mgÆmL
)1
protein and
reservoir [10% (w ⁄ v) monomethyl ether poly(ethlene glycol)
(M
r
= 2000), 0.1 m Mes (pH 5.2), 50 m m MgCl
2
]. Prior
to data collection, the crystals were stabilized using their
respective reservoir solution where the poly(ethlene glycol)
concentration had been increased to 28% (stabilizing

solution), followed by vitrification in liquid nitrogen. For
the ligand complexes, crystals were immersed for < 1 min
in a drop of stabilizing solution containing a grain of solid
ligand before being vitrified. All data sets were collected
using synchrotron radiation at MAX-lab (Lund, Sweden),
and processed using xds [42]. Phases were obtained either by
means of molecular replacement (phaser) [43] or Fourier
synthesis (fft, ccp4) [44] using the refined model of P2O
variant H167A as the starting model (PDB code 2IGO) [19].
Crystallographic refinement was performed with refmac5
[45], including anisotropic scaling, calculated hydrogen scat-
tering from riding hydrogen atoms and atomic displacement
parameter refinement using the translation, libration, screw-
rotation (TLS) model. The TLS models, comprising five to
eight TLS groups, were determined using the TLS Motion
Determination server (tlsmd) [46]. Corrections of the mod-
els were performed manually based on r
A
-weighted 2F
o
) F
c
and F
o
) F
c
electron density maps. The R
free
reflection sets
were kept throughout refinement. All model building was

performed with the software o [47] and coot [48]. Crystal
parameters and data collection details are given in Table 7.
All figures showing protein models were prepared with
pymol [49]. For calculation of the active-site volume, a
single tetramer consisting of protein and cofactor atoms was
generated for all models. In addition to these atoms, a plane
of atoms consisting of 150 carbon atoms (10 · 15 atoms,
1A
˚
atomic distance) was added for each subunit to allow
the active-site volume to be separated from the volume of
the large internal void buried in the homotetramer. The
planes were then placed to close off each active-site gorge
where it opens to the central void. The cavity calculation
was performed with voidoo [50] using a probe radius of
1.4 A
˚
and the ‘probe-occupied’ cavity type calculation on a
fine grid (0.24 A
˚
), which calculates the cavity occupied by a
rolling probe.
Acknowledgements
This work has received financial support from the
Austrian Science Fund (FWF, Translational Project
L213-B11) (to O.S. and D.H.); the Swedish Research
Council VR (2005-4721, 2008-4056) (to C.D. and
T.C.T.); and the Carl Tryggers Foundation (CTS07:79,
CTS08:78). We thank the beamline staff scientists at
MAX-lab (Lund, Sweden) for their support during

data collection.
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Supporting information
The following supplementary material is available:
Table S1. Nucleotide sequences of synthetic oligonucle-
otides used for the P2O variants.
This supplementary material can be found in the
online version of this article.
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O. Spadiut et al. Substrate-recognition loop mutations in P2O
FEBS Journal 277 (2010) 2892–2909 ª 2010 The Authors Journal compilation ª 2010 FEBS 2909