Báo cáo khoa học: Tryptophan 243 affects interprotein contacts, cofactor binding and stability in D-amino acid oxidase from Rhodotorula gracilis ppt

Tryptophan 243 affects interprotein contacts, cofactor
binding and stability in
D-amino acid oxidase from
Rhodotorula gracilis
Laura Caldinelli, Gianluca Molla, Mirella S. Pilone and Loredano Pollegioni
Department of Biotechnology and Molecular Sciences, University of Insubria, Varese, Italy
Oligomeric proteins are the rule rather than the excep-
tion. As recently reviewed by Mei et al. [1], a surpris-
ingly high number of proteins are made up of two
subunits and, in most of these ( 80%), the two sub-
units are identical. Recently, we investigated the role
of subunit interaction in homodimeric proteins by
using the enzyme d-amino acid oxidase (DAAO) (EC
1.4.3.3), from yeast, as a model tool. DAAO contains,
as a coenzyme, a noncovalently bound molecule of
FAD per 40 kDa protein monomer. DAAO is a com-
ponent of the glutathione reductase family, the FAD-
containing protein family that has been studied in
greatest detail. The protein members of this populous
class catalyze diverse reactions and adopt the Ross-
mann fold [2]. DAAO belongs to the second sub-
family, GR
2
, whose members only align well at their
N terminus ( 30 residues) [3].
Two major structural features have been proposed to
be responsible for the ‘head-to-tail’ mode of dimeriza-
tion of yeast DAAO (Fig. 1A–C), namely (a) electro-
static interactions between the positively charged
residues of the long loop (from Pro302 to Glu322), con-
necting b-strands F5 and F6 and the negatively charged

residues belonging to the a-helices I3¢ and I3¢¢ of the
other monomer and (b) the interactions in the core of
the dimer (where Trp243 is located, see below) [4–6].
The importance of the bF5–bF6 loop for the mono-
mer–monomer interaction has been validated by site-
directed mutagenesis, when a stable monomeric
holoenzyme was obtained by removing part of this loop
Keywords
cofactor binding; flavoprotein;
oligomerization state; rational design;
structural stability
Correspondence
L. Pollegioni, Dipartimento di Biotecnologie
e Scienze Molecolari, Universita
`
degli Studi
dell’Insubria, Via J. H. Dunant, 3–21100
Varese, Italy
Fax: +39 0332 421500
Tel: +39 0332 421506
E-mail:
(Received 10 October 2005, revised 18
November 2005, accepted 2 December
2005)
doi:10.1111/j.1742-4658.2005.05083.x
The flavoenzyme d-amino acid oxidase from Rhodotorula gracilis is a
homodimeric protein whose dimeric state has been proposed to occur as a
result of (a) the electrostatic interactions between positively charged resi-
dues of the bF5–bF6 loop of one monomer and negatively charged residues
belonging to the a-helices I3¢ and I3¢¢ of the other monomer, and (b) the

interaction of residues (e.g. Trp243) belonging to the two monomers at the
mixed interface region. The role of Trp243 was investigated by substituting
it with either tyrosine or isoleucine: both substitutions were nondisruptive,
as confirmed by the absence of significant changes in catalytic activity, but
altered the tertiary structure (yielding a looser conformation) and decreased
the stability towards temperature and denaturants. The change in
conformation interferes both with the interaction of the coenzyme to the
apoprotein moiety (although the kinetics of the apoprotein–FAD complex
reconstitution process are similar between wild-type and mutant d-amino
acid oxidases) and with the interaction between monomers. Our results
indicate that, in the folded holoenzyme, Trp243 is situated at a position
optimal for increasing the interactions between monomers by maximizing
van der Waals interactions and by efficiently excluding solvent.
Abbreviations
ANS, 1,8-anilinonaphtalene sulfonic acid; C
m
, midpoint concentration of urea required for unfolding; DAAO, D-amino acid oxidase; T
m
,
melting temperature.
504 FEBS Journal 273 (2006) 504–512 ª 2006 The Authors Journal compilation ª 2006 FEBS
[7,8]. Thermodynamic studies performed on monomeric
and dimeric DAAO forms showed that the shift to such
a monomeric form resulted in an enzyme with increased
sensitivity to thermal denaturation [6].
The importance of Trp243 in the monomer–mono-
mer interaction was envisaged by comparing the near-
UV CD spectra and the tryptophan fluorescence
between dimeric wild-type and monomeric DLOOP
proteins [6]: the observed differences may be indicative

of changes in the environment of Trp243 following
deletion of the bF5–bF6 loop and conversion to the
monomeric state because no other tryptophan residues
are buried on the surface of each monomer of the
DAAO dimer (Fig. 1B,C). Although the atoms of the
Trp243 side chain in each subunit are too remote to
allow a direct and strict contact between the two
residues at the dimer interface (the distance between
the a-carbons is  3.7 A
˚
and between the indole rings
is ‡ 7.7 A
˚
; see Fig. 1C), they account for a significant
region of the dimerization surface.
In order to gain further insight into the role of the
structural determinants of this prototypical flavopro-
tein, we investigated, by site-directed mutagenesis, the
influence of Trp243 on the functionality, the oligo-
meric state and the FAD binding of DAAO.
Results
Spectral properties of Trp243 DAAO mutants
Upon purification, the DAAO mutant, W243Y, was
found to retain the FAD coenzyme, as confirmed by
Fig. 1. Structural features of Rhodotorula gracilis D-amino acid oxidase (DAAO) (PDB code: 1c0p). (A) ‘Head-to-tail’ mode of monomer–mono-
mer interaction: the two subunits are presented in different colors (a, green; b, blue). The bF5–bF6 loop is depicted in red, the flavin cofactor
is in yellow, and the substrate
D-alanine is in purple. (B) Details of the residues involved in the monomer–monomer interaction at the ‘mixed
interface’ (blue, positively charged residues; red, negatively charged residues; white, apolar residues). For the sake of clarity, the two sub-
units have been separated. (C) Positioning of Trp243 (yellow) in the buried region at the dimer interface (no other tryptophan residues are

located in the buried surface between monomers). Amino acids belonging to the A-chain are labeled in black, while residues belonging to
the B-chain are labeled in blue. (D) Details of the structure depicting the interactions of strand F5 (blue), which follows the loop containing
Arg289 (the site of trypsinolysis in the unfolding intermediate) with the bab elements of the Rossmann fold (green), the C-terminal a-helix
containing the SKL sequence for peroxisomal targeting (red), and the region containing Trp243 (the loop following the bI7–aI3, dark blue).
L. Caldinelli et al. Structural determinants for dimerization of DAAO
FEBS Journal 273 (2006) 504–512 ª 2006 The Authors Journal compilation ª 2006 FEBS 505
the absorption spectrum in the visible region (an
E
274 nm
⁄ E
455 nm
ratio of  9.6 and an extinction coef-
ficient, at 455 nm, of 12 800 m
)1
Æcm
)1
was deter-
mined). In contrast, the W243I mutant was found to
exhibit a remarkably higher E
274nm
⁄ E
455nm
ratio (ran-
ging from 16 to 25, depending on the enzyme pre-
paration), indicating a significantly lower content of
flavin cofactor. As both mutants showed a purity
of ‡ 95% on SDS ⁄ PAGE, the latter result indicates
that the W243I mutant is purified as a mixture of
holo- and apoprotein. This conclusion is further
supported by the observation that a significantly

higher enzymatic activity is measured for the W243I
mutant after adding exogenous FAD to the assay
mixture.
The apoprotein forms of W243I and W243Y
DAAOs were obtained by dialysis, where a high yield
was obtained (‡ 60% of protein recovery). The binding
constant for the coenzyme was determined by titrating
the apoprotein with increasing amounts of FAD and
following the decrease in protein fluorescence: a K
d
value of 0.37 and 0.035 lm of the apoprotein–FAD
complex was obtained for the W243I and W243Y
mutants, respectively. The value determined for W243I
is 20-fold higher than obtained for the wild-type
RgDAAO protein [9] and fourfold higher than those
values determined for the monomeric DLOOP DAAO
mutant (Table 1) [7,8].
Far-UV CD spectra of the wild-type protein, and
of DLOOP and Trp243 DAAO mutants, did not
reveal any major change in the features related to
the secondary structure of the two mutants. In con-
trast, and as shown in Fig. 2, the near-UV CD spec-
tra of the different DAAO forms under investigation
are different. These alterations may be ascribed to a
different contribution from aromatic amino acid resi-
dues, which are responsible for most transitions in
the near-UV spectral region, in particular in terms
of altered mutual relationships between nearby struc-
tural elements. Alterations in protein conformation
are also made evident by changes in the fluorescence

of the indole moiety of tryptophan [10]: the fluores-
cence emission at 345 nm (following excitation at
280 nm) was higher for both W243Y and W243I
mutants than for dimeric wild-type and monomeric
DLOOP DAAOs (Table 1).
A sensitive marker of the folding state of a flavo-
enzyme is represented by the fluorescence of the FAD
cofactor, which is much lower in the holoenzyme-
bound form of DAAO with respect to free FAD [9].
Table 1. Comparison of dissociation constants determined for FAD, apparent kinetic parameters and fluorescence spectral properties for
wild-type and mutants of
D-amino acid oxidase (DAAO). a.u., Arbitrary units determined under the same experimental conditions, at a protein
concentration of 0.1 mgÆmL
)1
.
Enzyme form
K
FAD
d
(lM)
D-Alanine Fluorescence properties
V
max
(lmol O
2
Æmin
)1
Æmg
)1
protein)

K
m
(mM)
Protein
a.u. (nm)
Flavin
a.u.
Wild type 0.02 ± 0.01
a
110 ± 8
b
0.8 ± 0.2
b
135 (334) 12.7
(apoprotein) 940 (342)
DLOOP
c
0.12 ± 0.01 86 ± 2 1.5 ± 0.2 230 (338) 18.9
(apoprotein) 900 (342)
W243I 0.37 ± 0.03 95 ± 2
d
1.6 ± 0.1
d
340 (338) 9.4
(apoprotein) 465 (340)
W243Y 0.04 ± 0.01 108 ± 2 1.4 ± 0.1 370 (337) 12.3
(apoprotein) 715 (341)
a
As described previously [9].
b

As described previously [15].
c
As described previously [7].
d
These values were determined in the presence
of a large excess of free FAD (0.2 m
M) in the activity assay mixture.
Fig. 2. Near-UV CD spectra of wild-type (—), DLOOP (– - –), W243I
(- - - -), and W243Y (– – –)
D-amino acid oxidases (DAAOs). The pro-
tein concentration used was 0.4 mgÆmL
)1
in 50 mM potassium
phosphate, pH 7.5, containing 10% (v ⁄ v) glycerol and 2 m
M EDTA.
Structural determinants for dimerization of DAAO L. Caldinelli et al.
506 FEBS Journal 273 (2006) 504–512 ª 2006 The Authors Journal compilation ª 2006 FEBS
The emission at 526 nm, following excitation at
450 nm, was higher for the DLOOP mutant than for
wild-type DAAO (  19 versus 13 arbitrary units) [6]
and was similar among W243Y and wild-type DAAO
protein (Table 1). On the other hand, the lower value
determined for the purified W243I is caused by the
presence of a large amount of apoprotein in the puri-
fied sample.
In order to investigate the exposure of clusters of
buried hydrophobic residues, we studied the binding
of the fluorescent probe, 1,8-anilinonaphtalene sulfonic
acid (ANS), to hydrophobic side chains in proteins:
this binding results in a marked increase in ANS fluor-

escence yield for all DAAO forms, accompanied by
a blue-shift in the emission fluorescence spectra. An
empiric parameter (such as the ratio between the
change in fluorescence emission at 500 nm at satur-
ating ANS concentration) and the K
d
for ANS binding
were estimated [11]: values of 0.84, 0.78, and 0.21 arbi-
trary unitsÆlm
)1
for wild-type, W243Y and W243I
DAAOs were obtained, whereas the K
d
for ANS was
practically unchanged (140 ± 20 lm for wild-type and
Trp243 mutants). The low intensity of ANS fluores-
cence observed with the W243I mutant was caused by
the presence of 0.1 mm free FAD in the assay mixture,
which was added to avoid complications as a result of
apoprotein formation. Therefore, it is not possible to
compare the exposure of ANS-accessible hydrophobic
regions between the W243I mutant and the other
DAAO forms.
In the apoprotein form of all DAAO forms under
investigation, the protein conformation is looser and
the tryptophan side chains are transferred to a more
polar environment because when the cofactor is
removed, tryptophan emission is significantly higher
and a shift in the maximum emission is observed
(Table 1). The higher fluorescence intensity of the

apoprotein forms of wild-type and DLOOP DAAOs
compared with those of the W243 mutants probably
reflects the contribution of tryptophan exposure at
position 243 (see Table 1). Furthermore, similar K
d
values were determined for the ANS titration of
apoprotein forms of wild-type, W243Y and W243I
DAAOs (ranging from 24 to 31 lm): these values
are fivefold lower than determined for the corres-
ponding holoenzymes, and this change is paralleled
by a remarkably higher fluorescence emission at sat-
uration. The significant increase observed in the
empiric DF ⁄ K
d
ratio (7.2, 7.8 and 7.3 arbitrary unitsÆ
lm
)1
for W243Y, W243I and wild-type DAAO,
respectively) points to a greater exposure of hydro-
phobic regions in the apoprotein forms than in the
holoenzymes.
Kinetic properties of Trp243 DAAO mutants
The apparent steady-state kinetic parameters were
determined using d-alanine (the most frequently used
DAAO substrate) at a fixed (21%) oxygen concentra-
tion. In order to avoid problems arising from apopro-
tein formation, measurements with the W243I mutant
were performed in the presence of 0.2 mm FAD. With
both Trp243 mutant DAAOs, the apparent kinetic
parameters were modified only slightly: the most signi-

ficant change was an increase of approximately two-
fold in K
m
for d-alanine (Table 1).
Oligomerization state of Trp243 DAAO mutants
The oligomerization state of the Trp243 mutants was
investigated by gel-filtration chromatography. The
wild-type and W243Y enzymes behaved very similarly
during gel-filtration chromatography at 1 mgÆmL
)1
protein concentration. However, the elution volume
for the mutant enzyme is related to protein concentra-
tion: at 0.1 mgÆmL
)1
, the wild-type DAAO retained its
elution behavior, but the W243Y mutant eluted at
a higher elution volume (from a K
av
of 0.40 at
1mgÆmL
)1
to a K
av
of 0.45 at 0.1 mgÆmL
)1
). On the
other hand, the W243I mutant was found to exhibit an
elution volume corresponding to a monomeric form
of DAAO at all protein concentrations tested (0.2–
16 mgÆmL

)1
).
We previously reported that yeast DAAO is a stable
dimer and that it converts to a monomeric form in the
presence of 0.5 m NH
4
SCN [6]. Analogously, the elu-
tion volume of W243Y is also altered by thiocyanate,
although the conversion to a monomeric state was
achieved at a lower concentration of lipophilic ion (i.e.
0.2 m), further confirming the weaker interaction
between monomers.
Spectroscopic studies of thermal stability
Temperature ramp experiments were performed, as
reported previously [6], by following changes of var-
ious spectroscopic signals. Following the increase in
FAD fluorescence, the two Trp243 DAAO mutants
(that at 0.1 mgÆmL
)1
protein concentration are both
monomeric) show a different increase in flavin fluores-
cence and are less thermostable than wild-type DAAO
(showing a melting temperature (T
m
)ofupto 9 °C
lower for the W243I mutant than for the dimeric wild-
type DAAO, see Table 2). When the temperature-
induced loss of the tertiary structure was monitored by
following the increase in protein fluorescence and in
ANS fluorescence, the Trp243 mutants were still more

L. Caldinelli et al. Structural determinants for dimerization of DAAO
FEBS Journal 273 (2006) 504–512 ª 2006 The Authors Journal compilation ª 2006 FEBS 507
sensitive to temperature than the wild-type DAAO
(Table 2). Taken together, these data show that the
loss of the tertiary structure elements and of the flavin
cofactor occurs at lower temperatures in the two
Trp243 mutants than in the dimeric wild-type DAAO.
Urea-induced unfolding
The stability towards chemical denaturation of Trp243
mutants was compared with that of wild-type and
DLOOP DAAO forms using equilibrium unfolding
measurements and by following different spectroscopic
signals of these proteins after equilibration in the pres-
ence of increasing urea concentration. The change in
protein emission intensity (used as a probe of the glo-
bal unfolding) was only accurately measured for the
W243Y mutant because, for the W243I mutant, a high
scattering was observed in both the absence and pres-
ence of exogenous FAD. For both mutants, the chan-
ges were complete at 2 m urea and suggest a simple
two-state transition (see Fig. 3A for W243Y DAAO).
The midpoint concentration of urea required for
unfolding (C
m
) of the W243Y mutant is similar to that
determined for the monomeric DLOOP enzyme, which
is significantly lower than for wild-type DAAO
(Table 2). In contrast, the change in wavelength of
maximal emission as a function of urea concentration
exhibited significantly higher C

m
values (2.0 ± 0.2 m
for all DAAO forms; Fig. 3A). The lack of correlation
between the fluorescence intensity and the maximum
emission, two parameters that report on different
events (solvent exposition of the indole ring of trypto-
phan versus its distance from quenching moieties) sup-
ports the hypothesis that the unfolding process of
Trp243 mutants is complex.
Analogously to that reported previously for wild-
type DAAO [11], a biphasic dependence of flavin fluor-
escence intensity with increasing urea concentrations
was also observed for the W243Y mutant (see Fig. 3B
and Table 2), leading to the production of the unfold-
ing intermediate at a urea concentration similar to that
observed for the monomeric DLOOP mutant (a similar
experiment was not performed with the W243I mutant
because in the absence of added free flavin it is largely
present as apoprotein). At ‡ 7 m urea, the flavin fluor-
escence of the W243Y mutant attains values similar to
those observed for the DLOOP mutant and wild-type
DAAOs under similar conditions and corresponds to
that of the free flavin.
The binding of ANS was used to study the partial
exposure of hydrophobic patches upon loosening of
the protein tertiary structure at increasing urea concen-
trations. For wild-type and DLOOP DAAOs, two
transitions were seen in the intensity of ANS fluores-
cence at 500 nm in solutions containing 0.1 mm ANS
and increasing denaturant concentration [11]: an

increase in ANS fluorescence up to 2 m urea (paral-
leled by a change in the maximum ANS emission
wavelength) was followed by a quenching of ANS
fluorescence along with a shift of  20 nm in the maxi-
mum emission to a longer wavelength at higher urea
concentrations (Fig. 3C). Using the W243Y DAAO,
the ANS fluorescence was high (and the maximum
emission wavelength decreased) up to 4.5 m urea
(Fig. 3D), indicating that exposure of new hydropho-
bic ANS-binding surfaces in the mutant enzyme is
observed up to this concentration of urea. In all cases,
the surfaces progressively disappeared as urea concen-
trations were increased further.
Effects of urea on the associative behaviour
Upon preincubation with increasing concentrations of
urea (0–8 m), both dimeric wild-type and monomeric
DLOOP DAAOs eluted from size-exclusion chroma-
tography as multiple peaks with retention volumes
corresponding to DAAO aggregates comprising 2–10
subunits [11]. Under the same experimental conditions,
and at a protein concentration of 1 mgÆmL
)1
, the
W243I DAAO (which is monomeric in the absence of
urea) and the W243Y DAAO (which is dimeric in the
absence of urea), converted into aggregates of increas-
ing size at urea concentrations from 2 to 4 m, with a
Table 2. Comparison of parameters for temperature- and urea-
induced unfolding as determined by different approaches on the
wild-type and mutant proteins of

D-amino acid oxidase (DAAO).
Method Wild type
a,b
DLOOP
a,b
W243I W243Y
Temperature (°C)
Trp fluorescence 46.8 39.2 42.4 40.6
FAD fluorescence 47.5 42.3 38.5 41.2
ANS fluorescence 50.0 n.d. 41.7 40.2
Urea C
m
(M)
Trp fluorescence 1.4 (1.8) 1.0 (2.0) n.d. (1.9) 0.8 (2.0)
FAD fluorescence
c
1.8, 6.2 1.3, 5.6 n.d. 1.1, 5.0
In order to avoid the presence of apoprotein in the assay mixture,
the protein- and 1,8-anilinonaphtalene sulfonic acid (ANS) fluores-
cence of W243I was determined in the presence of 0.1 m
M FAD.
Melting temperature (T
m
) values were obtained by deriving the
spectroscopic signals and not corrected for delay effects. The SD
was within 0.2 °C for all values determined. The numbers in paren-
theses are the midpoint concentration of urea required for unfolding
(C
m
) determined from changes in the wavelength of maximum

emission as function of urea concentration.
a
As described previously [6].
b
As described previously [11].
c
These values were estimated using a three-state model, as des-
cribed previously [11,16].
Structural determinants for dimerization of DAAO L. Caldinelli et al.
508 FEBS Journal 273 (2006) 504–512 ª 2006 The Authors Journal compilation ª 2006 FEBS
concomitant loss of solubility. These aggregates con-
verted into soluble polymeric forms at urea concentra-
tions higher than 6 m, as evident by the increase in the
total area of the eluted peaks (up to  80% of the
value determined for the untreated DAAO). This result
is consistent with the surface hydrophobicity data pre-
sented in Fig. 3C,D, and indicates a difference in the
exposure of hydrophobic regions during the urea-
induced unfolding of W243 mutants compared with
the wild-type DAAO.
Kinetics of reconstitution of apoprotein with FAD
Because of the weaker binding of the FAD cofactor to
the apoprotein of the W243 mutants, we studied the
kinetics of reconstitution of the apoprotein–FAD com-
plexes under pseudo-first-order conditions (10- and
20-fold excess of FAD) by following the quenching of
protein fluorescence both under steady-state conditions
and using a stopped-flow instrument. The reaction
course was previously demonstrated to be biphasic
using wild-type and DLOOP DAAO apoproteins [12].

The quenching curves obtained with the W243Y apo-
protein are similar to those obtained for wild-type
DAAO (Fig. 4A). The observed first-order rate con-
stant of the fast phase is  1.0 ± 0.1Æs
)1
and that of
the slow phase is 0.013 ± 0.002Æs
)1
for all DAAO
forms. Under these experimental conditions, the weak
binding of FAD to W243I apoprotein in combination
with the small change in protein fluorescence between
the corresponding apoprotein and the holoenzyme
forms (see Table 1) represents a hindrance in acquiring
reasonable time-course fluorescence traces. Under the
same experimental conditions, the change in fluores-
cence intensity of the first phase corresponds to
40 ± 4% of the total change for the wild-type DAAO
and to 32 ± 3% for the W243Y mutant. This result
indicates that the quenching of the signal associated
with W243 largely belongs to the first phase (i.e. to the
rearrangement of the interaction between the flavin
Fig. 3. (A) Equilibrium denaturation curves of W243Y D-amino acid
oxidase (DAAO) detected by means of tryptophan fluorescence.
Samples of DAAO (0.02 mgÆmL
)1
) were equilibrated for 40 min in
the presence of increasing urea concentrations, and the fraction of
unfolded protein was determined from the fluorescence intensity at
 340 nm (s) and emission peak maximum (d). The reported val-

ues were corrected for the emission of the solution prior to adding
protein. Solid lines represent the best fit obtained using a two-state
denaturation model. (B) Comparison of equilibrium urea-denatura-
tion profiles of wild-type (d), DLOOP (h), and W243Y (m) DAAOs
detected by means of flavin fluorescence (see the legend of panel
A for details). Lines represent the best fit obtained using a three-
state denaturation model for wild-type (—) and W243Y (- - - -)
enzymes [11,16]. (C, D) 1,8-Anilinonaphtalene sulfonic acid (ANS)
fluorescence in the presence of wild-type (C) and W243Y (D)
DAAOs as a function of urea concentration: fluorescence intensity
at 500 nm (d) and wavelength of emission maximum (n). Samples
of DAAO (2.5 l
M ¼ 0.1 mgÆmL
)1
) were equilibrated for 40 min at
15 °C in the presence of increasing concentrations of urea, and the
fluorescence spectra were recorded after the addition of 100 l
M
ANS. The values reported have been corrected for the emission of
the solution prior to adding protein.
L. Caldinelli et al. Structural determinants for dimerization of DAAO
FEBS Journal 273 (2006) 504–512 ª 2006 The Authors Journal compilation ª 2006 FEBS 509
and the surrounding amino acid residues to form an
holoenzyme intermediate) [12]. In all cases, the enzyme
activity was regained during the second phase, as
defined by the fluorescence experiments (requiring
 95 ± 10 s for all apoproteins; Fig. 4B).
The reconstitution process is significantly faster in
the presence of 2 m urea (i.e. the denaturant concentra-
tion at which the unfolding intermediate is largely

produced) (Fig. 3B) [11]. The fluorescence changes
obtained for wild-type and W243Y DAAOs at 2 m
urea were observed during the dead-time of mixing
(£ 10 ms), and also the enzymatic activity is recovered
faster than in the absence of urea (requiring £ 40 s for
all DAAO forms, Fig. 4B). At 2 m urea,  15% of the
activity was recovered, corresponding to the residual
activity measured at this urea concentration during
chemical unfolding experiments [11]. Therefore, at the
urea concentration required to yield the unfolding
intermediate, the reconstitution process for the DAAO
apoprotein is different from that observed under native
conditions and it is not significantly modified by
Trp243 substitution.
Discussion
Both of the Trp243 substitutions introduced in
DAAO were nondisruptive, as confirmed by the
absence of major changes in catalytic activity, and
altered the protein in a different way. Some of the
properties of the W243Y mutant are similar to those
of the wild-type protein (e.g. the oligomeric state at
1mgÆmL
)1
protein concentration, the absorption
spectrum, the flavin fluorescence, the far-UV CD
spectrum, and the binding with FAD), whereas other
properties differ (e.g. the monomeric state at low
protein concentration, the protein fluorescence, the
near-UV CD spectrum, and the thermal stability).
Furthermore, the changes are more evident for the

W243I mutant: it has been purified as a monomer
whose interaction with the FAD coenzyme is signifi-
cantly decreased (Table 1).
Substitution of Trp243 modifies the protein confor-
mation such that it interferes with both the coenzyme
binding to the apoprotein moiety and with the acquisi-
tion of the tertiary structure required for the mono-
mer–monomer interaction, resulting in a lower stability
towards temperature and denaturants. Concerning the
monomer–monomer interaction, the structural pertur-
bation introduced by substitution of Trp243 with
isoleucine is sufficient to prevent dimerization of the
enzyme, although the bF5–bF6 dimerization loop is
still present in the mutant DAAO (Fig. 1A,B). Simi-
larly, a single tryptophan residue (Trp548 in the sub-
unit interface region) is also responsible for the
integrity of the quaternary structure of the cytosolic
malic enzyme [13]. Our results suggest that the ‘hole’
generated by the substitution of Trp243 could be filled
with water molecules that, in a hydrophobic environ-
ment, might be a major factor that prevents associ-
ation (or tight contact) of the two monomers. Such a
conclusion is supported by the results obtained for the
W243Y mutant, the most conservative substitution. In
fact, although the W243Y mutant binds the FAD
cofactor tightly and is dimeric, its oligomerization state
depends on the protein concentration, and its conver-
sion to a monomeric state is obtained at a significantly
lower thiocyanate concentration than for wild-type
DAAO (0.2 versus 0.5 m, respectively). We hypothesize

that Trp243 changes its position during flavin binding
Fig. 4. (A) Time course of protein fluorescence change at 340 nm
during the binding of FAD to wild-type (d) and W243Y (n) apopro-
teins at 15 °C and pH 7.5. Proteins were used at a concentration of
0.1 l
M (0.004 mgÆmL
)1
)in50mM potassium phosphate, pH 7.5,
containing 10% (v ⁄ v) glycerol and 2 m
M EDTA, and were reacted
with 10-fold excess free FAD. (B) Time course of activity recovery
during the binding of FAD to wild-type
D-amino acid oxidase
(DAAO) apoprotein at 15 °C and pH 7.5 in the presence (s)or
absence (d)of2
M urea. The activity was measured using an oxy-
gen-consumption assay under the same experimental conditions
used for the fluorescence analysis (see Fig. 4A).
Structural determinants for dimerization of DAAO L. Caldinelli et al.
510 FEBS Journal 273 (2006) 504–512 ª 2006 The Authors Journal compilation ª 2006 FEBS
to the DAAO apoprotein (in particular during the first
phase, see Fig. 4A), reaching a location that promotes
dimerization.
The second structural element fundamental for the
stability of yeast DAAO is represented by the coenzyme
binding: temperature ramp experiments demonstrated
that the apoprotein is less stable than the holoenzyme
and that flavin release triggers protein denaturation [6].
Indeed, limited proteolysis experiments have highlighted
the looser conformation of the apoprotein form [14].

Recently, we reported that the urea-induced unfolding
of DAAO is a three-state process, yielding an intermedi-
ate at  2 m urea [11]. The intermediate species lacks
the characteristic tertiary structure of native DAAO,
but has a significant secondary structure, retains flavin
binding, and shows an increased sensitivity to trypsin of
a specific site (Arg289) belonging to the loop preceding
the b-strand F5 of the FAD-binding domain. Strand F5
has been proposed as a specific connection during holo-
enzyme reconstitution between the FAD-binding
process and the exposure of Trp243 required for dimeri-
zation [11]. In fact, it is connected on one side (and
through the C-terminal a-helix containing the peroxi-
somal targeting signal) to the bab motif, known as the
dinucleotide-binding domain [2], and on the other side
to the long loop following the b-strand I7 containing
Trp243 (see Fig. 1D). The looser conformation of the
mutants DAAO, as evident by the higher protein fluor-
escence and by the modification of near-UV CD spec-
trum of their holoenzyme forms compared with the
wild-type, indicates that the tertiary structure modifica-
tion caused by Trp243 substitution (in particular with
an isoleucine) prevents the aforementioned interactions
from occurring.
In conclusion, the site-directed mutagenesis studies
on Trp243 in yeast DAAO indicate that (a) the inter-
actions at the monomer–monomer interface, which
result in tight packing, stabilize the protein by maxim-
izing van der Waals interactions and by efficiently
excluding solvent and (b) Trp243 substitution alters

the protein conformation required for efficient coen-
zyme binding.
Experimental procedures
Mutagenesis, enzyme expression and purification,
and apoprotein preparation
The mutant DAAO genes were generated by site-directed
mutagenesis using the QuikChangeÒ site-directed mutagen-
esis kit (Stratagene, LaJolla, CA, USA) and the recombin-
ant plasmid, pT7-HisDAAO, as template [15]. Both W243I
and W243Y DAAOs are produced as a fusion protein
because 13 additional residues are added at the N terminus
of the protein before the original methionine: the presence
of this short peptide has been demonstrated not to alter the
overall properties of the wild-type DAAO [15]. The Trp243
DAAO mutants were expressed using the strain
BL21(DE3)pLysS Escherichia coli as host and purified as
reported previously [15]; however, the W243I mutant was
better expressed when the cells were grown at 25 °C after
isopropyl thio-b-d-galactoside induction (up to 50 DAAO
unitsÆg
)1
cell paste). The overall yield of the purification
was  50% for both Trp243 mutants, similar to the yield
obtained with other DAAO forms. The apoprotein form of
both DAAO mutants was obtained by dialysis in the pres-
ence of 2 m KBr and 20% (v ⁄ v) glycerol [9], and can be
stored at )20 °C for months without any loss in ability to
reconstitute with FAD to give the corresponding holo-
enzyme.
Activity assay

DAAO activity was assayed with an oxygen electrode at
25 °C, as described previously [15], using 28 mmd-alanine
as substrate. One DAAO unit corresponds to the amount
of enzyme that converts 1 lmol of d-alanine per minute.
Size-exclusion chromatography
Size-exclusion chromatography was performed on a Super-
dex 200 H column (Amersham Biosciences, Piscataway NJ,
USA), at a flow rate of 0.5 mLÆmin
)1
and at room tempera-
ture. The elution buffer used was 50 mm potassium phos-
phate, pH 7.5, containing 5% (v ⁄ v) glycerol, 2 mm EDTA,
and the appropriate concentration of urea or NH
4
SCN.
The column was calibrated with suitable standard proteins.
Spectroscopy
All experiments were performed in 50 mm potassium phos-
phate buffer, pH 7.5, containing 10% (v ⁄ v) glycerol and
2mm EDTA, and at 15 °C. Fluorescence was measured in
a Jasco FP-750 instrument and at a protein concentration
of 0.1 mgÆmL
)1
. For temperature ramp experiments, the
instrument was equipped with a software-driven Peltier
temperature controller (to produce a 0.5 °CÆmin
)1
tempera-
ture gradient) [6]. Tryptophan emission spectra were recor-
ded from 300 to 400 nm using an excitation wavelength of

280 nm, and flavin emission spectra were recorded from
475 to 600 nm using an excitation wavelength of 450 nm.
Emission and excitation bandwidths were set at 10 and
20 nm, respectively. Kinetics of the apoprotein–FAD com-
plex formation were measured at a concentration of 0.1 lm
apoprotein (0.004 mgÆmL
)1
) and following the emission at
305 nm or at 340 nm (excitation at 280 nm). Reconstitution
kinetics were also determined by stopped-flow in a Bio-
L. Caldinelli et al. Structural determinants for dimerization of DAAO
FEBS Journal 273 (2006) 504–512 ª 2006 The Authors Journal compilation ª 2006 FEBS 511
Logic SFM-300 instrument (BioLogic, Claix, France) inter-
faced to the spectrofluorimeter. Reaction rates were calcula-
ted by fitting the traces to a sum of exponentials equation
using Biokine32 (BioLogic). ANS-binding experiments
were carried out at a protein concentration of 2.5 lm
(0.1 mgÆmL
)1
) [11]. Fluorescence emission spectra were
recorded in the 450–600 nm range using an excitation wave-
length of 370 nm. In general, the ANS binding was fol-
lowed at 500 nm but, because of the FAD-induced
quenching of ANS signal, its binding to W243I mutant was
estimated using the values at 478 nm, a wavelength at
which the change in fluorescence is not affected by the pres-
ence of the cofactor. CD spectra were recorded on a Jasco
J-810 spectropolarimeter (Jasco Europe, Cremella, Italy)
and analysed by means of Jasco software [6,11].
The unfolding equilibrium of DAAO was determined by

following the changes in flavin and protein fluorescence, as
detailed previously [11]. Protein fluorescence unfolding
curves were analyzed, according to a two-state mechanism,
using the apparent fraction of the unfolding form, and the
flavin fluorescence data were analyzed according to a three-
state denaturation pathway (N « I « U) [11,16].
Acknowledgements
We thank Dr Stefania Iametti for kindly helping with
the CD measurements, and Dr Luciano Piubelli for
helpful discussions. This work was supported by grants
from Fondazione Cariplo to LP, and from FAR 2004.
References
1 Mei G, Di Venere A, Rosato N & Finazzi-Agro A
(2005) The importance of being dimeric. FEBS J 272,
16–27.
2 Rossmann MG, Moras D & Olsen KW (1974) Chemical
and biological evolution of nucleotide-binding protein.
Nature 250, 194–199.
3 Dym O & Eisenberg D (2001) Sequence-structure analy-
sis of FAD-containing proteins. Protein Sci 10, 1712–
1728.
4 Umhau S, Pollegioni L, Molla G, Diederichs K, Welte
W, Pilone MS & Ghisla S (2000) The x-ray structure of
d-amino acid oxidase at very high resolution identifies the
chemical mechanism of flavin-dependent substrate dehy-
drogenation. Proc Natl Acad Sci USA 97, 12463–12468.
5 Pollegioni L, Diederichs K, Molla G, Umhau S, Welte
W, Ghisla S & Pilone MS (2002) Yeast d-amino acid
oxidase: structural basis of its catalytic properties.
J Mol Biol 324, 535–546.

6 Pollegioni L, Iametti S, Fessas D, Caldinelli L, Piubelli
L, Barbiroli A, Pilone MS & Bonomi F (2003) Contri-
bution of the dimeric state to the thermal stability of
the flavoprotein d-amino acid oxidase. Protein Sci 12,
1018–1029.
7 Piubelli L, Caldinelli L, Molla G, Pilone MS & Pollegioni
L (2002) Conversion of the dimeric d-amino acid oxidase
from Rhodotorula gracilis to a monomeric form. A
rational mutagenesis approach. FEBS Lett 526, 43–48.
8 Piubelli L, Molla G, Caldinelli L, Pilone MS & Polle-
gioni L (2003) Dissection of the structural determinants
involved in formation of the dimeric form of d-amino
acid oxidase from Rhodotorula gracilis: role of the size
of the bF5-bF6 loop. Protein Eng 16, 1063–1069.
9 Casalin P, Pollegioni L, Curti B & Pilone Simonetta M
(1991) A study on apoenzyme from Rhodotorula gracilis
d-amino acid oxidase. Eur J Biochem 197, 513–517.
10 Lakowicz JR (1999) Principles of Fluorescence Spectro-
scopy, 2nd edn, pp. 237–266. Kluwer ⁄ Plenum, New
York.
11 Caldinelli L, Iametti S, Barbiroli A, Bonomi F, Piubelli
L, Ferranti P, Picariello G, Pilone MS & Pollegioni L
(2004) Unfolding intermediate in the peroxisomal flavo-
protein d-amino acid oxidase. J Biol Chem 279, 28426–
28434.
12 Pollegioni L & Pilone MS (1996) On the holoenzyme
reconstitution process in native and truncated Rhodotor-
ula gracilis d-amino acid oxidase. Arch Biochem Biophys
332, 58–62.
13 Chang HC & Chang GG (2003) Involvement of single

residue tryptophan 548 in the quaternary structural sta-
bility of pigeon cytosolic malic enzyme. J Biol Chem
278, 23996–24002.
14 Pollegioni L, Ceciliani F, Curti B, Ronchi S & Pilone
MS (1995) Studies on the structural and functional
aspects of Rhodotorula gracilis d-amino acid oxidase by
limited trypsinolysis. Biochem J 310, 577–583.
15 Fantinato S, Pollegioni L & Pilone MS (2001) Engineer-
ing, expression and purification of a His-tagged chimeric
d-amino acid oxidase from Rhodotorula gracilis. Enzyme
Microbial Technol 29, 407–412.
16 Ayed A & Duckworth HW (1999) A stable intermediate
in the equilibrium unfolding of Escherichia coli citrate
synthase. Protein Sci 8, 1116–1126.
Structural determinants for dimerization of DAAO L. Caldinelli et al.
512 FEBS Journal 273 (2006) 504–512 ª 2006 The Authors Journal compilation ª 2006 FEBS