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 Kd value of 0.37 and
Trang 1binding 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, GR2, 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: loredano.pollegioni@uninsubria.it
(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 resiresi-dues 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; Cm, midpoint concentration of urea required for unfolding; DAAO, D -amino acid oxidase; Tm, melting temperature.
Trang 2[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).
Trang 3the absorption spectrum in the visible region (an
E274 nm⁄ E455 nm ratio of 9.6 and an extinction
coef-ficient, at 455 nm, of 12 800 m)1Æcm)1 was
deter-mined) In contrast, the W243I mutant was found to
exhibit a remarkably higher E274nm⁄ E455nm 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
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 Kd
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
(l M )
Vmax (lmol O 2 Æmin)1Æmg)1protein)
Km (m M )
Protein a.u (nm)
Flavin a.u.
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 m M potassium phosphate, pH 7.5, containing 10% (v ⁄ v) glycerol and 2 m M EDTA.
Trang 4The 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 Kdfor 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 Kd 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 Kd
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⁄ Kd ratio (7.2, 7.8 and 7.3 arbitrary unitsÆ
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 Kmfor 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 Kav of 0.40 at
1 mgÆmL)1 to a Kav 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 NH4SCN [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 (Tm) of up to 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
Trang 5sensitive 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 sugchan-gest a simple
two-state transition (see Fig 3A for W243Y DAAO)
The midpoint concentration of urea required for
unfolding (Cm) 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 Cm 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).
Temperature (C)
Urea Cm( 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
(Cm) determined from changes in the wavelength of maximum
emission as function of urea concentration.
a
As described previously [6]. bAs described previously [11].
c These values were estimated using a three-state model, as
des-cribed previously [11,16].
Trang 6concomitant 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.
Trang 7and 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
1 mgÆmL)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) in 50 m M 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) of 2 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).
Trang 8to 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 substitumodifica-tion (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 QuikChange 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 25C 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
25C, as described previously [15], using 28 mm d-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)1and 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 NH4SCN 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
2 mm EDTA, and at 15C 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 0.5CÆmin)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
Trang 9Bio-Logic SFM-300 instrument (BioBio-Logic, 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
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