We also have recently described channels that might allow access of oxygen to pockets at the active site of the flavoproteinD-amino acid oxi-dase DAAO that have a high affinity for dioxyge
Trang 1O2 diffusion pathways and enhancement of reactivity
Elena Rosini, Gianluca Molla, Sandro Ghisla and Loredano Pollegioni
Dipartimento di Biotecnologie e Scienze Molecolari, Universita` degli Studi dell’Insubria, and The Protein Factory, Centro Interuniversitario di Biotecnologie Proteiche, Politecnico di Milano and Universita` degli Studi dell’Insubria, Varese, Italy
Introduction
Flavins are highly versatile co-factors of flavoproteins
that catalyze a wide array of chemical and
photochemi-cal processes [1–3] As a prominent member of this
fam-ily, d-amino acid oxidase (DAAO, EC 1.4.3.3) is a
homodimeric enzyme found in all eukaryotic cells,
where it fulfils various roles [4] It is the archetype of the
oxidase⁄ dehydrogenase class of flavoproteins [1]; each
subunit contains one non-covalently bound FAD
mole-cule > 10 A˚ below the surface [5] DAAO catalyzes net
hydride transfer from the aC–H bond of neutral
d-amino acids (and of basic d-amino acids, but with lower efficiency) to FAD (on the Re side) in the reduc-tive half-reaction (Scheme 1a) and oxidation of reduced co-factor (FADH)) by O2in the oxidative half-reaction, forming H2O2as a product (Scheme 1b,c) [6,7]
With some notable exceptions [8], research into the mechanistic details of the reaction of (reduced) flavoprotein oxidases with dioxygen has long been neglected The reasons for this were mainly due to experimental limits, such as conversion of the species in
Keywords
flavoproteins; mutagenesis; oxidases;
oxygen diffusion; oxygen reactivity
Correspondence
L Pollegioni, Dipartimento di Biotecnologie
e Scienze Molecolari, Universita` degli Studi
dell’Insubria, Varese, Italy
Fax: +39 332 421500
Tel: +39 332 421506
E-mail: loredano.pollegioni@uninsubria.it
(Received 19 July 2010, revised 2
November 2010, accepted 20 November
2010)
doi:10.1111/j.1742-4658.2010.07969.x
Evidence is accumulating that oxygen access in proteins is guided and con-trolled We also have recently described channels that might allow access
of oxygen to pockets at the active site of the flavoproteinD-amino acid oxi-dase (DAAO) that have a high affinity for dioxygen and are in close prox-imity to the flavin With the goal of enhancing the reactivity of DAAO with oxygen, we have performed site-saturation mutagenesis at three posi-tions that flank the putative oxygen channels and high-affinity sites The most interesting variants at positions 50, 201 and 225 were identified by a screening procedure at low oxygen concentration The biochemical proper-ties of these variants have been studied and compared with those of wild-type DAAO, with emphasis on the reactivity of the reduced enzyme species with dioxygen The substitutions at positions 50 and 225 do not enhance this reaction, but mainly affect the protein conformation and stability However, the T201L variant shows an up to a threefold increase in the rate constant for reaction of O2 with reduced flavin, together with a fivefold decrease in the Km for dioxygen This effect was not observed when a valine is located at position 201, and is thus attributed to a specific alter-ation in the micro-environment of one high-affinity site for dioxygen (site B) close to the flavin that plays an important role in the storage of oxygen The increase in O2 reactivity observed for T201L DAAO is of great interest for designing new flavoenzymes for biotechnological applica-tions
Abbreviations
DAAO, D -amino acid oxidase (EC 1.4.3.3); E-Fl ox , oxidized enzyme form; E-Fl red , reduced enzyme form; E-Fl red –IA, reduced enzyme–imino acid complex; IA, imino acid.
Trang 2the absence of observable intermediates In oxidases,
the reaction of reduced flavin with O2proceeds through
an electron-transfer step that generates a caged radical
pair This is generally thought to be rate-limiting
Reduced flavoprotein oxidases (and monooxygenases)
react rapidly with dioxygen (exhibiting bimolecular rate
constants up to 106m)1Æs)1) and show no saturation
with O2 (i.e the Kd value for oxygen is much larger
than the maximal O2 concentration in solution) [6–9]
These enzymes appear to ‘consume’ O2 without
high-affinity binding
Because of the scarcity of information concerning
the reaction of flavoproteins with molecular oxygen,
we previously used a directed evolution approach to
enhance the affinity for dioxygen of DAAO,
specifi-cally to generate optimized enzyme variants for use in
biocatalysis or medical applications [10] The
S19G⁄ S120P ⁄ Q144R ⁄ K321M ⁄ A345V DAAO variant
has increased activity at low O2 concentrations,
result-ing from a 10-fold lower Km;O2 value, although the
rate constant for reduced flavin re-oxidation was only
marginally affected [10] Recently, however, important
advances have been made in the field: as with
heme-dependent enzymes [11–13], specific paths within the
protein matrix of flavoprotein oxidases have been
identified that serve to channel O2 to its destination
[14,15] We have described funnels that lead to
pock-ets at the active site of DAAO, in particular two
regions (sites A and B) that have the highest affinity
for dioxygen inside the protein and are in close
prox-imity to the Si side of the flavin, and determined the most likely diffusion pathways (Fig 1) [16] The energy required to place an O2molecule at site A or B
is 15 kJÆmol)1 lower than the corresponding energy required to place O2in the solvent; thus, the probabil-ity of finding a molecule of O2 at these sites is corre-spondingly higher This corresponds to a virtual [O2] that is 1000-fold higher than that in an equivalent solvent volume The local lower dielectric constant might play a main role in the apparent [O2] increase Site A is in close proximity with the C(4a) position of the isoalloxazine ring of FAD (at 3.5 A˚), an ideal location for efficient oxygen reactivity Site B is located
5 A˚ from the xylene ring of the flavin and is thus also suitable for electron transfer The importance of the predicted site A was tested experimentally by mutating Gly52, i.e by partially filling the space expected to be occupied by O2: the G52V DAAO vari-ant shows a 100-fold lower oxygen reactivity [16] The present study represents an extension of these previous studies with the goal of enhancing the reactivity of DAAO with dioxygen It is based on the predictions of the implicit ligand sampling analysis [16], which has identified several residues in the oxy-gen channels The most interesting substitutions at positions 50, 201 and 225 were identified by a screen-ing procedure at low oxygen concentration on mutant libraries prepared by site-saturation mutagenesis These studies identified a DAAO variant at position
201 that reacted more efficiently with dioxygen and
(a) E-Flox + AA E-Flox–AA E-Flred–IA E-Flred + IA
E-Flox–IA E-Flox–IA E-Flox
k1
k6
k3 k4
k–1
k2
k–2
k5
k–5
E-Flred + O2
E-Flred–IA + O2
(b)
(c)
(d) et/v = Φ0 + Φ D -Ala / [ D -Ala] + ΦO2 / [O2] + Φ D -ALa,O 2 / [ D -Ala]•[O2]
where: kcat = 1/Φ 0; Km,D -Ala = Φ D -Ala /Φ 0; Km,O2 = Φ O 2 /Φ 0
For wild-type DAAO (k4»k2), the expressions can be simplified:
kcat = [k2•k4/k2 + k4)] ≈ k 2
Km,O2 = [k4•(k2 + k–2)]/[k3•(k2 + k4)] ≈ k2/k3
Km,D-Ala = [k4•(k–1 + k2)]/[k1•(k2 + k4)] ≈ (k –1 + k2)/k1
et/v = [(k2 + k4)/(k2•k4)] + [(k–1 + k2)/(k1•k2 •[ D-Ala])] + [(k2 + k–2)/(k2•k3 •[O 2])] + [(k–1 + k–2)/(k1•k2•k3 •[ D -Ala] •[O 2 ])]
Scheme 1 Kinetic steps in the catalytic cycle proposed for DAAO [6,7]: (a) reductive half-reaction, (b,c) oxidative half-reaction, and (d) corre-lation between steady-state kinetic parameters and single rate constants, and their reduction for wild-type DAAO [7].
Trang 3provided further insight into the oxygen reactivity of
DAAO
Results
Identification of residues possibly involved in
oxygen binding
Recent computational implicit ligand sampling studies
have discovered a small channel (filled by several water
molecules) that leads from the bulk solvent to the Si
face of the flavin at the active site of yeast DAAO
(PDB code 1c0p; Fig 1A) and is fundamental for O2
access to the active site during turnover [16] The W50
and T201 residues are aligned along this channel, and,
in particular, the side chain of residue W50 is oriented
toward the outer part of the channel toward the bulk
solvent (Fig 1B) We have hypothesized that the
modi-fying size and polarity of this side chain could result in
major alterations in O2 accessibility through the
pro-posed channel A further important residue, T201, is
located in the inner part of the channel, close to the
benzene ring of FAD (3.7 A˚) and in close proximity to
the two regions with the highest affinity for oxygen
(sites A and B in Fig 1) [16] T201 is thus a further
candidate as a target for the study of O2 interactions
The third such residue is I225, the side chain of which,
together with the side chain of M213, forms a large
part of the DAAO active site roof; it is located at a
distance of 4.5 A˚ from the FAD C(4)=O locus
However, it is possible that mutations at this site could
also affect the activity⁄ substrate specificity of the
enzyme [16–18]
Libraries of DAAO variants at positions 50, 201
or 225 were generated by site-saturation mutagenesis,
and two or three variants for each single position showing altered oxygen reactivity were selected by a screening procedure performed at low oxygen con-centration (2.5%) The W50F, T201L and T201V variants were identified because of higher activities than the wild-type DAAO, the I225F and I225V variants were identified as the most active clones at this position 225, while the W50R and W50P vari-ants were isolated because they exhibit no activity
General properties of DAAO variants All the purified recombinant DAAO variants are homodimeric 80 kDa holoenzymes, as determined by gel-permeation chromatography and spectral analyses, and in the oxidized state show the typical spectrum of FAD-containing flavoproteins (i.e absorbance maxima
at 455 and 375 nm, an e455 nmof 12 600 m)1Æcm)1, and an A274 nm⁄ A455 nm ratio of 8.2; Appendix S1 and Fig S1) Free FAD is not found in all purified enzyme preparations, indicating preservation of the strong interaction between the co-factor and the apo-protein moiety The substitutions introduced at posi-tion 50 alter the tertiary structure of DAAO (Fig S2)
as well as the protein stability: the W50 DAAO vari-ants are less thermostable than wild-type enzyme (Table S1) The redox properties of the flavin co-factor are also altered by the W50P substitution: an Em of ) 207 ± 6 mV was determined for the W50P variant versus ) 109 mV for wild-type DAAO [19] The con-formation and flavin properties of DAAO variants at position 201 and 225 are not significantly affected by the substitutions introduced, with only a slight alter-ation in the ability to stabilize the flavin semiquinone species (Appendix S1)
Fig 1 (A) O2channel connecting bulk solvent to the Si face of FAD at the DAAO active site [16] Mutated residues described in the text, the product imino acid (IA) and FAD are shown using CPK representation Water molecules filling the channels (green) are shown using VdW representation (50% VdW radius) (B) Position of the mutated residues with respect to the O 2 high-affinity sites A (green) and B (blue) The product (imino pyruvate, IA) and the water molecules were modeled into the DAAO structure (PDB code 1c0p) as described previously [16].
Trang 4The apparent kinetic parameters of DAAO variants
were determined using d-alanine as the substrate by an
oxygen consumption assay at 21% oxygen saturation
and 25C (Appendix S1 and Table S2) In comparison
to wild-type DAAO, the most significant changes were
apparent for the W50P (lower kcat,app and higher
Km,app), W50R (lower d-alanine affinity) and I225F
(lower kcat,app) variants A comparison of the substrate
specificity of the I225F variant with respect to the
wild-type DAAO is shown in Table S3: these results
support the conclusion that the side chain at this
posi-tion contributes to the d-amino acid preference of
yeast DAAO
Kinetic mechanism of DAAO variants
Steady-state kinetics
Dependence of the catalytic activity of DAAO variants
on the oxygen and d-alanine concentrations was
assessed using the enzyme-monitored turnover method
[6,7,20] Air-saturated solutions of DAAO and
d-ala-nine were mixed in a stopped-flow instrument, and
absorbance spectra were recorded continuously in the
300–700 nm range at 15C [16] During turnover, all
DAAO variants are largely present in the oxidized
form, and the spectrum of the reduced enzyme is
observed only at the end of the observation time, i.e
when the O2 concentration becomes very low (Fig 2A
and Fig S3) This is consistent with the steps involving
oxidation of reduced DAAO by oxygen being faster
than those involved in reduction, as observed for
wild-type and other variants of DAAO [7,10,16] The results
of these steady-state measurements (at saturating O2
and d-alanine concentrations; Scheme 1 and Table 1)
show a decrease in kcat for all the variants with the
exception of the T201V DAAO; the variant with the
lowest kcat is the W50P DAAO (compare traces in
Fig S3A) The W50P and W50R variants show a
lower affinity for d-alanine compared to wild-type
DAAO, and a decrease in the Km;O2 value is apparent
for W50P, W50R, T201L and I225V enzymes For
Lineweaver–Burk (double-reciprocal) plots show a
set of parallel lines (not shown), secondary plots of
the reciprocals of the x and y intercepts from the
Lineweaver–Burk plot (apparent kcatand Km;O2,
respec-tively) against [d-alanine] show an unprecedented
sig-moidal dependence on d-amino acid concentration
(Fig 2B,C) The steady-state parameters for I225V
DAAO determined by using the extreme kinetic Hill
coefficient for cooperativity (h = 2.2) [21] are listed in
Table 1 In contrast, the I225F variant does not show
any sigmoidal behavior, but an 20-fold decrease
Fig 2 Steady-state kinetics of the I225V variant of DAAO (A) The kinetic data were determined by the enzyme-monitored turnover method (Fig S3), using D -alanine (at 0.4, 0.5, 0.8 and 1.0 m M ) and 0.25 m M oxygen [20], by monitoring the time course of the flavin oxidation state based on its absorbance at 455 nm [6,7] at pH 8.5 and 15 C (B,C) Tertiary plots of the reciprocal of the y intercepts (B) and the x intercepts (C) as calculated from Lineweaver–Burk plots obtained from the experimental traces shown in (A) Experimental values were fitted using a hyperbolic fit using a Hill coefficient of 2.2 [21].
Trang 5in kcat compared to wild-type DAAO is apparent
(Fig S3C)
Reductive half-reaction
The reductive half-reaction (Scheme 1a) was studied
for wild-type and variants of DAAO, using d-alanine
as the substrate under anaerobic conditions at 15C,
and monitoring the absorbance changes [7,15] In all
cases, the oxidized form of the enzyme is rapidly
converted to the reduced enzyme–imino acid complex
(E-Flred–IA in Scheme 1; phase 1, kobs1), an
intermedi-ate that is then converted at a slower rintermedi-ate into free
fully reduced enzyme (phase 2, kobs2) (Fig 3) For all
DAAO variants, the dependence of kobs1 values on
[d-alanine] shows curvature: there is ample evidence
for a hyperbolic dependence of kobs1 on[d-alanine] for
various DAAOs, and it represents a second-order
pro-cess (formation of an initial enzyme–substrate
com-plex), followed by a first-order reaction as shown in
Scheme 1a [6,7,22] As the data are satisfactorily fitted
by a rectangular hyperbola that intersects close to the
origin, the reduction step is practically irreversible (k)2
of 0) A significant change in the rate constant of
flavin reduction was observed only for W50P DAAO
(Table 2), in agreement with the observed decrease in
the kcat value For the I225F and I225V variants, the
k2 rate constant was two- to threefold slower than for
wild-type DAAO (Table 2): importantly, no indication
of sigmoidal behavior is evident
The rate for the observed second-phase kobs2,
corre-sponding to product dissociation from E-Flred–IA (k5in
Scheme 1a) does not depend on [d-alanine], and its
value is 1.3 ± 0.5 s)1 for wild-type and variants of
DAAO The main exception is the W50P variant, for
which the k5value is estimated to be£ 0.1 s)1(Table 2)
Table 1 Comparison of steady-state kinetic parameters for wild-type and variants of DAAO using D -alanine as substrate and at 15 C Data were obtained in 50 m M sodium pyrophosphate buffer, pH 8.5, 1% glycerol and 0.25 m M 2-mercaptoethanol The steady-state F parameters are defined in Scheme 1.
Lineweaver–Burk
plot behavior kcat(s)1) F D-alanine ( M Æs)1) Km,D -alanine (m M ) F O2 ( M Æs)1) K m;O2(m M )
F D-alanine,O2
( M2Æs)1· 10)9) Wild-type [10] Convergent 330 ± 30 (0.8 ± 0.1) · 10)5 2.6 ± 0.4 (5.0 ± 0.1) · 10)6 1.9 ± 0.1 3.0 ± 0.2 W50F Parallel 190 ± 20 (2.0 ± 0.3) · 10)5 3.9 ± 0.7 (7.7 ± 0.4) · 10)6 1.4 ± 0.5
W50R Parallel 62 ± 6 (1.7 ± 0.2) · 10)3 10.4 ± 1.5 (8.8 ± 1.7) · 10)6 0.54 ± 0.18
T201L Parallel 170 ± 15 (3.6 ± 0.5) · 10)5 6.2 ± 0.8 (2.3 ± 0.05) · 10)6 0.40 ± 0.05
T201V Parallel 365 ± 50 (1.7 ± 0.3) · 10)5 6.9 ± 0.7 (9.4 ± 0.1) · 10)6 3.9 ± 0.1
(sigmoidal
n = 2.2)
98 ± 6 (8.0 ± 0.4) · 10)5 0.9 ± 0.1 (11 ± 1) · 10)6 0.8 ± 0.1
G52V [16] Convergent 0.33 ± 0.03 (1.2 ± 0.2) · 10)5 0.036 ± 0.005 (4.4 ± 1.0) · 10)4 0.15 ± 0.03 12.1 ± 1.2
A
B
Fig 3 Reductive half-reaction of wild-type and variants of DAAO Comparison of time courses of flavin reduction followed at 455 nm (vertical bars = experimental data points) for W50 variants (A) and T201L ⁄ V and I225V variants (B) versus wild-type DAAO The enzymes ( 8 l M ) were reacted under anaerobic conditions with 0.25 m M D -alanine at pH 8.5 and 15 C ( D -alanine concentration of
5 m M for W50P) The rate constants were obtained by fitting using
a double exponential equation (continuous line) The rates are listed
in Table 2.
Trang 6Oxidative half-reaction
The (re)oxidation of reduced DAAO variants by
diox-ygen (Scheme 1b,c) was also studied using the
stopped-flow apparatus For this, anaerobic solutions of free
reduced enzyme were reacted with buffer solutions
equilibrated at various O2 concentrations (Scheme 1b),
and re-oxidation was monitored by following the
(re)appearance of absorption of the oxidized flavin
spe-cies The experimental traces at 455 nm (conversion of
the reduced enzyme form E-Flred into the oxidized
enzyme form E-Flox) are close to those of wild-type
for the W50F, W50P and T201V variants, slightly
slower for W50R and I225F variants, and appreciably
faster for T201L DAAO (Fig 4) The time course of
(re)oxidation is monophasic with the exception of
I225V The kobs values obtained for re-oxidation are
reported as a function of [O2], yielding a line that does
not indicate saturation with [O2] (data not shown); this
behaviour is assumed to reflect a second-order process
The slope of this linear fit yields the k6 rate constant,
which is approximately two- to threefold lower for
I225F and W50R DAAOs and threefold higher for the
T201L variant compared with wild-type DAAO (Fig 4
and Table 2) For the I225V variant, the experimental
traces of re-oxidation at 455 nm are better fitted using
a two-exponential equation (Fig 4C): a fast phase is followed by a second slower phase, with rate £ 3 s)1, and for which the value and amplitude ( 60% of the overall absorbance change) do not depend on oxygen concentration The kobs values for the faster phase of re-oxidation of the I225V variant show a linear depen-dence on [O2], with no indication of oxygen saturation: the k6 bimolecular rate constant is unchanged for
(Table 2)
A similar experiment was performed using the reduced enzyme E-Flred–IA complex (i.e the form present at high concentrations of ammonia and pyru-vate; Scheme 1c): in this case, the time course of re-oxidation is clearly biphasic A fast phase with an amplitude corresponding to 50% of the overall absorbance change at 455 nm is followed by a slower one, whose rate corresponds to that observed with free reduced DAAO at the same [O2] (Fig 4B) From this
we deduce that the first fast phase corresponds to (re)oxidation of the E-Flred–IA complex present at equilibrium (Scheme 1c) and the second phase corre-sponds to the re-oxidation of uncomplexed E-Flred (Scheme 1b) DAAO variants behave similarly to the wild-type enzyme: the re-oxidation is still faster for T201L than for T201V, W50F or wild-type DAAO,
Table 2 Rate constants for the reductive and the oxidative half-reaction of wild-type and variants of DAAO estimated from rapid reaction methods at 15 C For the reductive half-reaction, the parameters were obtained using D -alanine as substrate; for the oxidative half-reaction, the re-oxidation was started from the free reduced enzyme species or the imino acid complex (Scheme 1b,c) The rate constants refer to those defined in Scheme 1 Data were obtained in 50 m M sodium pyrophosphate buffer, pH 8.5, 1% glycerol and 0.25 m M 2-mercaptoetha-nol.
k obs1 ( k 2 ) (s)1) K d (k)1⁄ k 1 ) (m M ) k obs2 ( k 5 ) (s)1)
a k3from E-Flred–IA ( M )1Æs)1)· 10 5
b k6from E-Flred ( M )1Æs)1)· 10 4
(60% amplitude); 2.9 ± 0.1 (40% amplitude)
a Buffer as above containing 20 m M glucose, 20 m M pyruvate and 400 m M NH 4 Cl The rate constants of the second (slower) phase of flavin re-oxidation observed in the presence of imino acid and corresponding to re-oxidation of the free reduced enzyme form (k 6 in Scheme 1b) are shown in parentheses.
b Buffer as above containing 20 m M glucose.
c
The value estimated from simulation of the steady-state kinetics is shown in parentheses [16].
NF, not feasible (flavin re-oxidation following imino acid addition).
Trang 7and is approximately twofold slower for I225F and
I225V variants (Fig 4B and Table 2) However, the
same experiment was not feasible using W50P or
W50R DAAO variants because flavin was re-oxidized
after adding ammonia and pyruvate to enzyme that had been anaerobically reduced using an up to 10-fold molar excess of d-alanine or because flavin was re-oxi-dized when the photoreduced W50R DAAO was mixed with the imino acid solution or with classical inhibitors, such as benzoate and anthranilate The reoxidation of E-Flred–IA was also not feasible using G52 DAAO variants [16]
Therefore, the T201L substitution increases the oxy-gen reactivity of yeast DAAO, a change that does not modify the kcatvalue (i.e the maximal activity at satu-rating concentration of both substrates) but does affect activity at low oxygen concentrations These results also indicate that two forms of the free reduced enzyme species exist for I225V DAAO, and that these forms affect turnover at low substrate concentrations, i.e when there is competition between E-Flredand the E-Flred–IA complex for O2-induced re-oxidation (Scheme 1)
Discussion
The biochemical (and structural) basis of the capacity of flavoenzymes to react with dioxygen is still poorly understood, but represents a very interesting issue in flavoenzymology Trajectories and sites of high affinity for O2in the yeast DAAO have recently been identified
by molecular dynamics simulations and implicit ligand sampling methods [16]: a specific dynamic channel for
O2 diffusion leads from the solvent to the flavin Si side (i.e the opposite side with respect to the substrate⁄ prod-uct binding site; Fig 1) In a previous study, we investi-gated the role of the residue G52: in the G52V variant, the valine side chain occupies the site that has the high-est O2 affinity in wild-type DAAO (site A in Fig 1), and the reactivity of reduced G52V DAAO with O2 is considerably decreased, as well as the turnover number [16] Here we have focused on three additional residues that are potentially involved in oxygen migration as they flank the putative O2high-affinity sites
The substitution of W50 (a residue close to the tun-nel entrance; Fig 1A) with R or P significantly desta-bilizes DAAO, as is evident from the 10 C lower melting temperatures (Table S1), and modifies the CD and fluorescence spectra (Fig S2) Unpredictably (as this residue is distant from the isoalloxazine ring of the flavin), the redox properties of the W50P variant are also significantly altered, for example there is no stabil-ization of the flavin anionic semiquinone and the mid-point potential is 100 mV more negative than in wild-type DAAO This substitution also significantly alters the kinetic properties of the flavo-oxidase: compared to wild-type DAAO, the W50P variant has
A
B
C
Fig 4 Oxidative half-reaction of wild-type and variants of DAAO.
(A) Comparison of time courses of the (re)oxidation of reduced
wild-type and W50 variants of DAAO followed at 455 nm upon
mix-ing of 10 l M reduced enzyme with 153 l M oxygen (vertical
bars = experimental data points) Conditions were as described in
Experimental procedures The mono-exponential fit of the
experi-mental data is shown as a continuous line (B) Comparison of
re-oxidation of E-Flred (vertical bars) and the E-Flred–IA complex
(crosses; obtained by adding 20 m M pyruvate and 400 m M
ammo-nium chloride) wild-type and T201L DAAOs by 153 l M oxygen A
mono-exponential fit was used for the re-oxidation of E-Flredand a
bi-exponential equation was used for re-oxidation of the E-Fl red –IA
complex (C) Comparison of re-oxidation of E-Flred wild-type and
I225V DAAOs by 0.6 m M oxygen The experimental data points for
I225V were fitted using a mono-exponential equation (dashed line)
or a bi-exponential equation (solid line): the latter gave a better
reproduction The rates are listed in Table 2.
Trang 8a much lower ( 100-fold) maximal activity, an
12-fold lower Km for oxygen and an increased Km
for d-alanine; the binding of the competitive inhibitor
benzoate is also negatively affected by this substitution
(Table S2) The change in turnover number for W50P
DAAO is mainly due to the slower rate of the
reduc-tive half-reaction (the rate constant for flavin reduction
is decreased 100-fold; Table 2), but the rate of flavin
re-oxidation is slightly decreased for W50R DAAO
only ( 1.8-fold slower; Table 2), indicating a change
in the rate-limiting step for catalysis in this latter
variant
The substitutions introduced at position 225
decreased the enzyme activity (kcat 20-fold lower for
the I225F variant; Table 1), but Km;O2 and the rate
constants for the reduced flavin re-oxidation are only
modified to a limited extent (Tables 1 and 2) The
I225V variant shows an unprecedented sigmoidal
behavior in the kcatversus [d-alanine] and Km;O2 versus
[d-alanine] plots (Fig 2B,C) The biphasic re-oxidation
process observed using the free reduced enzyme species
(Fig 4C) possibly explains this, suggesting the
exis-tence of two alternative configurations (whose ratio is
close to 1, based on the amplitude of the observed
phases of re-oxidation), one of which shows a very
slow reactivity with dioxygen (£ 3 s)1; Table 2)
Simi-lar behavior was previously observed for the F359W
variant of Streptomyces cholesterol oxidase [23], and
was related to kinetic cooperativity known as the
mne-monic model [21] This occurs when the free enzyme
exists in at least two conformations that can react with
the substrate at different rates: the mnemonic model
for a two-substrate, two-product reaction sequence
dis-plays kinetic cooperativity with respect to the first
sub-strate but no cooperativity with respect to the other
substrate In fact, this unusual behavior of I225V
DAAO is lost in the presence of the imino acid
prod-uct The reaction of the reduced enzyme forms with
oxygen is negatively affected by substitution of I225:
the most significant change is an approximately
three-fold decrease in k3 as observed for the I225F variant
versus wild-type DAAO
With regard to position 201 (close to both
high-affinity sites A and B; Fig 1), the activity of the
T201V variant resembles that of wild-type DAAO (at
both 21% and saturating oxygen concentrations;
Table 1 and Table S2) However, introduction of
leu-cine results in a slight decrease in kcat and k2 values,
and, more interestingly, an approximately fivefold
decrease in Km;O2, which is accompanied by faster
re-oxidation of the corresponding reduced enzyme
species, up to threefold higher using the E-Flred–IA
complex (Fig 4B and Tables 1 and 2)
Functional data on flavo-oxidase variants designed
on the basis of molecular dynamic simulations of O2 diffusion have been reported recently for alditol oxi-dase [14] Its 3D structure was determined at 1.1 A˚ resolution [24], and indicated five putative pathways that bring O2 molecules in front of reduced FAD co-factor and a small cavity that may contain O2: sym-metrically to that observed for DAAO, this site is located on the Re side of the FAD co-factor, while substrate⁄ product exchange occurs on the Si side The changes in kinetic parameters for the A105G variant
of alditol oxidase were minor (see below) Site-directed mutagenesis designed to block individual routes had little effect on the kcat⁄ Km ratio in copper-containing amine oxidase [13], but significantly affected choles-terol oxidases [23,25] These results suggest that multi-ple pathways are employed by dioxygen to reach the active site, as also suggested by our results for DAAO
Of the residues modified in DAAO, G52 appeared to play the major role in O2 reactivity (up to a 100-fold decrease in k3 and k6 rate constants for G52V com-pared to the wild-type enzyme; Table 2), while W50 and I225 appeared to mainly affect the conformation
of the flavoprotein On the other hand, an increase in the rate constant for reduced flavin re-oxidation was observed for the T201L DAAO variant Sites A and B are in close proximity ( 8 A˚ apart), and are con-nected through a pathway that has a low activation energy barrier [16]: site B appears to increase the effec-tive dioxygen concentration in the proximity of site A From a structural point of view, the T201L mutation results in substitution of a small and polar side chain
by a large hydrophobic one whose d-methyl groups are very close to site B and the FAD xylene ring (Fig 5A,B) Local alteration of the hydrophobicity close to site B could increase the affinity of this site for
O2, and, as a consequence, the reactivity of the T201L DAAO variant with molecular oxygen A similar effect was not observed with the T201V variant, as the dis-tance between the valine side chain c-methyl groups and site B is expected to be larger than that in T201L DAAO ( 3.5 A˚ versus 2.0 A˚; Fig 5B,C)
The threefold increase in oxygen reactivity observed for the T201L DAAO is a meaningful difference, as improvements in oxygen reactivity of a similar extent for ‘efficient flavo-oxidases’ are uncommon Limited changes have been observed for various flavo-oxidases (e.g a 1.5-fold increase for the A105G variant of alditol oxidase and a 2.2-fold increase for the E475Q variant of cholesterol oxidase) [14,25] The opposite alterations in
O2reactivity for variants at position 52 and 201 confirm the importance of inferred oxygen channels in DAAO, and are in agreement with the ‘storage’ role proposed
Trang 9for site B Our results give direction to enhance the
robustness of existing O2-consuming flavo-oxidases in
order to design new catalysts for novel biotechnological
applications, e.g for evolution of a flavo-oxidase useful
in enzyme pro-drug cancer therapy [10]
Experimental procedures
Site-saturation mutagenesis and enzyme
expression and purification
Site-saturation mutagenesis at positions 50, 201 and 225
was performed on DAAO cDNA subcloned into
pT7-His-DAAO as template [18] using a QuikChange site-directed
mutagenesis kit (Stratagene, La Jolla, CA, USA) and a set
of degenerate synthetic oligonucleotides The PCR products
were used to transform JM109 Escherichia coli cells, and
then the recombinant plasmids were transferred into
BL21(DE3)pLysS E coli cells; these clones were used for
the screening procedure
DAAO variants with an altered enzymatic activity at low
O2concentration (2.5% = 30 lm) were identified using the
screening procedure described previously [10] and the
AtmosBag incubation system (Sigma Aldrich, St Louis,
MO, USA) on 250 clones for each position Introduction
of the mutations was confirmed by automated DNA
sequencing Recombinant clones encoding DAAO variants
selected using the screening procedure were grown and
purified as described previously [10] The purified DAAO
preparations were then equilibrated with 50 mm potassium
phosphate buffer, pH 7.5, 10% glycerol, 2 mm EDTA and
5 mm 2-mercaptoethanol As with wild-type DAAO, the
purified variants were > 90% pure according to SDS⁄ PAGE
analysis (data not shown)
The expression of W50F and W50R variants was similar
to that of the wild-type DAAO ( 1.5 mg enzyme per g cell
paste), but with a lower volumetric yield ( 6 mg protein
per liter of fermentation broth versus 11 mg for the
wild-type) A significantly lower yield was achieved for the W50P variant DAAO (0.6 mg per g cell paste and 2 mgÆL)1
of fermentation broth) The expression of DAAO mutants
at positions 201 and 225 was similar to that of the wild-type DAAO ( 2 mg enzyme per g cell paste and
11 mgÆL)1of fermentation broth)
Spectral properties
Extinction coefficients of the oxidized form of DAAO vari-ants were determined by heat denaturation of the enzymes (at 95C for 10 min) and using the absorption coefficient for free FAD of 11.3 mm)1Æcm)1 Semiquinone formation was achieved by light irradiation (using a 250 W lamp at a distance of 20 cm) with anaerobic enzyme solutions ( 10 lm) containing 5 mm EDTA and 0.5 lm 5-deazafla-vin [26] The amount of the thermodynamically stable sem-iquinone form was evaluated after incubation for 24 h at
4C or after adding 5 lm benzyl viologen to the enzyme solution [27] Redox potentials were estimated by the dye equilibration method [19,28] The dissociation constants for sulfite and benzoate binding to DAAO ( 10 lm) were assessed spectrophotometrically by following the changes in absorbance at 455 nm and 497 nm, respectively, that accompany complex formation: Kd values were estimated based on [29]
Protein fluorescence measurements were obtained between 300 and 400 nm, with excitation at 280 nm; flavin emission spectra were recorded from 475 to 600 nm, with excitation at 450 nm Fluorescence measurements were performed using a Jasco FP-750 instrument (Cremello, Italy) at 15C and 0.1 mgÆmL)1protein concentration, and corrected for buffer contributions Temperature-ramp experiments were performed as reported previously [30] using a software-driven, Peltier-equipped fluorometer in which a temperature gradient could be reproduced (0.5CÆmin)1) Circular dichroism (CD) spectra were recorded at 15C using a Jasco J-810 spectropolarimeter and analyzed by means of Jasco software The cell path
Fig 5 Detail of the residues surrounding oxygen high-affinity site B (A) Wild-type DAAO, (B) T201V DAAO, and (C) T201L DAAO Steric hindrance of atoms is shown as molecular surface colored by atom type The distance between the residue 201 side chain and the center
of site B is shown as a dotted line Residue 201 is labeled in bold IA: imino acid product (imino pyruvate) modeled into the DAAO active site (PDB code 1c0p) as described previously [16] Models of DAAO mutants were prepared using VMD [31] Molecular surfaces were calculated using the MSMS program [32].
Trang 10length was 1 cm for measurements above 250 nm and
0.1 cm for measurements in the 190–250 nm region [30]
Activity assays and stopped-flow measurements
DAAO activity was assayed using an oxygen electrode at
pH 8.5 and 25C with 28 mm d-alanine and air saturation
([O2] = 0.253 mm) [10] One DAAO unit is defined as the
amount of enzyme that converts 1 lmol of d-alanine per
minute at 25C
Steady-state and pre-steady-state stopped-flow
experi-ments were performed in 50 mm sodium pyrophosphate,
pH 8.5, containing 1% v⁄ v glycerol and 0.25 mm
2-mercap-toethanol, at 15C in a BioLogic SFM-300 instrument
equipped with a J&M diode array detector (BioLogic,
Grenoble, France) as described previously [7,10]
Steady-state kinetic parameters were determined by the
enzyme-monitored turnover technique, mixing equal volumes of
8–10 lm air-saturated enzyme with an air-saturated
solution of d-alanine The time courses at 455 nm reflect
the conversion of oxidized into reduced enzyme species,
and indicate the rate of catalysis as a continuous function
of oxygen concentration (the limiting substrate) [20]
For reductive half-reaction experiments, the oxidized
enzyme form was reacted with increasing d-alanine
concen-trations in the absence of dioxygen (the final solutions
con-tained 100 mm glucose, 0.1 lm glucose oxidase and 30 nm
catalase) For study of the oxidative half-reaction, reduced
enzyme forms were reacted with solutions of appropriate O2
concentrations Two reduced enzyme forms were used: (a)
free reduced DAAO (E-Flred, generated by reacting oxidized
DAAO with a small excess of d-alanine), and (b) the reduced
DAAO–imino acid complex (E-Flred–IA generated as above
but in the presence of 400 mm NH4Cl and 20 mm pyruvate
to generate imino pyruvate) Reaction rates for both the
reductive and oxidative half-reaction (Scheme 1) were
esti-mated from traces extracted at 455 and 530 nm by fitting
using the application Biokine32 (BioLogic) and one or
two exponential terms (e.g for a bi-exponential fit:
y= A e)k1t+ B e)k2t+ C, where A and B are amplitudes
and C is an initial absorbance value) The determined rate
constants were used to simulate the half-reactions and to
estimate the kinetic steps that cannot be detected
experimen-tally using Specfit software (Spectrum Software Associates,
Chapel Hill, NC)
Acknowledgements
This work was supported by grants from Fondo di
Ateneo per la Ricerca (University of Insubria, Varese,
Italy) We are grateful for the support of the
Consor-zio Interuniversitario per le Biotecnologie, and the
Centro di Ricerca in Biotecnologie per la Salute
Umana (University of Insubria)
References
1 Massey V & Hemmerich P (1980) Active-site probes of flavoproteins Biochem Soc Trans 8, 246–257
2 Massey V (1995) Introduction: flavoprotein structure and mechanism FASEB J 9, 473–475
3 Bornemann S (2002) Flavoenzymes that catalyse reac-tions with no net redox change Nat Prod Rep 19, 761– 772
4 Pollegioni L, Piubelli L, Sacchi S, Pilone MS & Molla
G (2007) Physiological functions of d-amino acid oxidases: from yeast to humans Cell Mol Life Sci 64, 1373–1394
5 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 dehydrogenation Proc Natl Acad Sci USA 97, 12463– 12468
6 Porter DJ, Voet JG & Bright HJ (1977) Mechanistic features of the d-amino acid oxidase reaction studied by double stopped flow spectrophotometry J Biol Chem
252, 4464–4473
7 Pollegioni L, Langkau B, Tischer W, Ghisla S & Pilone
MS (1993) Kinetic mechanism of d-amino acid oxidases from Rhodotorula gracilis and Trigonopsis variabilis
J Biol Chem 268, 13850–13857
8 Klinman JP (2007) How do enzymes activate oxygen without inactivating themselves? Acc Chem Res 40, 325–333
9 Mattevi A (2006) To be or not to be an oxidase: chal-lenging the oxygen reactivity of flavoenzymes Trends Biochem Sci 31, 276–283
10 Rosini E, Pollegioni L, Ghisla S, Orru R & Molla G (2009) Optimization of d-amino acid oxidase for low substrate concentrations – towards a cancer enzyme therapy FEBS J 276, 4921–4932
11 Furse KE, Pratt DA, Schneider C, Brash AR, Porter
NA & Lybrand TP (2006) Molecular dynamics simula-tions of arachidonic acid-derived pentadienyl radical intermediate complexes with COX-1 and COX-2: insights into oxygenation regio- and stereoselectivity Biochemistry 45, 3206–3218
12 Hiromoto T, Fujiwara S, Hosokawa K & Yamaguchi
H (2006) Crystal structure of 3-hydroxybenzoate hydroxylase from Comamonas testosteroni has a large tunnel for substrate and oxygen access to the active site
J Mol Biol 364, 878–896
13 Johnson BJ, Cohen J, Welford RW, Pearson AR, Schulten K, Klinman JP & Wilmot CM (2007) Explor-ing molecular oxygen pathways in Hansenula polymor-phacopper-containing amine oxidase J Biol Chem 282, 17767–17776
14 Baron R, Riley C, Chenprakhon P, Thotsaporn K, Winter RT, Alfieri A, Forneris F, van Berkel WJH,