On the reaction of D-amino acid oxidase with dioxygen: O2 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 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 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 controlled We also have recently described channels that might allow access of oxygen to pockets at the active site of the flavoprotein D-amino acid oxidase (DAAO) that have a high affinity for dioxygen and are in close proximity to the flavin With the goal of enhancing the reactivity of DAAO with oxygen, we have performed site-saturation mutagenesis at three positions 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 properties of these variants have been studied and compared with those of wildtype DAAO, with emphasis on the reactivity of the reduced enzyme species with dioxygen The substitutions at positions 50 and 225 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 alteration 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 applications Introduction Flavins are highly versatile co-factors of flavoproteins that catalyze a wide array of chemical and photochemical processes [1–3] As a prominent member of this family, 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 reductive half-reaction (Scheme 1a) and oxidation of reduced co-factor (FADH)) by O2 in the oxidative half-reaction, forming H2O2 as 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 Abbreviations DAAO, D-amino acid oxidase (EC 1.4.3.3); E-Flox, oxidized enzyme form; E-Flred, reduced enzyme form; E-Flred–IA, reduced enzyme–imino acid complex; IA, imino acid 482 FEBS Journal 278 (2011) 482–492 ª 2010 The Authors Journal compilation ª 2010 FEBS Modulation of O2 reactivity in D-amino acid oxidase E Rosini et al k1 (a) E-Flox + AA k–1 (b) E-Flred + O2 (c) E-Flred–IA + O2 k6 k5 k2 E-Flox–AA E-Flred–IA k–2 k–5 E-Flred + IA E-Flox k3 E-Flox–IA k4 E-Flox–IA (d) et/v = Φ0 + ΦD-Ala / [D-Ala] + ΦO2 / [O2] + ΦD-ALa,O2 / [D-Ala]•[O2] et/v = [(k2 + k4)/(k2•k4)] + [(k–1 + k2)/(k1•k2•[D-Ala])] + [(k2 + k–2)/(k2•k3•[O2])] + [(k–1 + k–2)/(k1•k2•k3•[D-Ala]•[O2])] where: kcat = 1/Φ0; Km,D-Ala = ΦD-Ala/Φ0; Km,O2 = ΦO2/Φ0 For wild-type DAAO (k4»k2), the expressions can be simplified: kcat = [k2•k4/k2 + k4)] ≈ k2 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 Scheme Kinetic steps in the catalytic cycle proposed for DAAO [6,7]: (a) reductive half-reaction, (b,c) oxidative half-reaction, and (d) correlation between steady-state kinetic parameters and single rate constants, and their reduction for wild-type DAAO [7] the absence of observable intermediates In oxidases, the reaction of reduced flavin with O2 proceeds 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 106 m)1Ỉs)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 highaffinity 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, specifically 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, resulting 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 hemedependent 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 pockets 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 O2 molecule at site A or B is  15 kJỈmol)1 lower than the corresponding energy required to place O2 in the solvent; thus, the probability of finding a molecule of O2 at these sites is correspondingly 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 ˚  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 variant 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 oxygen channels The most interesting substitutions at positions 50, 201 and 225 were identified by a screening 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 FEBS Journal 278 (2011) 482–492 ª 2010 The Authors Journal compilation ª 2010 FEBS 483 Modulation of O2 reactivity in D-amino acid oxidase E Rosini et al A B Fig (A) O2 channel 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 O2 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] provided 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 modifying size and polarity of this side chain could result in major alterations in O2 accessibility through the proposed 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, 484 and two or three variants for each single position showing altered oxygen reactivity were selected by a screening procedure performed at low oxygen concentration (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 variants 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 nm of  12 600 m)1Ỉcm)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 apoprotein moiety The substitutions introduced at position 50 alter the tertiary structure of DAAO (Fig S2) as well as the protein stability: the W50 DAAO variants 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 ± mV was determined for the W50P variant versus ) 109 mV for wild-type DAAO [19] The conformation and flavin properties of DAAO variants at position 201 and 225 are not significantly affected by the substitutions introduced, with only a slight alteration in the ability to stabilize the flavin semiquinone species (Appendix S1) FEBS Journal 278 (2011) 482–492 ª 2010 The Authors Journal compilation ª 2010 FEBS Modulation of O2 reactivity in D-amino acid oxidase E Rosini et al The apparent kinetic parameters of DAAO variants were determined using d-alanine as the substrate by an oxygen consumption assay at 21% oxygen saturation and 25 °C (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 position 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-alanine were mixed in a stopped-flow instrument, and absorbance spectra were recorded continuously in the 300–700 nm range at 15 °C [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 wildtype and other variants of DAAO [7,10,16] The results of these steady-state measurements (at saturating O2 and d-alanine concentrations; Scheme 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 the I225V DAAO, although the corresponding 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 kcat and Km;O2 , respectively) against [d-alanine] show an unprecedented sigmoidal 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 In contrast, the I225F variant does not show any sigmoidal behavior, but an  20-fold decrease Fig 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 mM) and 0.25 mM 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] FEBS Journal 278 (2011) 482–492 ª 2010 The Authors Journal compilation ª 2010 FEBS 485 Modulation of O2 reactivity in D-amino acid oxidase E Rosini et al Table 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 mM sodium pyrophosphate buffer, pH 8.5, 1% glycerol and 0.25 mM 2-mercaptoethanol The steady-state F parameters are defined in Scheme Lineweaver–Burk plot behavior Wild-type [10] W50F W50P W50R T201L T201V I225F I225V G52V [16] Convergent Parallel Parallel Parallel Parallel Parallel Parallel Parallel (sigmoidal n = 2.2) Convergent kcat (s)1) FD-alanine (MỈs)1) 330 ± 190 ±  4.0 62 ± 170 ± 365 ± 15 ± 98 ± (0.8 (2.0 (3.6 (1.7 (3.6 (1.7 (8.6 (8.0 30 20 15 50 2.1 0.33 ± 0.03 ± ± ± ± ± ± ± ± 0.1) 0.3) 0.3) 0.2) 0.5) 0.3) 0.4) 0.4) · · · · · · · · 10)5 10)5 10)3 10)3 10)5 10)5 10)5 10)5 (1.2 ± 0.2) · 10)5 in kcat compared to wild-type DAAO is apparent (Fig S3C) Km,D-alanine (mM) 2.6 3.9 14.4 10.4 6.2 6.9 1.2 0.9 ± ± ± ± ± ± ± ± 0.4 0.7 3.4 1.5 0.8 0.7 0.1 0.1 0.036 ± 0.005 FO2 (MỈs)1) (5.0 (7.7 (50 (8.8 (2.3 (9.4 (85 (11 ± ± ± ± ± ± ± ± 0.1) · 10)6 0.4) · 10)6 4) · 10)6 1.7) · 10)6 0.05) · 10)6 0.1) · 10)6 4) · 10)6 1) · 10)6 (4.4 ± 1.0) · 10)4 Km;O2 (mM) 1.9 1.4 0.16 0.54 0.40 3.9 1.2 0.8 ± ± ± ± ± ± ± ± FD-alanine,O2 (M2Ỉs)1 · 10)9) 0.1 0.5 0.04 0.18 0.05 0.1 0.4 0.1 3.0 ± 0.2 0.15 ± 0.03 12.1 ± 1.2 A 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 15 °C, 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 intermediate that is then converted at a slower rate 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 process (formation of an initial enzyme–substrate complex), 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, corresponding to product dissociation from E-Flred–IA (k5 in 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 k5 value is estimated to be £ 0.1 s)1 (Table 2) 486 B Fig 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 ( lM) were reacted under anaerobic conditions with 0.25 mM D-alanine at pH 8.5 and 15 °C (D-alanine concentration of mM for W50P) The rate constants were obtained by fitting using a double exponential equation (continuous line) The rates are listed in Table FEBS Journal 278 (2011) 482–492 ª 2010 The Authors Journal compilation ª 2010 FEBS Modulation of O2 reactivity in D-amino acid oxidase E Rosini et al Table 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 Data were obtained in 50 mM sodium pyrophosphate buffer, pH 8.5, 1% glycerol and 0.25 mM 2-mercaptoethanol Reductive half-reaction Oxidative half-reaction a )1 kobs1 ( k2) (s ) Wild-type W50F W50P W50R T201L T201V I225F I225V c G52V ‡ 250 170 ± 3.5 ± 130 ± 250 ± ‡ 250 79 ± 160 ± 30 0.4 20 15 21 ‡ 550 )1 Kd (k)1 ⁄ k1) (mM) 2.1 1.2 14.6 2.3 3.1 2.6 0.5 1.7 ± ± ± ± ± ± ± ± 0.5 0.3 2.6 0.3 0.2 0.4 0.1 0.3 kobs2 ( k5) (s ) 1.2 1.7 £ 0.1 0.7 1.8 0.8 1.3 1.4 2 ± 0.2 ± 0.2 ± ± ± ± ± 0.2 0.5 0.2 0.2 0.2 1.6 ± 0.2 k3 from E-Flred–IA (M)1Ỉs)1) · 105 2.4 ± 2.3 ± n.f n.f 4.2 ± 1.4 ± 1.4 ± 1.0 ± 0.3 [3.0 · 104] 0.1 [2.9 · 104] 0.4 [9.0 · 104] 0.2 [3.8 · 104] 0.1 [0.7 · 104] 0.1 NF (0.024 ± 0.008) b k6 from E-Flred (M)1Ỉs)1) · 104 3.6 ± 0.5 3.8 ± 0.2 4.0 ± 0.5 2.1 ± 0.3 11.0 ± 0.5 2.9 ± 0.2 1.2 ± 0.2 Biphasic: £ s)1 (60% amplitude); 2.9 ± 0.1 (40% amplitude) 0.046 ± 0.002 a Buffer as above containing 20 mM glucose, 20 mM pyruvate and 400 mM NH4Cl 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 (k6 in Scheme 1b) are shown in parentheses b Buffer as above containing 20 mM 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) Oxidative half-reaction The (re)oxidation of reduced DAAO variants by dioxygen (Scheme 1b,c) was also studied using the stoppedflow 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 species 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 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 £ s)1, and for which the value and amplitude ( 60% of the overall absorbance change) not depend on oxygen concentration The kobs values for the faster phase of re-oxidation of the I225V variant show a linear dependence on [O2], with no indication of oxygen saturation: the k6 bimolecular rate constant is unchanged for I225V variants compared to wild-type DAAO (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 pyruvate; 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 corresponds 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, FEBS Journal 278 (2011) 482–492 ª 2010 The Authors Journal compilation ª 2010 FEBS 487 Modulation of O2 reactivity in D-amino acid oxidase E Rosini et al 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-oxidized 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 oxygen reactivity of yeast DAAO, a change that does not modify the kcat value (i.e the maximal activity at saturating 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-Flred and the E-Flred–IA complex for O2-induced re-oxidation (Scheme 1) A B C Discussion Fig 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 mixing of  10 lM reduced enzyme with 153 lM oxygen (vertical bars = experimental data points) Conditions were as described in Experimental procedures The mono-exponential fit of the experimental 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 mM pyruvate and 400 mM ammonium chloride) wild-type and T201L DAAOs by 153 lM oxygen A mono-exponential fit was used for the re-oxidation of E-Flred and a bi-exponential equation was used for re-oxidation of the E-Flred–IA complex (C) Comparison of re-oxidation of E-Flred wild-type and I225V DAAOs by 0.6 mM 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 and 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 488 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 O2 in 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 ⁄ product binding site; Fig 1) In a previous study, we investigated the role of the residue G52: in the G52V variant, the valine side chain occupies the site that has the highest 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 O2 high-affinity sites The substitution of W50 (a residue close to the tunnel entrance; Fig 1A) with R or P significantly destabilizes 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 stabilization of the flavin anionic semiquinone and the midpoint 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 FEBS Journal 278 (2011) 482–492 ª 2010 The Authors Journal compilation ª 2010 FEBS E Rosini et al a 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 reductive 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 and 2) The I225V variant shows an unprecedented sigmoidal behavior in the kcat versus [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 existence 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 (£ s)1; Table 2) Similar behavior was previously observed for the F359W variant of Streptomyces cholesterol oxidase [23], and was related to kinetic cooperativity known as the mnemonic 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 displays kinetic cooperativity with respect to the first substrate 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 product The reaction of the reduced enzyme forms with oxygen is negatively affected by substitution of I225: the most significant change is an approximately threefold decrease in k3 as observed for the I225F variant versus wild-type DAAO With regard to position 201 (close to both highaffinity 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 and Table S2) However, introduction of leucine 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 and 2) Modulation of O2 reactivity in D-amino acid oxidase 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: symmetrically 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 cholesterol oxidases [23,25] These results suggest that multiple 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 compared 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 ( A apart), and are connected through a pathway that has a low activation energy barrier [16]: site B appears to increase the effective 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 distance 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 O2 reactivity 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 FEBS Journal 278 (2011) 482–492 ª 2010 The Authors Journal compilation ª 2010 FEBS 489 Modulation of O2 reactivity in D-amino acid oxidase A E Rosini et al B C Fig 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] for 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] type) A significantly lower yield was achieved for the W50P variant DAAO (0.6 mg per g cell paste and mgỈL)1 of fermentation broth) The expression of DAAO mutants at positions 201 and 225 was similar to that of the wildtype DAAO ( mg enzyme per g cell paste and  11 mgỈL)1 of fermentation broth) Experimental procedures Spectral properties Site-saturation mutagenesis and enzyme expression and purification Extinction coefficients of the oxidized form of DAAO variants were determined by heat denaturation of the enzymes (at 95 °C for 10 min) and using the absorption coefficient for free FAD of 11.3 mm)1Ỉcm)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 mm EDTA and 0.5 lm 5-deazaflavin [26] The amount of the thermodynamically stable semiquinone form was evaluated after incubation for 24 h at °C or after adding 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 15 °C and 0.1 mgỈmL)1 protein 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 (0.5 °CỈmin)1) Circular dichroism (CD) spectra were recorded at 15 °C using a Jasco J-810 spectropolarimeter and analyzed by means of Jasco software The cell path Site-saturation mutagenesis at positions 50, 201 and 225 was performed on DAAO cDNA subcloned into pT7-HisDAAO 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 O2 concentration (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, mm EDTA and 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 ( mg protein per liter of fermentation broth versus 11 mg for the wild- 490 FEBS Journal 278 (2011) 482–492 ª 2010 The Authors Journal compilation ª 2010 FEBS E Rosini et al length was 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 25 °C 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 lmol of d-alanine per minute at 25 °C Steady-state and pre-steady-state stopped-flow experiments were performed in 50 mm sodium pyrophosphate, pH 8.5, containing 1% v ⁄ v glycerol and 0.25 mm 2-mercaptoethanol, at 15 °C in a BioLogic SFM-300 instrument equipped with a J&M diode array detector (BioLogic, Grenoble, France) as described previously [7,10] Steadystate kinetic parameters were determined by the enzymemonitored 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 concentrations in the absence of dioxygen (the final solutions contained 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 estimated 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 experimentally 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 Consorzio Interuniversitario per le Biotecnologie, and the Centro di Ricerca in Biotecnologie per la Salute Umana (University of Insubria) Modulation of O2 reactivity in D-amino acid oxidase References Massey V & Hemmerich P (1980) Active-site probes of flavoproteins Biochem Soc Trans 8, 246–257 Massey V (1995) Introduction: flavoprotein structure and mechanism FASEB J 9, 473–475 Bornemann S (2002) Flavoenzymes that catalyse reactions with no net redox change Nat Prod Rep 19, 761– 772 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 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 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 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 Klinman JP (2007) How enzymes activate oxygen without inactivating themselves? 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the spectral properties related to protein conformation for W50 variants of DAAO Fig S3 Steady-state measurements of oxygen consumption by DAAO variants Table S1 Comparison of melting temperatures as determined by various approaches for the wild-type and W50 variants of DAAO Table S2 Comparison of the ligand-binding properties and apparent steady-state kinetic parameters on d-alanine as substrate determined for wild–type and variants of DAAO Table S3 Comparison of the substrate specificity of wild-type and I225F DAAOs Appendix S1 Biochemical properties of DAAO variants This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 278 (2011) 482–492 ª 2010 The Authors Journal compilation ª 2010 FEBS ... larger than the maximal O2 concentration in solution) [6–9] These enzymes appear to ‘consume’ O2 without highaffinity binding Because of the scarcity of information concerning the reaction of flavoproteins... 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... higher using the E-Flred–IA complex (Fig 4B and Tables and 2) Modulation of O2 reactivity in D-amino acid oxidase Functional data on flavo -oxidase variants designed on the basis of molecular dynamic