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

Importance of the gating segment in the substrate-recognition loop of pyranose 2-oxidase Oliver Spadiut 1, *, Tien-Chye Tan 1,2, *, Ines Pisanelli 3 , Dietmar Haltrich 3 and Christina Divne 1,2 1 KTH Biotechnology, Royal Institute of Technology, Stockholm, Sweden 2 Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden 3 Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU – University of Natural Resources and Applied Life Sciences, Vienna, Austria Keywords active-site loop; alanine-scanning mutagenesis; crystal structure; pyranose 2-oxidase; site-saturation mutagenesis Correspondence C. Divne, KTH Biotechnology, Royal Institute of Technology, Albanova, Roslagstullsbacken 21, SE-106 91 Stockholm, Sweden Fax: +46 8 5537 8468 Tel: +46 8 5537 8296 E-mail: divne@biotech.kth.se or D. Haltrich; Food Biotechnology Laboratory, Department of Food Science and Technology, BOKU – University of Natural Resources and Applied Life Sciences, A-1190 Vienna, Austria Fax: +43 1 47654 6251 Tel: +43 1 47654 6140 E-mail: dietmar.haltrich@boku.ac.at *These authors contributed equally to this work Database The atomic coordinates and structure factors for the models are available in the Protein Data Bank database under the accession numbers 3K4J (H450Q), 3K4K (F454N), 3K4L (F454N 2FG ), 3K4M (Y456W 2FG ) and 3K4N (F454A ⁄ S455A ⁄ Y456A) (Received 22 February 2010, revised 2 May 2010, accepted 6 May 2010) doi:10.1111/j.1742-4658.2010.07705.x Pyranose 2-oxidase from Trametes multicolor is a 270 kDa homotetrameric enzyme that participates in lignocellulose degradation by wood-rotting fungi and oxidizes a variety of aldopyranoses present in lignocellulose to 2-ketoaldoses. The active site in pyranose 2-oxidase is gated by a highly conserved, conformationally degenerate loop (residues 450–461), with a conformer ensemble that can accommodate efficient binding of both elec- tron-donor substrate (sugar) and electron-acceptor substrate (oxygen or quinone compounds) relevant to the sequential reductive and oxidative half-reactions, respectively. To investigate the importance of individual resi- dues in this loop, a systematic mutagenesis approach was used, including alanine-scanning, site-saturation and deletion mutagenesis, and selected variants were characterized by biochemical and crystal-structure analyses. We show that the gating segment ( 454 FSY 456 ) of this loop is particularly important for substrate specificity, discrimination of sugar substrates, turn- over half-life and resistance to thermal unfolding, and that three conserved residues (Asp 452 , Phe 454 and Tyr 456 ) are essentially intolerant to substitu- tion. We furthermore propose that the gating segment is of specific impor- tance for the oxidative half-reaction of pyranose 2-oxidase when oxygen is the electron acceptor. Although the position and orientation of the slow substrate 2-deoxy-2-fluoro-glucose when bound in the active site of pyra- nose 2-oxidase variants is identical to that observed earlier, the substrate- recognition loop in F454N and Y456W displays a high degree of confor- mational disorder. The present study also lends support to the hypothesis that 1,4-benzoquinone is a physiologically relevant alternative electron acceptor in the oxidative half-reaction. Abbreviations ABTS, 2,2¢-azinobis(3-ethylbenz-thiazolinesulfonic acid; 2FG, 2-deoxy-2-fluoro- D-glucose; BQ, 1,4-benzoquinone; Fc + , ferrocenium ion; P2O, pyranose 2-oxidase; PDB, Protein Data Bank; TLS, translation, libration, screw-rotation. 2892 FEBS Journal 277 (2010) 2892–2909 ª 2010 The Authors Journal compilation ª 2010 FEBS Introduction Pyranose 2-oxidase (P2O; pyranose:oxygen 2-oxidore- ductase; EC 1.1.3.10) from Trametes multicolor (syno- nym Trametes ochracea) is a 270 kDa, 8a-(N3)-histidyl flavinylated, homotetrameric flavoprotein oxidase found in the fungal hyphal periplasmic space [1–5]. P2O has been suggested to perform a dual function during lignin degradation by wood-rotting fungi, by providing H 2 O 2 for ligninolytic enzymes [6,7] and reducing quinones as part of the extracellular quinone redox cycling machinery [8]. In some white-rot basid- iomycetes, P2O participates in a secondary meta- bolic pathway in which d-glucose is first converted to 2-keto-d-glucose (2-arabino-hexos-2-ulose; d-gluco- sone; (Scheme 1) and then further oxidized by aldos-2-ulose dehydratase to the b-pyrone antibiotic cortalcerone [9,10]. During the reductive half-reaction, P2O catalyses the oxidation at the C2 of several aldopyranoses (Scheme 1) present in lignocellulose to the correspond- ing 2-ketoaldoses, accompanied by electron transfer to FAD, yielding the reduced flavin, FADH 2 [11]. In the oxidative half-reaction, the cofactor is re-oxidized by an electron acceptor (e.g. O 2 or quinone compounds), producing either H 2 O 2 or reduced quinone [1,8]. The reaction mechanism is of the Ping Pong Bi Bi type [12], which is common in flavoprotein oxidoreductases [13,14]. In analogy with the hydride-transfer mecha- nism proposed for flavoproteins in general [15,16], and in particular for the reductive half-reaction of Phanero- chaete chrysosporium cellobiose dehydrogenase [17,18], which is the closest relative of P2O among glucose- methanol-choline family members, His 548 in P2O is suitably positioned to act as a general base to deproto- nate the equatorial substrate 2-OH group, with support from an O2–Asn 593 N d2 hydrogen bond, accompanied by transfer of the axial 2-hydrogen atom as a hydride from C2 to the flavin N5 atom [5,19]. The two half- reactions catalysed by P2O (i.e. oxidation of electron donor and reduction of electron acceptor) have differ- ent prerequisites with respect to the local chemical and structural environment. Our crystal structures of a closed state of wild-type P2O with the competitive inhibitor acetate bound (Fig. 1A) [5], and an open state with a slow sugar substrate bound (Fig. 1B) [19], have shown that a conserved substrate-recognition loop 450 HRDAFSYGAVQQ 461 is highly dynamic and offers a conformational gating mechanism to P2O. In T. multicolor P2O, b-d-glucose is oxidized regi- oselectively at C2 without traces of 3-keto product. For the related Peniophora gigantea P2O, however, oxidation at C3 can take place as a side reaction when the glucose 2-OH group is either absent (e.g. 2-deoxy-d-glucose) or modified (e.g. 2-keto-d-glucose) [11]. This was also observed for the T. multicolor P2O Scheme 1. Substrates (electron acceptors and donors) discussed in the text. O. Spadiut et al. Substrate-recognition loop mutations in P2O FEBS Journal 277 (2010) 2892–2909 ª 2010 The Authors Journal compilation ª 2010 FEBS 2893 mutant H167A with the slow substrate 2-deoxy-2-flu- oro-d-glucose (2FG) (Scheme 1, Fig. 1B) [19]. In the H167A variant, the flavinylation ligand His 167 has been mutated to an alanine and the FAD is noncova- lently bound. It was also shown that the flavin reduc- tion was significantly slower than in the wild-type, which enabled structure determination of an ordered complex with 2FG [19]. The ability of the enzyme to oxidize some substrates at both C2 and C3 requires that the sugar can bind in two productive binding modes. On the basis of the in silico modelling of glu- cose, we have suggested that these two binding modes are related by a 180° rotation about an axis running through two points in the pyranose ring (one point between C5 and O5, and the other between C2 and C3), producing almost isosteric substrate-binding modes [19]. Although d-galactose (Scheme 1) is a relatively poor substrate for wild-type P2O (6% relative activity compared to d-glucose) [8], it is struc- turally very similar to d-glucose, differing only by the axial C4 hydroxyl group (equatorial in glucose), and thus, the C4 position must be important for the substrate selectivity mechanism in P2O. We have shown that d-galactose cannot be well accommodated in the P2O active site as a result of possible steric hindrance between the axial O4 and the side chain of Thr 169 [20,21]. Engineering of P2O for improved d-galactose turnover is also highly relevant for indus- trial purposes because C2 oxidation of d-galactose yields 2-keto-d-galactose, which can be further reduced at C1 to give the low caloric sweetener d-tagatose [22]. In the present study, we report the results obtained from a systematic mutagenesis approach that aimed to investigate the importance of key amino acids in the substrate-recognition loop of T. multicolor P2O by means of alanine-scanning, site-saturation and deletion mutagenesis, as well as the characterization of mutants by biochemical and crystal-structure analysis. We dis- cuss the catalytic competence and stability of the mutants and their preference for electron donors and electron acceptors in light of the steady-state kinetics, stability and structural data presented. The finding of the present study demonstrate that the gating segment of the substrate-recognition loop is of particular importance for P2O function and substrate specificity, as well as for catalytic and thermal stability at elevated temperatures. AB Fig. 1. Overall P2O subunit structure of the closed and open state. Subunit structure of (A) wild-type P2O in complex with acetate (PDB code 1TT0) with the substrate-recognition loop closed [5] and (B) the P2O variant H167A in complex with 2FG (PDB code 2IGO) where the loop is completely open [19]. The H167A mutant has the flavinylation ligand His 167 mutated to alanine, and the FAD is noncovalently bound. The Rossmann domain is coloured pink, and the substrate-binding domain is shown in light blue. Loops are shown in beige and the FAD cofactor is shown in yellow. The ‘head domain’, which is not present in related glucose-methanol-choline members, is shown in light green. The substrate-recognition loop (residues 450–461) is highlighted as a purple coil, with the side chains that form the gating segment (i.e. Phe 454 , Ser 455 and Tyr 456 ) shown as stick representations. The ligands (coloured bright green), acetate in the closed form and 2FG in the open state, are bound at the re face of the isoalloxazine ring. Substrate-recognition loop mutations in P2O O. Spadiut et al. 2894 FEBS Journal 277 (2010) 2892–2909 ª 2010 The Authors Journal compilation ª 2010 FEBS Results Site-saturation, alanine-scanning and deletion mutagenesis Site-saturation mutagenesis was employed to generate a library of T. multicolor P2O variants targeting the substrate-recognition loop, combined with high- throughput screening of mutants in a 96-well plate format. A procedure for generating and screening P2O variants targeting position 450 has been described previously [23]. We used this approach to generate enzyme variants by targeting the positions 452, 454 and 456. The P2O mutant library covered > 95% of all possible combinations of variants at the selected positions (His 450 , Asp 452 , Phe 454 and Tyr 456 ), and high- throughput screening included 360 colonies for each position, which were tested for activity towards two electron-donor substrates: d-glucose and d-galactose. The statistics show that a large number of mutations at these positions result in inactive P2O variants: 60%, 44%, 56% and 48% inactive variants for His 450 , Asp 452 , Phe 454 and Tyr 456 , respectively (site-saturation mutagenesis at position 450 has been reported sepa- rately) [23]. This demonstrates the importance of the targeted loop residues for enzymatic activity and ⁄ or proper folding and stability. In addition, alanine-scan- ning and site-directed mutagenesis were performed. A set of mutants that displayed interesting characteris- tics with respect to d-glucose or d-galactose turnover were selected and subjected to more detailed analysis. These included the single-substitution variants H450Q, F454P, F454N and Y456W; two multiple-alanine mutants targeting the gating segment (F454A ⁄ Y456A and F454A ⁄ S455A ⁄ Y456A); and one deletion mutant lacking the FSY segment ( D454–456). Typical yields of mutant P2Os were in the range 15–30 mgÆL )1 culture medium, although the D454–456 and alanine mutants were expressed at significant lower levels (0.2– 0.8 mgÆL )1 ) and overexpression was accompanied by an increase in inclusion-body formation. Kinetic characterization of mutants Apparent kinetic constants were determined for the loop variants as a preliminary means to assess the effect of these mutations on the P2O-catalysed reaction; accordingly, one of the substrates of P2O (either the electron acceptor or donor) was fixed, with the other one being varied, and the obtained data were fit to the Michaelis–Menten equation for a single substrate. For all variants, the apparent k app cat values with d-glucose and O 2 (fixed at a concentration of 256 lm, air satura- tion) as substrates, k cat[Glc ⁄ O2] , decreased dramatically compared to the wild-type enzyme, and K m[Glc] values increased by a factor in the range 1.9–3.3 (Table 1), resulting in decreased catalytic efficiency constants (k cat[Glc ⁄ O2] ⁄ K m[Glc] ). The only exceptions are Y456W, a conservative mutation with one bulky hydrophobic amino acid replacing another one, which shows a simi- lar k cat[Glc ⁄ O2] , albeit with a doubled K m[Glc] and an associated three-fold decrease in k cat[Glc ⁄ O2] ⁄ K m[Glc] ,as well as H450Q, where k cat[Glc ⁄ O2] was decreased by a factor of 2. Consistently, turnover numbers for the elec- tron donor ⁄ acceptor substrate pair d-galactose ⁄ O 2 for the variants H450Q and Y456W were comparable to that of the wild-type. Other variants, in particular the Ala mutants and D454-456, showed three- to 12-fold lower k cat[Gal ⁄ O2] , and up to six-fold elevated K m[Gal] values (Table 1). Mutations in the gating segment affected sugar substrate specificity significantly. The wild-type enzyme displays a clear preference for d-glucose over d-galac- tose, as indicated by a selectivity ratio of  160, with Table 1. Apparent steady-state kinetic constants of T. multicolor P2O wild-type and mutants with D-glucose (0.1–50 mM)orD-galactose (0.1–200 m M) as electron donor and O 2 (air) under saturation as electron acceptor. Variant Glc ⁄ O 2 Gal ⁄ O 2 K m [Glc] (m M) k cat [Glc ⁄ O 2 ] (s )1 ) k cat ⁄ K m [Glc] ( M )1 Æs )1 ) K m [Gal] (m M) k cat [Gal ⁄ O 2 ] (s )1 ) k cat [Gal ⁄ O 2 ] ⁄ K m [Gal] ( M )1 Æs )1 ) Wild-type 0.76 ± 0.05 33 ± 0 43 · 10 3 6.1 ± 0.3 1.7 ± 0 0.27 · 10 3 H450Q 2.5 ± 0.3 17 ± 1 7.0 · 10 3 34 ± 7 2.0 ± 0.1 0.059 · 10 3 F454P 1.8 ± 0.3 0.99 ± 0.04 0.54 · 10 3 13 ± 1 0.30 ± 0.01 0.024 · 10 3 F454N 1.5 ± 0.1 12 ± 0 8.2 · 10 3 26 ± 2 1.2 ± 0 0.046 · 10 3 Y456W 1.7 ± 0.3 26 ± 1 15 · 10 3 29 ± 2 1.5 ± 0 0.053 · 10 3 F454A ⁄ Y456A 1.5 ± 0.2 7.1 ± 0.2 4.7 · 10 3 10 ± 1 0.64 ± 0.02 0.062 · 10 3 F454A ⁄ S455A ⁄ Y456A 2.1 ± 0.3 0.20 ± 0.01 0.094 · 10 3 13 ± 3 0.14 ± 0.01 0.011 · 10 3 D454–456 1.4 ± 0 3.1 ± 0 2.2 · 10 3 7.7 ± 0.9 0.29 ± 0.01 0.038 · 10 3 O. Spadiut et al. Substrate-recognition loop mutations in P2O FEBS Journal 277 (2010) 2892–2909 ª 2010 The Authors Journal compilation ª 2010 FEBS 2895 the ratio of the specificity constants [24] for the two substrates being [(k cat[Glc ⁄ O2] ⁄ K m[Glc] ) ⁄ (k cat[Gal ⁄ O2] ⁄ K m[Gal] )]. Most of the mutations in the gating segment reduced the selectivity ratio. The lowest ratio [(k cat[Glc ⁄ O2] ⁄ K m[Glc] ) ⁄ (k cat[Gal ⁄ O2] ⁄ K m[Gal] )] of  8.4 was observed for the triple-alanine mutant, which, however, still retains some preference for d-glucose over d-galactose. By contrast, the replacements F454N or Y456W show increased [(k cat[Glc ⁄ O2] ⁄ K m[Glc] ) ⁄ (k cat[Gal ⁄ O2] ⁄ K m[Gal] )] values of  180 and 290, respectively. This effect on substrate selectivity is even more pronounced when considering the disaccharide melibiose (D-Gal-a(1 fi 6)-D-Glc; Scheme 1). Melibiose is a rather poor substrate for P2O, mainly because of its very high K app m value (K m[Mel] = 1530 mm and k cat = 7.6 for wild-type; Table 2), and the selectivity ratio [(k cat[Glc ⁄ O2] ⁄ K m[Glc] ) ⁄ (k cat[Mel ⁄ O2] ⁄ K m[Mel] )] for wild-type is  8700, indicat- ing very strong discrimination of P2O in favour of the monosaccharide substrate d-glucose over the disaccha- ride melibiose, with oxygen as acceptor. Again, muta- tions in the gating segment of the substrate loop reduced the substrate selectivity for all of the variants to values in the range 12–1630. The lowest [(k cat[Glc ⁄ O2] ⁄ K m[Glc] ) ⁄ (k cat[Mel ⁄ O2] ⁄ K m[Mel] )] ratios of 93 and 12 were found for the F454P and triple-Ala mutant, respectively. Furthermore, apparent kinetic constants were deter- mined for the reduction of the two-electron acceptor 1,4-benzoquinone (BQ) to hydroquinone (Scheme 1, Table 3), and for the reduction of the 1-electron accep- tor ferrocenium (Fc + ) to ferrocene (Scheme 1, Table 4), with either d-glucose or d-galactose at satu- rating concentrations. Variant Y456W is characterized by improved BQ and Fc + binding, as indicated by lower K app m values, and increased k app cat values, resulting in an  2.5-fold higher k cat[BQ ⁄ Glc] ⁄ K m[BQ] and k cat[BQ ⁄ Gal] ⁄ K m[BQ] relative to wild-type. The triple-Ala and loop-deletion mutants also show considerably lower K m[BQ] with Glc as electron donor, although with an associated decrease in k cat[BQ ⁄ Glc] values. Similar results were obtained for the reduction of BQ with d-galac- tose, and for Fc + with d-glucose or d-galactose as sat- urating electron-donor substrates. In addition, H450Q, F454N and Y456W show improved kinetic properties Table 2. Apparent steady-state kinetic constants of T. multicolor P2O wild-type and mutants with melibiose (5.0–500 mM) as electron donor and O 2 (air) under saturation as electron acceptor. Variant Mel ⁄ O 2 Substrate selectivity K m [Mel] (mM) k cat [Mel ⁄ O 2 ](s )1 ) k cat [Mel ⁄ O 2 ] ⁄ K m [Mel] ( M )1 Æs )1 ) (k cat [Glc ⁄ O 2 ] ⁄ K m [Glc]) ⁄ (k cat [Mel ⁄ O 2 ] ⁄ K m [Mel]) Wild-type 1500 ± 300 7.6 ± 1.3 5.0 8700 H450Q 390 ± 50 3.3 ± 0.2 8.4 830 F454P 50 ± 4 0.29 ± 0.01 5.8 93 F454N 240 ± 40 2.7 ± 0.2 11.3 720 Y456W 260 ± 10 4.4 ± 0.1 16.6 920 F454A ⁄ Y456A 350 ± 110 1.3 ± 0.2 3.7 1300 F454A ⁄ S455A ⁄ Y456A 23 ± 4 0.19 ± 0.01 8.0 12 D454–456 210 ± 60 0.28 ± 0.04 1.3 1600 Table 3. Apparent steady-state kinetic constants of T. multicolor P2O wild-type and mutants with BQ (0.01–1.5 mM) as electron acceptor and D-glucose or D-galactose at saturation (100 mM each) as electron donor. Variant BQ ⁄ Glc BQ ⁄ Gal K m [BQ] (l M) k cat [BQ ⁄ Glc] (s )1 ) k cat [BQ ⁄ Glc] ⁄ K m [BQ] ( M )1 Æs )1 ) K m [BQ] (l M) k cat [BQ ⁄ Gal] (s )1 ) k cat [BQ ⁄ Gal] ⁄ K m [BQ] ( M )1 Æs )1 ) Wild-type 140 ± 20 160 ± 10 1.2 · 10 6 27 ± 4 3.8 ± 0.1 0.14 · 10 6 H450Q 240 ± 80 220 ± 30 0.94 · 10 6 13 ± 2 3.0 ± 0.1 0.23 · 10 6 F454P 72 ± 29 30 ± 4 0.42 · 10 6 7.1 ± 1.0 2.0 ± 0.1 0.28 · 10 6 F454N 52 ± 13 130 ± 10 2.6 · 10 6 5.2 ± 0.8 2.7 ± 0.1 0.52 · 10 6 Y456W 72 ± 15 220 ± 10 3.0 · 10 6 10 ± 1 3.3 ± 0.1 0.33 · 10 6 F454A ⁄ Y456A 29 ± 4 61 ± 2 2.1 · 10 6 78 ± 21 2.9 ± 0.3 0.037 · 10 6 F454A ⁄ S455A ⁄ Y456A 29 ± 11 15 ± 1 0.52 · 10 6 8.9 ± 1.0 1.2 ± 0 0.13 · 10 6 D454–456 41 ± 4 25 ± 1 0.60 · 10 6 37 ± 19 1.2 ± 0.2 0.032 · 10 6 Substrate-recognition loop mutations in P2O O. Spadiut et al. 2896 FEBS Journal 277 (2010) 2892–2909 ª 2010 The Authors Journal compilation ª 2010 FEBS for Fc + (lower K m and higher k cat values) both with glucose and galactose as saturating substrate compared to wild-type. Both BQ and Fc + are considerably larger molecules compared to O 2 , and, most likely, shorten- ing the loop or introducing smaller side chains pro- motes the reaction with the larger electron-acceptor substrates. Heat inactivation, pH optima, UV-visible spectra ThermoFAD analysis The half-life of P2O activity (i.e. the time during which the enzyme remains active) was measured for wild-type and mutants at constant pH (6.5) at 60 or 70 °C. The inactivation constant, k in , and the half-life of activity, s 1 ⁄ 2 , were determined (Table 5). On the basis of [ln(residual activity) versus time] plots, all mutants show first-order inactivation kinetics (Fig. 2). H450Q shows pronounced destabilization (Fig. 2A), whereas the other variants show similar or improved stability (Fig. 2A,B). The substitutions Phe 454 fi Asn, and Tyr 456 fi Trp result in a 29- and 34-fold increase in s 1 ⁄ 2 values at 60 °C, respectively. Interestingly, the ala- nine-substituted variants are also more stable at 60 °C, with a 12-fold and 23-fold increase in s 1 ⁄ 2 for F454A ⁄ Y456A and F454A ⁄ S455A ⁄ Y456A, respec- tively. Some stabilization is also seen for D454–456 (four- to five-fold increase) at 60 °C. A similar trend of increased heat inactivation half-life was observed for all variants at 70 °C (Fig. 2C). All variants show pH optima at pH 6.5 (data not shown), suggesting that the altered kinetics is not inti- mately correlated with changes in pH profile. All enzymes also display typical flavoprotein UV-visible spectra with absorption maxima k max at 345 and 456 nm (data not shown), and reduction of the enzymes with d-glucose and sodium dithionite in the absence of oxygen resulted in the disappearance of the absorption peak at 456 nm that was expected for the fully reduced state. FAD was not released upon trichloroacetic acid treatment, demonstrating that, despite extensive mutagenesis in the vicinity of the FAD-binding pocket, the mutants remain properly fla- vinylated (not shown). The thermal stability was inves- tigated using the ThermoFAD technique to derive thermal unfolding transition values (T m ). The T m val- ues are summarized in Table 6. Of the variants ana- lyzed, all but two mutants show slightly decreased T m values (1–3 °C). Y456W and F454A⁄ Y456A show improved T m values by 5.5 and 1.5 °C, respectively. Overall monomer structure of loop variants The mutants analyzed structurally include the unbound forms of H450Q, F454N and F454A ⁄ S455A ⁄ Y456A, and F454N or Y456W with bound 2FG. Data for the triple-Ala mutant were obtained to medium-low resolu- tion (2.75 A ˚ ), and the model is included mainly to evalu- ate the backbone conformation of the substrate loop. Table 4. Apparent steady-state kinetic constants of T. multicolor P2O wild-type and mutants with Fc + (0.005–1.5 mM) as electron acceptor and D-glucose or D-galactose under saturation (100 mM each) as electron donor. Variant Fc + ⁄ Glc Fc + ⁄ Gal K m [Fc + ] (l M) k cat [Fc + ⁄ Glc] (s )1 ) k cat [Fc + ⁄ Glc] ⁄ K m [Fc + ] ( M )1 Æs )1 ) K m [Fc + ] (l M) k cat [Fc + ⁄ Gal] (s )1 ) k cat [Fc + ⁄ Gal] ⁄ K m [Fc + ] ( M )1 Æs )1 ) Wild-type 400 ± 110 210 ± 30 0.51 · 10 6 100 ± 70 6.6 ± 1.8 0.063 · 10 6 H450Q 380 ± 110 470 ± 70 1.25 · 10 6 49 ± 15 17 ± 2 0.34 · 10 6 F454P 140 ± 60 67 ± 12 0.48 · 10 6 23 ± 14 4.5 ± 0.7 0.20 · 10 6 F454N 350 ± 210 420 ± 110 1.21 · 10 6 25 ± 10 7.3 ± 1.1 0.29 · 10 6 Y456W 240 ± 40 400 ± 30 1.64 · 10 6 41 ± 9 7.3 ± 0.4 0.18 · 10 6 F454A ⁄ Y456A 150 ± 30 74 ± 5 0.50 · 10 6 16 ± 7 2.9 ± 0.3 0.18 · 10 6 F454A ⁄ S455A ⁄ Y456A 280 ± 70 110 ± 10 0.37 · 10 6 15 ± 9 5.4 ± 0.7 0.36 · 10 6 D454–456 300 ± 60 15 ± 1 0.050 · 10 6 11 ± 1 1.3 ± 0.1 0.12 · 10 6 Table 5. Heat inactivation half-life of T. multicolor P2O wild-type and mutants at 60 and 70 °C. k in , inactivation constant; s 1 ⁄ 2 , half- life; ND, not determined. Variant 60 °C70°C k in (min )1 ) s 1 ⁄ 2 (60 °C) (min) k in (min )1 ) s 1 ⁄ 2 (70 °C) (min) Wild-type )58 · 10 )3 12 )9.9 0.07 H450Q )86 · 10 )3 8.1 ND ND F454P )67 · 10 )3 10 ND ND F454N )2.0 · 10 )3 350 )1.8 0.39 Y456W )1.7 · 10 )3 410 )1.1 0.63 F454A ⁄ Y456A )4.8 · 10 )3 140 )1.1 0.64 F454A ⁄ S455A ⁄ Y456A )2.5 · 10 )3 280 )1.2 0.60 D454–456 )13 · 10 )3 55 ND ND O. Spadiut et al. Substrate-recognition loop mutations in P2O FEBS Journal 277 (2010) 2892–2909 ª 2010 The Authors Journal compilation ª 2010 FEBS 2897 All structures show satisfactory model statistics (Table 7), and are very similar overall to the previously determined crystal structures of T. multicolor P2O [5,19,25,26], demonstrating that the mutations do not result in significant structural changes beyond the targeted region. In the unbound form of variants H450Q, F454N and F454A ⁄ S455A ⁄ Y456A, the sub- strate loop is in the open conformation. Therefore, structural comparisons are made with H167A rather than the wild-type because H167A has the substrate- recognition loop in the fully open conformation [19]. This is in agreement with our earlier observation that the substrate loop tends to be in the open state either when the active site is unoccupied, or when sugar (elec- tron donor) is bound. However, in the former case, we typically observe varying degrees of disorder of the loop, whereas the loop becomes well defined when sugar substrate is bound [19,25,26]. The same is observed in the present study where the unbound variants display varying degrees of fluctuation of the open state, which is manifested as weak but interpretable electron density indicative of multiple conformers. Furthermore, the two 2FG-bound F454N and Y456W models show the open state of the substrate-recognition loop. Structure of the Y456W-2FG complex Despite the larger tryptophan side chain, the substrate loop in Y456W 2FG (Fig. 3A) assumes the same open state as observed in H167A 2FG [Protein Data Bank (PDB) code 2IGO] [19]. However, the electron density is weak for the Trp 456 indole ring, as well as for the sub- strate loop, suggesting that the larger side chain induces local disorder and suboptimal side-chain packing. Despite this local disorder, the 2FG molecule is orderly bound in an orientation identical to that in H167A 2FG , corresponding to the C3-oxidation binding mode (Fig. 3B) (i.e. oriented for oxidation at the substrate C3 atom). The only notable difference is that the side chain of Asp 452 assumes a different conformation, offering the possibility of a hydrogen bond between its Od2 carboxylic oxygen and the glucosyl O1 of 2FG, thus replacing the 2FG O1-Gln 448 Ne2 interaction observed in H167A 2FG [19]. The Tyr 456 side chain of the open state in H167A 2FG is located some 13 A ˚ from the A B C Fig. 2. Inactivation kinetics of P2O wild-type and mutants. Inactiva- tion at 60 °C (pH 6.5). (A) , wild-type; , H450Q; , F454P; , F454N; , Y456W. (B) , wild-type; , F454A ⁄ Y456A; , F454A ⁄ S455A ⁄ Y456A; , D454–456. (C) Same as in (A) and (B), but at 70 °C. Table 6. Melting temperature T m of T. multicolor P2O wild-type and mutants. ND, not determined. Variant T m (°C) Wild-type 63.5 H450Q 60.5 F454P 62.0 F454N 62.5 Y456W 69.0 F454A ⁄ Y456A 65.0 F454A ⁄ S455A ⁄ Y456A 62.5 D454–456 ND Substrate-recognition loop mutations in P2O O. Spadiut et al. 2898 FEBS Journal 277 (2010) 2892–2909 ª 2010 The Authors Journal compilation ª 2010 FEBS Table 7. Data collection and crystallographic refinement statistics. Statistics for the high-resolution shell are given in parentheses. H450Q F454N F454N 2FG Y456W 2FG F454A ⁄ S455A ⁄ Y456A Data collection Cell constants: a, b, c (A ˚ ); b (°) 101.91, 101.91, 120.47 102.06, 102.06, 119.69 101.57, 101.57, 250.05 168.19, 103.14, 168.71; 106.467 101.58, 101.58, 250.00 Space group P4 2 2 1 2 P4 2 2 1 2 P4 3 2 1 2 P2 1 P4 3 2 1 2 Number of molecules ⁄ a.s.u. 1 1 2 8 2 Beamline, k (A ˚ ) I911-2, 1.0379 I911-2, 1.0379 I911-2, 1.0379 I911-2, 1.0379 I911-2, 1.0379 Resolution range (A ˚ ) 29–2.0 (2.10–2.00) 30–1.60 (1.70–1.60) 30–1.75 (1.80–1.75) 30–2.20 (2.30–2.20) 30–2.75 (2.80–2.75) Unique reflections 43 397 (5 784) 83 548 (13 637) 132 005 (10 540) 273 077 (32 973) 34 840 (1776) Multiplicity 14.5 (14.5) 12.9 (9.9) 7.3 (7.3) 2.7 (2.4) 6.8 (6.3) Completeness (%) 99.8 (99.9) 99.9 (99.6) 99.9 (99.9) 97.2 (94.6) 99.7 (99.8) <I ⁄ rI> 17.3 (5.2) 18.1 (3.1) 15.4 (2.5) 7.6 (1.5) 11.8 (2.6) R sym a (%) 18.7 (85.9) 10.0 (86.0) 10.2 (88.3) 12.9 (82.7) 19.2 (88.3) Refinement Resolution range (A ˚ ) 30–2.0 (2.11–2.00) 30–1.60 (1.69–1.60) 30–1.75 (1.84–1.75) 30–2.20 (2.32–2.20) 30–2.75 (2.90–2.75) Completeness % 99.9 (100) 99.9 (99.5) 99.9 (99.9) 97.4 (94.9) 99.8 (99.8) R factor b ⁄ work reflns 15.8 ⁄ 41 420 18.6 ⁄ 81 587 17.6 ⁄ 130 031 19.4 ⁄ 270 272 19.6 ⁄ 32 909 R free ⁄ free reflns 20.0 ⁄ 1990 21.3 ⁄ 1961 20.9 ⁄ 1974 25.8 ⁄ 2798 27.4 ⁄ 1931 Nonhydrogen atoms 4891 5021 9836 38 783 9264 Mean B (A ˚ 2 ) protein all ⁄ mc ⁄ sc 13.3 ⁄ 12.3 ⁄ 14.4 15.1 ⁄ 14.0 ⁄ 16.2 16.0 ⁄ 14.9 ⁄ 17.1 22.9 ⁄ 22.3 ⁄ 23.5 25.9 ⁄ 25.8 ⁄ 26.0 Mean B (A ˚ 2 ) solvent ⁄ number of molecules 22.4 ⁄ 309 27.1 ⁄ 437 25.2 ⁄ 726 23.9 ⁄ 1930 24.0 ⁄ 150 rmsd bond lengths (A ˚ ), angles (°) 0.022, 1.86 0.020, 1.84 0.021, 2.00 0.021, 1.98 0.018, 1.81 Ramachandran c favoured ⁄ allowed (%) 98.1 ⁄ 100 97.7 ⁄ 100 97.6 ⁄ 100 96.2 ⁄ 100 93.3 ⁄ 99.5 PDB accession code 3K4J 3K4K 3K4L 3K4M 3K4N a R sym =[R hkl R i |I ) <I>| ⁄ R hkl R i |I |] · 100%. b R factor = R hkl ||F o | ) |F c || ⁄ R hkl |F o |. c Ramachandran analysis according to MOLPROBITY [24a]. O. Spadiut et al. Substrate-recognition loop mutations in P2O FEBS Journal 277 (2010) 2892–2909 ª 2010 The Authors Journal compilation ª 2010 FEBS 2899 2FG molecule, where it forms hydrogen bonds to Gln 365 Ne2 and one water molecule. In the open state of Y456W 2FG , the Trp 456 side chains adopts the same position and conformation as the tyrosine, and also makes the same edge-to-face ring stacking interaction with Phe 454 as observed in H167A 2FG . The tryptophan side chain is unable to form the tyrosine Og hydrogen bonds, although a new hydrogen bond is possible between Trp 456 Ne1 and Asp 101 Od2 (Fig. 3C). The observation that the loop is highly ordered in the H167A 2FG complex was attributed to two principal factors: first, that the H167A variant is redox impaired (i.e. removal of the covalent His 167 -FAD bond reduces the oxidative power of FAD) and, second, that 2FG is a very slow substrate for P2O [i.e. reduction by 2FG of wild-type P2O (k obs = 0.0064 min )1 ) and of H167A (k obs = 0.000027 min )1 )] [19]. Because H167A 2FG and Y456W 2FG bind the same slow substrate, the difference is therefore mainly attributed to the introduction of the larger tryptophan side chain at position 456, which may alter the conformational ensemble accessible to the loop, and to the intact His 167 -FAD bond, which retains the oxidative power of FAD in Y456W to allow higher 2FG turnover rates. Structure of the F454N-2FG complex In the F454N 2FG complex, more pronounced changes occur in the substrate-recognition loop (Fig. 4). The electron density is of very high quality for the overall protein, including the 2FG molecule bound in the same C3-oxidation mode as in H167A 2FG [19] and A B C Fig. 3. Active-site structure of the Y456W mutant with bound 2FG. (A) Active-site loop conformation in Y456W 2FG (yellow) superim- posed onto H167A 2FG (green) [19]. The comparison of the mutant is made with H167A rather than the wild-type because the sub- strate-recognition loop is open in H167A and the same ligand is bound. The 2FG molecule is bound for oxidation at C3. (B) Close-up of region around 2FG. Superposition of models as in (A). For clarity, no water molecules are shown. (C) Details of the interactions made by Trp 456 (Y456W) and Tyr 456 (H167A). The overall loop conforma- tion, the bound ligand, the flavin cofactor and most active-site resi- dues are strikingly similar in the two complexes. Small backbone changes at position 452 induce a different conformation of the Asp side chain, and the interactions made by Tyr 456 are abolished by the mutation. The Trp side chain is instead stabilized by a hydrogen bond to Asp 101 . Fig. 4. Active-site structure of the F454N mutant with bound 2FG. Loop conformation in F454N 2FG (yellow) superimposed onto H167A 2FG (green) [19]. The 2FG molecule is bound for oxidation at C3. The Phe fi Asn replacement at position 454 induces significant changes in the 452–454 backbone and side chains without affecting the position or orientation of the bound 2FG molecule. Substrate-recognition loop mutations in P2O O. Spadiut et al. 2900 FEBS Journal 277 (2010) 2892–2909 ª 2010 The Authors Journal compilation ª 2010 FEBS Y456W 2FG (present study). Nonetheless, the substrate loop in F454N 2FG is disordered beyond the mutated residue 454, lacking interpretable electron density for the segment 455–460. Thus, the Phe 454 fi Asn replace- ment appears to have a large influence on the local conformation of the loop compared to other variants for which structural data have been analyzed. We have reported previously that, in the open loop conforma- tion in the C3 oxidation mode (H167A 2FG ), residues 454 and 456 do not form specific interactions with the sugar substrate but, rather, they are folded away from the active site [19]. It is therefore particularly interest- ing to note that Asn 454 assumes a position not previ- ously observed in 2FG complexes of P2O, and that its side-chain amide group approaches the exocylic O6 hydroxyl group of the substrate, but does not come close enough to form a hydrogen bond (Fig. 4). In a structure superposition, the distance between the Ca position of Asn 454 in F454N 2FG and that of Phe 454 in the closed wild-type acetate complex (WT ACT ; PDB code 1TT0) [5] is  1.5 A ˚ . The lack of density beyond position 454 does not allow further analysis of this loop conformer, indicating that, even in the presence of orderly bound substrate analogue, the loop under- goes significant dynamic fluctuations. Despite the unu- sual position of Asn 454 , Asp 452 is positioned as in the Y456W mutant, forming a potentially tight interaction with 2FG O1 (distance 2FG O1–Asp 452 Od2, 2.6 A ˚ ). Structure of H450Q In the WT ACT complex, Arg 451 , the residue immedi- ately following the mutated histidine in H450Q has two well defined alternative side-chain conformations, each of which appears appropriately stabilized. In the first conformation, two interactions are possible: Arg 451 Ng1–Asp 470 Od2 and Arg 451 Ng2–water. In the second conformation, the possible interactions are: Arg 451 Ng1–Ser 465 O and Arg 451 Ng2–water. Thus, it appears that the arginine can alternate between these two alternative conformations when the loop is in the fully closed state. In the open loop state of H167A 2FG , the latter conformation is prevalent (with a Ser 465 interaction). H450Q assumes the same open loop con- formation as H167A 2FG ; however, in H450Q, we observe backbone displacements of 1 A ˚ at positions 451 and 452, and the Arg 451 side chain assumes a dif- ferent conformation than those observed in the fully closed and open states, participating in a different set of side-chain interactions (Fig. 5): Arg 451 Ng1–Asp101 Od1, Arg 451 Ng1–Tyr 456 Og and Arg 451 Ng1–water. The changes introduced in the region 450–452 are also manifested as a somewhat weak density for the side-chain carboxylate group of Asp 452 , most likely because the carboxylate group lacks interaction possi- bilities in H450Q. In both the WT ACT and H167A 2FG complexes, Asp 452 can participate in side-chain interac- tions with nearby residues and solvent (WT ACT : Asp 452 Od2–Lys 91 Nf, Asp 452 Od2–Ala 453 N, Asp 452 Od1–two water molecules; H167A 2FG : Asp 452 Od1–Asp 470 Od2, Asp 452 Od1–Arg 472 Ng1). Comparison of the active-site volumes To determine whether there are changes in the volume of the active site as a result of mutations that may explain the ability to accommodate substrates other than glucose, the cavity volumes were calculated for the mutant models, and compared with those of the closed state in WT ACT (PDB code 1TT0) [5] and the open state in H167A 2FG (PDB code 2IGO) [19]. The substrate-recognition loop in F454N with bound 2FG is heavily disordered in the region 455–460 and, because these residues were not modelled, the volume could not be calculated for this mutant. In all models, the substrate-recognition loop is in the open state, which means that the gorge leading to the active site is open to the large internal void at the homotetramer centre from which the active sites are accessible. This complicates any attempt at computation of the Fig. 5. Active-site structure in the H450Q mutant without ligand. Changes of the 450–452 backbone region (in particular, Asp 452 and Arg 451 ) in H450Q (yellow) compared to H167A 2FG (green) [19]. Hydrogen bonds formed by Arg 451 are coloured yellow in H450Q, and green in H167A. The mutation at position 450 leads to back- bone perturbation accompanied by compensatory stabilizing interac- tions formed by Arg 451 . Although no sugar is bound in H450Q, the overall structure of the substrate-recognition loop is similar to that of the open sugar-binding state of H167A 2FG . O. Spadiut et al. Substrate-recognition loop mutations in P2O FEBS Journal 277 (2010) 2892–2909 ª 2010 The Authors Journal compilation ª 2010 FEBS 2901 [...]... to the gating mechanism, are likely to be represented in the conformational ensemble One segment of the substrate-recognition loop undergoes a particularly large rearrangement during the transition from the closed to the open state (Fig 1) [19] This region, which we refer to as the gating segment, involves only the tip of the loop (454FSY456) but, nonetheless, appears to play a most prominent role in. .. 3A), result˚ ing in a minimum edge-to-edge distance of 14 A between the 2FG glucosyl and Trp456 indole rings On the basis of the position of the side chain and its remote location from the ligand in the Y456W2FG structure, the Trp456 side chain would be unable to form any interaction with either the mono- or disaccharides in the open C3 oxidation state Whether this is true also for the open C2 oxidation... limited by the steps of flavin reduction and decay of the C4a-hydroperoxyFAD, which is formed as a transient intermediate in the oxidative half-reaction of P2O [12] It has also been proposed that movement of the active-site loop in the presence of oxygen facilitates the release of the oxidized sugar product in this reaction P2O was the first flavoprotein oxidase for which the C4a hydroperoxyflavin intermediate... concentration of 5% (v ⁄ v) and the enzymes denatured at 100 °C (10 min) Denatured protein was partioned from the supernatant by centrifugation, and the flavin absorption spectra of the supernatants were recorded ThermoFAD analysis The ThermofluorÒ-based ThermoFAD method [41] was used to monitor protein unfolding for analysis of thermal stability of wild-type and mutants This method takes advantage of the intrinsic... possible to assess in the absence of a structure represent- Substrate-recognition loop mutations in P2O ing this mode However, considering the inherent plasticity and conformational degeneracy of the P2O substrate-recognition loop, additional conformers should be possible, especially in light of the new and unexpected loop conformation of F454N2FG (Fig 4) In previous modelling of d-glucose in position for... mutations in the gating segment are affecting mainly the oxidative half-reaction when oxygen is the electron acceptor However, to obtain further insight regarding this, more detailed studies are necessary, including pre-steady-state kinetics of the two half reactions Substrate-recognition loop mutations in P2O We do not propose that O2 is a non-natural electron acceptor, but rather that the enzyme... the open form of the loop interacts with the hydrophobic part of cholesterol and is thus responsible for substrate specificity [34] Similarly, the loop of P2O in its open state, and some of the residues in the gating segment appear to play an essential role in sugar substrate recognition and binding This is evident when comparing the 2904 sugar-substrate selectivity for the wild-type and the variants studied... Tyr456 of the substrate-recognition loop Relative to the wild-type, Y456W shows increased app app Km , but only slightly lower kcat values for all substrates tested In the open state of the loop in H167A2FG, where 2FG is bound for oxidation at C3, ˚ the aromatic side chain of Tyr456 is some 13 A away from the sugar [19] In Y456W, the loop assumes the same conformation as in H167A2FG (Fig 3A), result˚ ing... (i.e that of an oxidase and of a quinone reductase), depending on the metabolic state of the fungus The quinone compounds to which P2O would be exposed (typically BQ and substituted BQ) are similar in size to the aldopyranose substrates Therefore, we do not expect the loop to be in the closed state during the oxidative half-reaction with quinones as acceptors The closed state is probably mainly relevant... suggested that the structural features possibly facilitating the formation and stabilization of this intermediate include an elongated, mainly hydrophobic cavity, which is formed at the re side of the isoalloxazine ring upon closure of the substrate loop, and which is large enough to accommodate a peroxide group at the C4a position [33] When considering the importance of the active-site loop for this . demonstrating that, despite extensive mutagenesis in the vicinity of the FAD-binding pocket, the mutants remain properly fla- vinylated (not shown). The thermal stability was inves- tigated using the ThermoFAD. result- ing in a minimum edge-to-edge distance of 14 A ˚ between the 2FG glucosyl and Trp 456 indole rings. On the basis of the position of the side chain and its remote location from the ligand in the. mm sodium dithionite in the absence of oxygen, and recording of the spectra of the reduced states was repeated. To investi- gate the effect of the mutations on the flavinylation state of the P2O variants,