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A Conserved Active-site Threonine Is Important for Both Sugar and Flavin Oxidations of Pyranose 2-Oxidase*

  • Warintra Pitsawong
    Footnotes
    Affiliations
    Department of Biochemistry and Center of Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
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  • Jeerus Sucharitakul
    Affiliations
    Department of Biochemistry, Faculty of Dentistry, Chulalongkorn University, Henri-Dunant Road, Patumwan, Bangkok 10300, Thailand
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  • Methinee Prongjit
    Affiliations
    Department of Biochemistry and Center of Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
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  • Tien-Chye Tan
    Footnotes
    Affiliations
    Division of Glycoscience, Kungliga Tekniska Högskolan Biotechnology, Royal Institute of Technology, Albanova University Center, SE-106 91 Stockholm, Sweden
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  • Oliver Spadiut
    Affiliations
    Division of Glycoscience, Kungliga Tekniska Högskolan Biotechnology, Royal Institute of Technology, Albanova University Center, SE-106 91 Stockholm, Sweden
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  • Dietmar Haltrich
    Footnotes
    Affiliations
    Department of Food Science and Technology, Bodenkultur-University of Natural Resource and Applied Life Sciences, A-1190 Vienna, Austria
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  • Christina Divne
    Footnotes
    Affiliations
    Division of Glycoscience, Kungliga Tekniska Högskolan Biotechnology, Royal Institute of Technology, Albanova University Center, SE-106 91 Stockholm, Sweden
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  • Pimchai Chaiyen
    Correspondence
    To whom correspondence should be addressed: Dept. of Biochemistry and Center of Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand. Tel.: 66-2-201-5596; Fax: 66-2-354-7174;
    Affiliations
    Department of Biochemistry and Center of Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
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  • Author Footnotes
    5 The abbreviations used are: P2Opyranose 2-oxidaseABTS2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium saltλwavelengthWTwild typeMes4-morpholineethanesulfonic acid.
    * This work was supported in part by the Thailand Research Fund through Grants BRG5180002 (to P. C.), MRG4980117 (to J. S.), and PHD/0151/2547 of the Royal Golden Jubilee Ph.D. program (to M. P.) and by the Faculty of Science, Mahidol University (to P. C.) and from the Faculty of Dentistry Chulalongkorn University (to J. S.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental text, Tables S1 and S2, and Figs. S1–S3.
    1 Supported by the Development and Promotion of Science and Technology Talent Project, Thailand.
    2 Supported by the Swedish Research Council Formas, the Swedish Research Council Vetenskapsrådet, the Carl Tryggers Foundation, and the Swedish Foundation for Strategic Research through the Swedish Center for Biomimetic Fiber Engineering (through Biomime).
    3 Supported by a grant from the Austrian Research Foundation (Fonds zur Förderung der wissenschaftlichen Forschung Translational Project L213-B11).
Open AccessPublished:January 20, 2010DOI:https://doi.org/10.1074/jbc.M109.073247
      Pyranose 2-oxidase (P2O)
      The abbreviations used are: P2O
      pyranose 2-oxidase
      ABTS
      2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt
      λ
      wavelength
      WT
      wild type
      Mes
      4-morpholineethanesulfonic acid.
      catalyzes the oxidation by O2 of d-glucose and several aldopyranoses to yield the 2-ketoaldoses and H2O2. Based on crystal structures, in one rotamer conformation, the threonine hydroxyl of Thr169 forms H-bonds to the flavin-N5/O4 locus, whereas, in a different rotamer, it may interact with either sugar or other parts of the P2O·sugar complex. Transient kinetics of wild-type (WT) and Thr169 → S/N/G/A replacement variants show that d-Glc binds to T169S, T169N, and WT with the same Kd (45–47 mm), and the hydride transfer rate constants (kred) are similar (15.3–9.7 s−1 at 4 °C). kred of T169G with d-glucose (0.7 s−1, 4 °C) is significantly less than that of WT but not as severely affected as in T169A (kred of 0.03 s−1 at 25 °C). Transient kinetics of WT and mutants using d-galactose show that P2O binds d-galactose with a one-step binding process, different from binding of d-glucose. In T169S, T169N, and T169G, the overall turnover with d-Gal is faster than that of WT due to an increase of kred. In the crystal structure of T169S, Ser169 Oγ assumes a position identical to that of Oγ1 in Thr169; in T169G, solvent molecules may be able to rescue H-bonding. Our data suggest that a competent reductive half-reaction requires a side chain at position 169 that is able to form an H-bond within the ES complex. During the oxidative half-reaction, all mutants failed to stabilize a C4a-hydroperoxyflavin intermediate, thus suggesting that the precise position and geometry of the Thr169 side chain are required for intermediate stabilization.

      Introduction

      Pyranose 2-oxidase (P2O,
      Supported by a grant from the Austrian Research Foundation (Fonds zur Förderung der wissenschaftlichen Forschung Translational Project L213-B11).
      pyranose:oxygen 2-oxidoreductase, EC 1.1.3.10) from Trametes multicolor is a homotetrameric enzyme with each subunit carrying one FAD covalently linked to Nϵ2 (N3) of His167 via the FAD 8α-methyl group (
      • Halada P.
      • Leitner C.
      • Sedmera P.
      • Haltrich D.
      • Volc J.
      ). P2O catalyzes the oxidation by molecular oxygen of d-glucose (d-Glc) and several aldopyranoses at the C2 position to yield the corresponding 2-ketoaldoses and hydrogen peroxide (
      • Leitner C.
      • Volc J.
      • Haltrich D.
      ). The overall catalytic reaction can be divided into two half-reactions (Scheme 1) obeying a Ping-Pong type mechanism at pH 7 (
      • Prongjit M.
      • Sucharitakul J.
      • Wongnate T.
      • Haltrich D.
      • Chaiyen P.
      ): a reductive half-reaction in which the protein-bound flavin receives a hydride equivalent from a sugar substrate to produce the reduced FAD (FADH) and the 2-keto-sugar, and an oxidative half-reaction in which two electrons are transferred from the reduced flavin to O2 to form H2O2 (
      • Leitner C.
      • Volc J.
      • Haltrich D.
      ,
      • Prongjit M.
      • Sucharitakul J.
      • Wongnate T.
      • Haltrich D.
      • Chaiyen P.
      ).
      We reported the first detection of a C4a-hydroperoxyflavin intermediate for a flavoprotein oxidase (
      • Sucharitakul J.
      • Prongjit M.
      • Haltrich D.
      • Chaiyen P.
      ). This flavin intermediate has not been previously observed for flavoprotein oxidases but has been limited to reactions of flavoprotein monooxygenases (
      • Palfey B.A.
      • Massey V.
      ,
      • Ballou D.P.
      • Entsch B.
      • Cole L.J.
      ,
      • van Berkel W.J.
      • Kamerbeek N.M.
      • Fraaije M.W.
      ). Besides P2O, an intermediate in flavoprotein oxidases has been detected only in the crystalline forms of choline oxidase (1.86 Å) (
      • Orville A.M.
      • Lountos G.T.
      • Finnegan S.
      • Gadda G.
      • Prabhakar R.
      ) and nitroalkane oxidase (in the presence of cyanide) (
      • Héroux A.
      • Bozinovski D.M.
      • Valley M.P.
      • Fitzpatrick P.F.
      • Orville A.M.
      ), and in the mutant form, C42S, of an NADH oxidase (
      • Mallett T.C.
      • Claiborne A.
      ). In the case of glucose oxidase from Aspergillus niger, the generation of a flavin semiquinone-superoxide radical pair using pulse radiolysis resulted in formation of a putative C4a-hydroperoxyflavin intermediate (
      • Ghisla S.
      • Massey V.
      ). Studies on the P2O reductive half-reaction indicate that P2O binds d-Glc according to a two-step binding mechanism: first an initial complex is formed, followed by an isomerization step to form an active Michaelis ES complex (P2O·glucose). Interestingly, this complex shows higher absorbance at 395 nm than does the oxidized enzyme, which is unique among flavoprotein oxidases (
      • Prongjit M.
      • Sucharitakul J.
      • Wongnate T.
      • Haltrich D.
      • Chaiyen P.
      ).
      P2O belongs to the large family of glucose-methanol-choline oxidoreductases (
      • Albrecht M.
      • Lengauer T.
      ,
      • Hallberg B.M.
      • Leitner C.
      • Haltrich D.
      • Divne C.
      ) with the catalytic side chains His548–Asn593 positioned at the re-face of the flavin isoalloxazine ring (
      • Hallberg B.M.
      • Leitner C.
      • Haltrich D.
      • Divne C.
      ). The active site is gated by a dynamic active-site loop (residues 451–461), which has been shown to harbor several residues important for substrate binding and catalysis (
      • Hallberg B.M.
      • Leitner C.
      • Haltrich D.
      • Divne C.
      ,
      • Kujawa M.
      • Ebner H.
      • Leitner C.
      • Hallberg B.M.
      • Prongjit M.
      • Sucharitakul J.
      • Ludwig R.
      • Rudsander U.
      • Peterbauer C.
      • Chaiyen P.
      • Haltrich D.
      • Divne C.
      ,
      • Salaheddin C.
      • Spadiut O.
      • Ludwig R.
      • Tan T.C.
      • Divne C.
      • Haltrich D.
      • Peterbauer C.
      ). Comparison of P2O structures in sugar-free and sugar-bound forms reveals the presence of at least two well defined active-site loop conformations, each of which is relevant to one of the half-reactions (
      • Kujawa M.
      • Ebner H.
      • Leitner C.
      • Hallberg B.M.
      • Prongjit M.
      • Sucharitakul J.
      • Ludwig R.
      • Rudsander U.
      • Peterbauer C.
      • Chaiyen P.
      • Haltrich D.
      • Divne C.
      ). The structure of a less active P2O mutant (H167A), in complex with a slow substrate (2-deoxy-2-fluoro-d-glucose (
      • Kujawa M.
      • Ebner H.
      • Leitner C.
      • Hallberg B.M.
      • Prongjit M.
      • Sucharitakul J.
      • Ludwig R.
      • Rudsander U.
      • Peterbauer C.
      • Chaiyen P.
      • Haltrich D.
      • Divne C.
      )) or the reaction product (2-keto-d-glucose (
      • Bannwarth M.
      • Heckmann-Pohl D.
      • Bastian S.
      • Giffhorn F.
      • Schulz G.E.
      )), shows the active-site loop in an open conformation. The open loop state is able to accommodate spatially the sugar substrate, and thus this conformation is relevant for the reductive half-reaction (
      • Prongjit M.
      • Sucharitakul J.
      • Wongnate T.
      • Haltrich D.
      • Chaiyen P.
      ,
      • Kujawa M.
      • Ebner H.
      • Leitner C.
      • Hallberg B.M.
      • Prongjit M.
      • Sucharitakul J.
      • Ludwig R.
      • Rudsander U.
      • Peterbauer C.
      • Chaiyen P.
      • Haltrich D.
      • Divne C.
      ). The wild-type structure of P2O in complex with the small competitive inhibitor acetate revealed the loop in a different well defined conformational state (
      • Hallberg B.M.
      • Leitner C.
      • Haltrich D.
      • Divne C.
      ). This state is characterized as fully closed, and the active site is solvent-inaccessible and provides a more hydrophobic substrate environment than does the open state. Thus, this structural state provides a suitable environment for the reaction of the reduced enzyme and oxygen, and can spatially accommodate the C4a-hydroperoxy-FAD intermediate during the oxidative half-reaction (
      • Sucharitakul J.
      • Prongjit M.
      • Haltrich D.
      • Chaiyen P.
      ,
      • Hallberg B.M.
      • Leitner C.
      • Haltrich D.
      • Divne C.
      ,
      • Kujawa M.
      • Ebner H.
      • Leitner C.
      • Hallberg B.M.
      • Prongjit M.
      • Sucharitakul J.
      • Ludwig R.
      • Rudsander U.
      • Peterbauer C.
      • Chaiyen P.
      • Haltrich D.
      • Divne C.
      ).
      We have discussed previously that the conserved residue Thr169 occupies an important position in the P2O active site, positioned immediately “above” the flavin, on the flavin si-side, where it forms a hydrogen bond to the flavin N5 and O4 atoms when the loop is in the closed conformation (
      • Hallberg B.M.
      • Leitner C.
      • Haltrich D.
      • Divne C.
      ,
      • Kujawa M.
      • Ebner H.
      • Leitner C.
      • Hallberg B.M.
      • Prongjit M.
      • Sucharitakul J.
      • Ludwig R.
      • Rudsander U.
      • Peterbauer C.
      • Chaiyen P.
      • Haltrich D.
      • Divne C.
      ). Flavin-N5-protein interactions are recurrent features in structures of many flavoprotein oxidases (
      • Fraaije M.W.
      • Mattevi A.
      ), and little is known about the functional effects of this H-bonding interaction. When the active-site loop assumes the open conformation in P2O, Thr169 discards its flavin interaction by means of a rotamer change (
      • Kujawa M.
      • Ebner H.
      • Leitner C.
      • Hallberg B.M.
      • Prongjit M.
      • Sucharitakul J.
      • Ludwig R.
      • Rudsander U.
      • Peterbauer C.
      • Chaiyen P.
      • Haltrich D.
      • Divne C.
      ). The position of the Thr169 side chain relative to the sugar substrate and FAD also provides a simple, straightforward explanation for the poor performance of d-galactose (d-Gal) as a substrate. The oxidation of d-Gal by P2O is industrially useful, because it produces the intermediate for further synthesis of d-tagatose, a low caloric and non-glycemic sweetener (
      • Haltrich D.
      • Leitner C.
      • Neuhauser W.
      • Nidetzky B.
      • Kulbe K.D.
      • Volc J.
      ). In wild-type P2O (WT), the axial O4 hydroxyl group of d-Gal is probably unfavorable due to steric clashes with the Thr169 side chain (
      • Kujawa M.
      • Ebner H.
      • Leitner C.
      • Hallberg B.M.
      • Prongjit M.
      • Sucharitakul J.
      • Ludwig R.
      • Rudsander U.
      • Peterbauer C.
      • Chaiyen P.
      • Haltrich D.
      • Divne C.
      ). The kinetics of the binding and reaction mechanism of P2O with d-Gal have not been rationalized in detail, because transient kinetics of the reaction have not been investigated.
      In this report, steady-state and transient kinetics of Thr169 variants, T169S, T169N, T169G, and T169A, with d-Glc and d-Gal were investigated. Rapid kinetics of WT with d-Gal are also reported. These results provide mechanistic insights regarding the role of Thr169 in the reductive and oxidative half-reactions. In addition, three-dimensional structures of T169G and T169S were solved and used in conjunction with kinetic data for interpretation of the roles of Thr169 in catalysis of P2O.

      DISCUSSION

      Based on our kinetic data presented here and its strategic position in the P2O active site (
      • Kujawa M.
      • Ebner H.
      • Leitner C.
      • Hallberg B.M.
      • Prongjit M.
      • Sucharitakul J.
      • Ludwig R.
      • Rudsander U.
      • Peterbauer C.
      • Chaiyen P.
      • Haltrich D.
      • Divne C.
      ,
      • Hallberg B.M.
      • Leitner C.
      • Haltrich D.
      • Divne C.
      ), it is clear that Thr169 is an intriguing and important amino acid in catalysis by P2O. Our findings indicate that a side chain at position 169 capable of forming H-bonds near the productive P2O·sugar complex is needed for efficient flavin reduction and identify mutants that can help improving application of P2O in d-tagatose synthesis. Stabilization of C4a-hydroperoxyflavin in P2O also requires the presence of threonine at position 169. Thr169 has been observed to assume two rotamer conformations in P2O: a rotamer with the Oγ pointing away from the flavin and toward amino acids in the dynamic active-site loop, as seen when the loop is open to allow sugar binding (Fig. 7A), and another rotamer where Oγ is pointing toward the flavin and offers H-bond(s) to the N5/O4 locus when the substrate loop is closed (Fig. 7B) (
      • Kujawa M.
      • Ebner H.
      • Leitner C.
      • Hallberg B.M.
      • Prongjit M.
      • Sucharitakul J.
      • Ludwig R.
      • Rudsander U.
      • Peterbauer C.
      • Chaiyen P.
      • Haltrich D.
      • Divne C.
      ). Based on available crystal structures, the Oγ atom of Thr169 in P2O assumes a position equivalent to that of Thr266 in electron transfer flavoprotein from Methylophilus methylotrophus (
      • Roberts D.L.
      • Frerman F.E.
      • Kim J.J.
      ); Ser254 in electron transfer flavoprotein from the methylotrophic bacterium W3A1 (
      • Yang K.Y.
      • Swenson R.P.
      ); Ser101 in choline oxidase (
      • Quaye O.
      • Lountos G.T.
      • Fan F.
      • Orville A.M.
      • Gadda G.
      ); Ser106 in alditol oxidase (
      • Forneris F.
      • Heuts D.P.
      • Delvecchio M.
      • Rovida S.
      • Fraaije M.W.
      • Mattevi A.
      ); Asp170 in vanillyl alcohol oxidase (
      • van den Heuvel R.H.
      • Fraaije M.W.
      • Mattevi A.
      • van Berkel W.J.
      ); the backbone amide group of Gly120 in Streptomyces or Brevibacterium cholesterol oxidase (
      • Yue Q.K.
      • Kass I.J.
      • Sampson N.S.
      • Vrielink A.
      ). The observed distances between atoms of the proteins and the flavin-N5/O4 locus indicate that they are mostly within H-bonding distance. Hydrogen-bonding interactions in the vicinity of the flavin N5/O4 locus are recurrent features in flavoenzymes (
      • Fraaije M.W.
      • Mattevi A.
      ). Despite the commonality of these interactions, little is known about the precise functional roles and mechanistic implications. In electron transfer flavoprotein from methylotrophic bacterium W3A1, the H-bond to N5 has been suggested to increase Emo′ of the enzyme (
      • Yang K.Y.
      • Swenson R.P.
      ). Clearly, more detailed studies on other flavoproteins are needed to determine whether the mechanistic implications of Thr169 in P2O, especially in flavin reduction, can be generalized to other flavoenzymes.
      Our results suggest that a protein side chain capable of forming H-bonds near the productive P2O·sugar complex is required for efficient flavin reduction. Results with d-Glc as a substrate indicate that the replacement of Thr169 with Asn and Ser has a negligible effect on the reductive half-reaction. The Kd values for the binding of d-Glc in WT, T169S, and T169N are almost identical. In addition, T169S and T169N all form an active P2O·Glc complex characterized by an absorbance increase at 395 nm (
      • Prongjit M.
      • Sucharitakul J.
      • Wongnate T.
      • Haltrich D.
      • Chaiyen P.
      ). The Emo′ values (Table 1) and the rates of flavin reduction (kred, Table 2) of WT, T169S, and T169N are also in the same range, indicating that the oxidative power of these mutants is preserved. Interestingly, although the Emo′ of T169G was significantly higher than the other mutant enzymes (−1 mV versus −105 mV or lower), the flavin reduction by d-Glc was affected inversely to the thermodynamic changes, i.e. >10-fold decrease in kred (Table 2). It has been shown for the flavoenzyme 2-methyl-3-hydroxypyridine-5-carboxylic monooxygenase that, despite similar Emo′ values, flavin reduction rates can differ up to 1600-fold due to changes in the precise geometry of the reactants (
      • Chaiyen P.
      • Brissette P.
      • Ballou D.P.
      • Massey V.
      ,
      • Chaiyen P.
      ). This suggests that the binding mode of d-Glc in T169G is different from that in WT, T169S, and T169N, thus resulting in lower efficiency of the hydride transfer. In T169A, the flavin reduction by d-Glc is significantly impaired (Table 2). The small absorbance increase at 395 nm accompanying the binding of d-Glc to T169G and T169A also supports the idea that substrate binding is different than in WT, T169N, and T169S. We may conclude that efficient hydride transfer from the sugar C2 to flavin N5 during the reductive half-reaction requires an enzyme conformational state that is compatible with Thr, Ser, and Asn at the position 169.
      Stopped-flow experiments of WT-P2O with d-Gal show that, unlike the binding of d-Glc, P2O binds d-Gal in one step. Replacement of Thr169 by Ser, Asn, and Gly increased flavin reduction rates when compared with that of WT without increasing the binding affinity. This result can be interpreted as d-Gal binds to the mutants with the geometry allowing the reactants to carry out the hydride-transfer reaction more optimal than in the WT. This is likely due to the relief of steric hindrance close to the axial C4 hydroxyl group of d-Gal, as predicted by our previous structural analysis (
      • Kujawa M.
      • Ebner H.
      • Leitner C.
      • Hallberg B.M.
      • Prongjit M.
      • Sucharitakul J.
      • Ludwig R.
      • Rudsander U.
      • Peterbauer C.
      • Chaiyen P.
      • Haltrich D.
      • Divne C.
      ). In T169A, flavin reduction by d-Gal is very slow, resulting in an almost inactive enzyme. As for the reaction with d-Glc discussed above, these data suggest that, by substituting Thr169 with Ala, the reductive half-reaction is significantly impaired.
      The crystal structures of T169S and T169G mutants also support the idea that the H-bonding ability of a side chain at position 169 is likely to be required for efficient flavin reduction. The structure of T169S shows that the Oγ of Ser assumes the same position as Oγ of Thr169 in the sugar-bound state of H167A P2O where the Thr169 side chain is pointing away from the flavin N5/O4 locus (Figs. 6A and 7C). Thus, by allowing identical positioning of the Thr/Ser Oγ, the reductive half-reaction remains unperturbed. In T169G, which lacks the possibility of forming a H-bond, two water molecules are found near the flavin N5/O4, offering the possibility of H-bonds. In T169G, the rate of flavin reduction by d-Glc decreased, but the rate with d-Gal increased when compared with that of the WT (Table 2). This mutant is thus quite competent in the reductive half-reaction, maybe due to the possibility of solvent-mediated H-bonds to the productive P2O·Gal complex. Although the structure of T169N is not available, the Asn side chain should also be able to H-bond to important groups in the productive ES complex. Hence, by maintaining the H-bonding ability at position 169 (as in T169S, T169N, and T169G), efficient flavin reduction can be maintained.
      Thr169 is important for formation of a C4a-hydroperoxy-FAD intermediate. Our previous study with the WT has shown that P2O is thus far unique among flavoprotein oxidases for its ability to stabilize the C4a-hydroperoxy-FAD during the oxidative half-reaction (
      • Sucharitakul J.
      • Prongjit M.
      • Haltrich D.
      • Chaiyen P.
      ). It was suggested that the cavity at the active site of this enzyme can provide the required space for accommodating a peroxide group at the flavin C4a position (
      • Sucharitakul J.
      • Prongjit M.
      • Haltrich D.
      • Chaiyen P.
      ). In other flavoprotein oxidases, the active sites may be too restricted to allow formation of the intermediate (
      • Sucharitakul J.
      • Prongjit M.
      • Haltrich D.
      • Chaiyen P.
      ,
      • Forneris F.
      • Heuts D.P.
      • Delvecchio M.
      • Rovida S.
      • Fraaije M.W.
      • Mattevi A.
      ,
      • Alfieri A.
      • Fersini F.
      • Ruangchan N.
      • Prongjit M.
      • Chaiyen P.
      • Mattevi A.
      ). We could not detect the C4a-hydroperoxy-FAD intermediate in any of the Thr169 variants. All mutants reacted with oxygen in a second-order fashion without a reverse rate constant, similar to other flavoprotein oxidases (
      • Mattevi A.
      ,
      • Gibson Q.H.
      • Swoboda B.E.
      • Massey V.
      ). The closed loop conformation observed in the wild-type P2O-acetate complex (1TT0 (
      • Hallberg B.M.
      • Leitner C.
      • Haltrich D.
      • Divne C.
      )) shows Thr169 Oγ1 interacting with the flavin N5/O4 locus, offering the possibility of H-bonds to N5 and O4 (Fig. 7B). In the structure of T169S, although the position of Ser169 Oγ (Fig. 6) is different from the position of Thr169 Oγ1 in the closed loop structure of WT (1TT0 (
      • Hallberg B.M.
      • Leitner C.
      • Haltrich D.
      • Divne C.
      )), a similar H-bond as in WT may be possible if loop movement occurs during the oxidative half-reaction. However, the presence of a group capable of making a H-bond to the flavin N5/O4 is unlikely to be the main factor for stabilizing the intermediate, because in T169S, no intermediate was detected. A possible explanation may be that, in the closed conformational state, the position of Thr169 provides an occluded reaction chamber that helps to control precisely the reactivity of the reduced flavin and oxygen to ensure the formation of the flavin-C4a intermediate prior to the subsequent H2O2 elimination.
      The steady-state kinetic data are in agreement with the kinetic model interpreted from stopped-flow data and suggest that T169S and T169N can be considered to be good biocatalysts. With d-Glc as a substrate, kcat of T169S is higher than the kcat of WT and other mutants. In all mutants, the effect on kcat followed the trend seen for kred (T169S > T169N > T169G > T169A). In WT, the overall turnover with d-Glc is governed by two steps: flavin reduction (kred) and C4a-hydroperoxyflavin decay (
      • Prongjit M.
      • Sucharitakul J.
      • Wongnate T.
      • Haltrich D.
      • Chaiyen P.
      ). Because in all of the mutants the oxidative half-reaction is second-order without an intermediate (Fig. 5), the reaction is limited only by flavin reduction (kred). Therefore, kcat of T169S with d-Glc is higher than the value of WT (Table 1). These data also imply that, if the reaction is equilibrated with high concentration of oxygen, T169S can be employed as a better catalyst than WT when using d-Glc as a substrate. When using d-Gal as a substrate, kcat of T169S and T169N are 2- and 4-fold higher than the value of WT, due to the increase in kred (rate-limiting step) of the mutants (Fig. 3 and TABLE 1, TABLE 2). These two mutants would also be better biocatalysts than the WT in d-Gal oxidation for producing d-tagatose. Effects of the mutations based on steady-state parameters reported in Table 1 are different from kcat and KmGlc,Gal values reported before (
      • Salaheddin C.
      • Spadiut O.
      • Ludwig R.
      • Tan T.C.
      • Divne C.
      • Haltrich D.
      • Peterbauer C.
      ,
      • Spadiut O.
      • Leitner C.
      • Tan T.C.
      • Ludwig R.
      • Divne C.
      • Haltrich D.
      ) where the values were from steady-state assay under air saturation. Deviations of the values reported in previous studies (
      • Salaheddin C.
      • Spadiut O.
      • Ludwig R.
      • Tan T.C.
      • Divne C.
      • Haltrich D.
      • Peterbauer C.
      ,
      • Spadiut O.
      • Leitner C.
      • Tan T.C.
      • Ludwig R.
      • Divne C.
      • Haltrich D.
      ) from the actual kinetic parameters in Table 1 are due to high values of KmO2, especially for the mutants T169N and T169G; the concentration of oxygen under air-saturation (0.26 mm) is below the KmO2 values of all the mutants (Table 1).
      All variants except T169A showed parallel-line patterns when d-Glc or d-Gal were used as substrates. This is similar to the reaction of WT with d-Glc as a substrate (
      • Prongjit M.
      • Sucharitakul J.
      • Wongnate T.
      • Haltrich D.
      • Chaiyen P.
      ). The kinetic mechanism of WT at pH 7.0 is Ping-Pong where the 2-keto-d-sugar product is released prior to the oxygen reaction. The parallel-line pattern can also be explained by a model in which the sugar product remains bound during the reaction of molecular oxygen, but the reduction step is essentially irreversible (k3k−3) (
      • Prongjit M.
      • Sucharitakul J.
      • Wongnate T.
      • Haltrich D.
      • Chaiyen P.
      ). Interestingly, the steady-state kinetics of T169A with d-Glc or d-Gal as substrates show an intersecting-line pattern instead of a parallel-line pattern, suggesting that, for T169A, regardless of the sugar used, the reaction of O2 with the reduced enzyme may occur while the 2-keto-d-sugar remains bound to the enzyme. These data also imply that the reverse rate constant (k−3 in Fig. 4A) of T169A is significant, in agreement with its low Emo′. The ternary-complex pattern indicates that a 2-keto-sugar product resulting from the reductive half-reaction fails to dissociate and remains bound to the active site during the reaction with O2 in the oxidative half-reaction. One possible explanation is that space allocation at the re-side of the isoalloxazine ring is increased in T169A compared with the WT and other mutants investigated. A shift in the reaction mechanism from the typical Ping-Pong type to a ternary-complex mechanism was also observed in the reaction of P2O at pH 8 or above (
      • Rungsrisuriyachai K.
      • Gadda G.
      ).
      In conclusion, our results suggest that the residue at position 169 is important for both half-reactions in P2O catalysis. Efficient hydride transfer from the sugar C2 to flavin N5 during the reductive half-reaction requires a residue at this position that can maintain a H-bonding within the ES complex, i.e. Thr (WT), Ser, Asn, and Gly (H-bond possibly maintained by solvent). When compared with the WT, the overall turnover of T169S and T169N with d-Gal was faster due to higher rate constants of the flavin reduction. In addition, the side chain of Thr169 is crucial for stabilizing the C4a-hydroperoxy-FAD during the oxidative half-reaction and cannot be substituted by other residues.

      Acknowledgments

      We thank Janewit Wongratana for constructing plasmids for expression of T169S, T169N, and T169A and the beam-line staff scientists at MAX-lab (Lund, Sweden) for support during data collection. We thank Bruce Palfey for critical reading of the manuscript.

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