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• W2071957028 abstract "The active site of mannitol 2-dehydrogenase from Pseudomonas fluorescens (PfM2DH) is connected with bulk solvent through a narrow protein channel that shows structural resemblance to proton channels utilized by redox-driven proton pumps. A key element of the PfM2DH channel is the “mobile” Glu292, which was seen crystallographically to adopt distinct positions up and down the channel. It was suggested that the “down → up” conformational change of Glu292 could play a proton relay function in enzymatic catalysis, through direct proton shuttling by the Glu or because the channel is opened for water molecules forming a chain along which the protons flow. We report evidence from site-directed mutagenesis (Glu292 → Ala) substantiated by data from molecular dynamics simulations that support a role for Glu292 as a gate in a water chain (von Grotthuss-type) mechanism of proton translocation. Occupancy of the up and down position of Glu292 is influenced by the bonding and charge state of the catalytic acid base Lys295, suggesting that channel opening/closing motions of the Glu are synchronized to the reaction progress. Removal of gatekeeper control in the E292A mutant resulted in a selective, up to 120-fold slowing down of microscopic steps immediately preceding catalytic oxidation of mannitol, consistent with the notion that formation of the productive enzyme-NAD+-mannitol complex is promoted by a corresponding position change of Glu292, which at physiological pH is associated with obligatory deprotonation of Lys295 to solvent. These results underscore the important role of conformational dynamics in the proton transfer steps of alcohol dehydrogenase catalysis. The active site of mannitol 2-dehydrogenase from Pseudomonas fluorescens (PfM2DH) is connected with bulk solvent through a narrow protein channel that shows structural resemblance to proton channels utilized by redox-driven proton pumps. A key element of the PfM2DH channel is the “mobile” Glu292, which was seen crystallographically to adopt distinct positions up and down the channel. It was suggested that the “down → up” conformational change of Glu292 could play a proton relay function in enzymatic catalysis, through direct proton shuttling by the Glu or because the channel is opened for water molecules forming a chain along which the protons flow. We report evidence from site-directed mutagenesis (Glu292 → Ala) substantiated by data from molecular dynamics simulations that support a role for Glu292 as a gate in a water chain (von Grotthuss-type) mechanism of proton translocation. Occupancy of the up and down position of Glu292 is influenced by the bonding and charge state of the catalytic acid base Lys295, suggesting that channel opening/closing motions of the Glu are synchronized to the reaction progress. Removal of gatekeeper control in the E292A mutant resulted in a selective, up to 120-fold slowing down of microscopic steps immediately preceding catalytic oxidation of mannitol, consistent with the notion that formation of the productive enzyme-NAD+-mannitol complex is promoted by a corresponding position change of Glu292, which at physiological pH is associated with obligatory deprotonation of Lys295 to solvent. These results underscore the important role of conformational dynamics in the proton transfer steps of alcohol dehydrogenase catalysis. The alcohol dehydrogenase (ADH) 2The abbreviations used are: ADHalcohol dehydrogenasePfM2DHmannitol 2-dehydrogenase from P. fluorescensKIEkinetic isotope effectMDmolecular dynamicsPDBprotein data baser.m.s.root mean square. reaction (Equation 1) is a key transformation of biological catalysis. It is exploited by the living cell in a wide variety of metabolic pathways (1.de Smidt O. du Preez J.C. Albertyn J. The alcohol dehydrogenases of Saccharomyces cerevisiae. A comprehensive review.FEMS Yeast Res. 2008; 8: 967-978Crossref PubMed Scopus (174) Google Scholar, 2.Höög J.O. Ostberg L.J. Mammalian alcohol dehydrogenases, a comparative investigation at gene and protein levels.Chem. Biol. Interact. 2011; 191: 2-7Crossref PubMed Scopus (40) Google Scholar, 3.Meijers R. Cedergren-Zeppezauer E.S. Messerschmidt A. Cygler M. Bode W. Handbook of metalloproteins. 1st Ed. John Wiley & Sons, Ltd., Chichester, United Kingdom2004: 5-33Google Scholar, 4.Metzler D.E. Metzler D.E. Biochemistry: The Chemical Reactions of Living Cells. 2nd Ed. Academic Press, San Diego2001: 765-836Google Scholar) and has important practical applications in the synthesis of pharmaceuticals (5.Roberts S.M. Use of enzymes as catalysts to promote key transformations in organic synthesis.Philos. Trans. R. Soc. Lond. B Biol. Sci. 1989; 324: 577-587Crossref Google Scholar, 6.Hummel W. New alcohol dehydrogenases for the synthesis of chiral compounds.Adv. Biochem. Eng. Biotechnol. 1997; 58: 145-184PubMed Google Scholar, 7.Faber K. Biotransformations in Organic Chemistry. 6th Ed. Springer-Verlag, Berlin2004Crossref Google Scholar, 8.Hall M. Bommarius A.S. Enantioenriched compounds via enzyme-catalyzed redox reactions.Chem. Rev. 2011; 111: 4088-4110Crossref PubMed Scopus (177) Google Scholar). The overall enzymatic conversion proceeds with release of one proton for each molecule NAD(P)H formed upon substrate oxidation (Equation 1).Alcohol+NAD(P)+↔carbonyl+NAD(P)H+H+(Eq. 1) alcohol dehydrogenase mannitol 2-dehydrogenase from P. fluorescens kinetic isotope effect molecular dynamics protein data base root mean square. The catalytic chemistry of ADH enzymes involves hydride transfer to NAD(P)+ coupled to a multistep proton transfer functioning in the abstraction of proton from alcohol substrate and delivery of proton to solvent (9.Plapp B.V. Kohen A. Limbach H.H. Isotope Effects in Chemistry and Biology. CRC Press, Boca Raton, FL2006: 811-836Google Scholar, 10.Pettersson G. Liver alcohol dehydrogenase.CRC Crit. Rev. Biochem. 1987; 21: 349-389Crossref PubMed Scopus (5) Google Scholar). Proton translocation from the ADH active site to bulk water usually occurs through a succession of “proton hops” along a wire of conducting groups, the so-called proton relay, which are connected by hydrogen bonds (9.Plapp B.V. Kohen A. Limbach H.H. Isotope Effects in Chemistry and Biology. CRC Press, Boca Raton, FL2006: 811-836Google Scholar, 11.Klimacek M. Hellmer H. Nidetzky B. Catalytic mechanism of Zn2+-dependent polyol dehydrogenases: kinetic comparison of sheep liver sorbitol dehydrogenase with wild-type and Glu154 → Cys forms of yeast xylitol dehydrogenase.Biochem. J. 2007; 404: 421-429Crossref PubMed Scopus (11) Google Scholar, 12.Koumanov A. Benach J. Atrian S. Gonzàlez-Duarte R. Karshikoff A. Ladenstein R. The catalytic mechanism of Drosophila alcohol dehydrogenase. Evidence for a proton relay modulated by the coupled ionization of the active site lysine/tyrosine pair and a NAD+ ribose OH switch.Proteins. 2003; 51: 289-298Crossref PubMed Scopus (28) Google Scholar, 13.Inoue J. Tomioka N. Itai A. Harayama S. Proton transfer in benzyl alcohol dehydrogenase during catalysis. Alternate proton-relay routes.Biochemistry. 1998; 37: 3305-3311Crossref PubMed Scopus (23) Google Scholar, 14.Filling C. Berndt K.D. Benach J. Knapp S. Prozorovski T. Nordling E. Ladenstein R. Jörnvall H. Oppermann U. Critical residues for structure and catalysis in short-chain dehydrogenases/reductases.J. Biol. Chem. 2002; 277: 25677-25684Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar, 15.Milburn C.C. Lamble H.J. Theodossis A. Bull S.D. Hough D.W. Danson M.J. Taylor G.L. The structural basis of substrate promiscuity in glucose dehydrogenase from the hyperthermophilic archaeon Sulfolobus solfataricus.J. Biol. Chem. 2006; 281: 14796-14804Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). This proton translocation is often regarded as a process in rapid equilibrium. However, kinetic studies of horse liver ADH have shown that the proton transfer can become rate-limiting for the overall enzymatic reaction (16.Kovaleva E.G. Plapp B.V. Deprotonation of the horse liver alcohol dehydrogenase-NAD+ complex controls formation of the ternary complexes.Biochemistry. 2005; 44: 12797-127808Crossref PubMed Scopus (29) Google Scholar), emphasizing how important it is to consider proton transfer in the ADH mechanism. As in the horse liver enzyme, the overall proton transfer in the different ADHs 3Horse liver ADH represents the group of Zn+-dependent ADHs of the medium-chain dehydrogenase/reductase protein family (3.Meijers R. Cedergren-Zeppezauer E.S. Messerschmidt A. Cygler M. Bode W. Handbook of metalloproteins. 1st Ed. John Wiley & Sons, Ltd., Chichester, United Kingdom2004: 5-33Google Scholar). ADHs from the protein families of short-chain dehydrogenases/reductases (68.Ladenstein R. Winberg J.O. Benach J. Medium- and short-chain dehydrogenase/reductase gene and protein families. Structure-function relationships in short-chain alcohol dehydrogenases.Cell. Mol. Life Sci. 2008; 65: 3918-3935Crossref PubMed Scopus (32) Google Scholar), long-chain dehydrogenases/reductases (69.Klimacek M. Kavanagh K.L. Wilson D.K. Nidetzky B. Pseudomonas fluorescens mannitol 2-dehydrogenase and the family of polyol-specific long-chain dehydrogenases/reductases. Sequence-based classification and analysis of structure-function relationships.Chem. Biol. Interact. 2003; 143: 559-582Crossref PubMed Scopus (19) Google Scholar), and aldo-keto reductases (70.Hyndman D. Bauman D.R. Heredia V.V. Penning T.M. The aldo-keto reductase superfamily homepage.Chem. Biol. Interact. 2003; 143: 621-631Crossref PubMed Scopus (261) Google Scholar) utilize a metal-independent mechanism of catalysis. The proton transfer in these non-Zn2+ ADHs is not well understood. must probably be viewed as a dynamic process in which protein conformational changes at different time and length scales are coupled to the actual protonation/deprotonation events (16.Kovaleva E.G. Plapp B.V. Deprotonation of the horse liver alcohol dehydrogenase-NAD+ complex controls formation of the ternary complexes.Biochemistry. 2005; 44: 12797-127808Crossref PubMed Scopus (29) Google Scholar, 17.Plapp B.V. Conformational changes and catalysis by alcohol dehydrogenase.Arch. Biochem. Biophys. 2010; 493: 3-12Crossref PubMed Scopus (77) Google Scholar). The molecular characterization of dynamic features of the proton transfer is a current theme of fundamental importance in mechanistic enzymology (17.Plapp B.V. Conformational changes and catalysis by alcohol dehydrogenase.Arch. Biochem. Biophys. 2010; 493: 3-12Crossref PubMed Scopus (77) Google Scholar, 18.Leferink N.G. Han C. Antonyuk S.V. Heyes D.J. Rigby S.E. Hough M.A. Eady R.R. Scrutton N.S. Hasnain S.S. Proton-coupled electron transfer in the catalytic cycle of Alcaligenes xylosoxidans copper-dependent nitrite reductase.Biochemistry. 2011; 50: 4121-4131Crossref PubMed Scopus (57) Google Scholar, 19.Heyes D.J. Levy C. Sakuma M. Robertson D.L. Scrutton N.S. A twin-track approach has optimized proton and hydride transfer by dynamically coupled tunneling during the evolution of protochlorophyllide oxidoreductase.J. Biol. Chem. 2011; 286: 11849-11854Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 20.Reece S.Y. Nocera D.G. Proton-coupled electron transfer in biology. Results from synergistic studies in natural and model systems.Annu. Rev. Biochem. 2009; 78: 673-699Crossref PubMed Scopus (346) Google Scholar, 21.Luo J. Bruice T.C. Dynamic structures of horse liver alcohol dehydrogenase (HLADH). Results of molecular dynamics simulations of HLADH-NAD(+)-PhCH(2)OH, HLADH-NAD(+)-PhCH(2)O(−), and HLADH-NADH-PhCHO.J. Am. Chem. Soc. 2001; 123: 11952-11959Crossref PubMed Scopus (55) Google Scholar, 22.Siegbahn P.E. Blomberg M.R. Quantum chemical studies of proton-coupled electron transfer in metalloenzymes.Chem. Rev. 2010; 110: 7040-7061Crossref PubMed Scopus (161) Google Scholar, 23.Hammes-Schiffer S. Comparison of hydride, hydrogen atom, and proton-coupled electron transfer reactions.Chemphyschem. 2002; 3: 33-42Crossref PubMed Scopus (95) Google Scholar, 24.Schwartz S.D. Hynes J.T. Klinman J.T. Limbach H.H. Schowen R.L. Hydrogen-Transfer Reactions. WILEY-VCH, Weinheim2007: 1209-1239Google Scholar). Such as in the classical ADHs (Equation 1), coupling of hydride transfer to a multistep proton transfer is a distinctive feature of the catalytic mechanism of various other NAD(P)+-dependent dehydrogenases (24.Schwartz S.D. Hynes J.T. Klinman J.T. Limbach H.H. Schowen R.L. Hydrogen-Transfer Reactions. WILEY-VCH, Weinheim2007: 1209-1239Google Scholar, 25.Ferrer S. Silla E. Tunon I. Oliva M. Moliner V. Williams I.H. Dependence of enzyme reaction mechanism on protonation state of titratable residues and QM level description: lactate dehydrogenase.Chem. Commun. 2005; 47: 5873-5875Crossref Scopus (19) Google Scholar), as well as alcohol oxidases utilizing FAD or FMN as cofactor to promote hydride-transfer oxidation of their substrates (18.Leferink N.G. Han C. Antonyuk S.V. Heyes D.J. Rigby S.E. Hough M.A. Eady R.R. Scrutton N.S. Hasnain S.S. Proton-coupled electron transfer in the catalytic cycle of Alcaligenes xylosoxidans copper-dependent nitrite reductase.Biochemistry. 2011; 50: 4121-4131Crossref PubMed Scopus (57) Google Scholar, 19.Heyes D.J. Levy C. Sakuma M. Robertson D.L. Scrutton N.S. A twin-track approach has optimized proton and hydride transfer by dynamically coupled tunneling during the evolution of protochlorophyllide oxidoreductase.J. Biol. Chem. 2011; 286: 11849-11854Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 26.Basran J. Hothi P. Masgrau L. Sutcliffe M.J. Scrutton N.S. Hynes J.T. Klinman J.T. Limbach H.H. Schowen R.L. Hydrogen-Transfer reactions. WILEY-VCH, Weinheim, Germany2007: 1341-1359Google Scholar, 27.Frederick K.K. Ballou D.P. Palfey B.A. Protein dynamics control proton transfers to the substrate on the His72 → Asn mutant of p-hydroxybenzoate hydroxylase.Biochemistry. 2001; 40: 3891-3899Crossref PubMed Scopus (24) Google Scholar, 28.Frederick K.K. Palfey B.A. Kinetics of proton-linked flavin conformational changes in p-hydroxybenzoate hydroxylase.Biochemistry. 2005; 44: 13304-13314Crossref PubMed Scopus (12) Google Scholar, 29.Furuichi M. Suzuki N. Dhakshnamoorhty B. Minagawa H. Yamagishi R. Watanabe Y. Goto Y. Kaneko H. Yoshida Y. Yagi H. Waga I. Kumar P.K. Mizuno H. X-ray structures of Aerococcus viridans lactate oxidase and its complex with d-lactate at pH 4.5 show an α-hydroxyacid oxidation mechanism.J. Mol. Biol. 2008; 378: 436-446Crossref PubMed Scopus (30) Google Scholar). Elucidation of the involvement of the proton relay system in the steps of the catalytic cycle presents a key scientific problem for each of these enzymes. Mannitol 2-dehydrogenase from the bacterium Pseudomonas fluorescens (PfM2DH) 4The enzyme belongs to a family of polyol-specific long-chain dehydrogenases/reductases and utilizes a metal-independent mechanism of catalysis (69.Klimacek M. Kavanagh K.L. Wilson D.K. Nidetzky B. Pseudomonas fluorescens mannitol 2-dehydrogenase and the family of polyol-specific long-chain dehydrogenases/reductases. Sequence-based classification and analysis of structure-function relationships.Chem. Biol. Interact. 2003; 143: 559-582Crossref PubMed Scopus (19) Google Scholar). shows a proton shuttle system that is quite noticeable among ADHs and mechanistically related oxidoreductases. Crystal structures of PfM2DH have revealed a narrow channel that is accessible to water and connects the active site with bulk solvent (30.Kavanagh K.L. Klimacek M. Nidetzky B. Wilson D.K. Crystal structure of Pseudomonas fluorescens mannitol 2-dehydrogenase binary and ternary complexes. Specificity and catalytic mechanism.J. Biol. Chem. 2002; 277: 43433-43442Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The PfM2DH channel shares various features of molecular anatomy (Fig. 1) with the canonical proton channels of redox-driven proton pumps such as cytochrome c oxidase (31.Namslauer A. Brzezinski P. Structural elements involved in electron-coupled proton transfer in cytochrome c oxidase.FEBS Lett. 2004; 567: 103-110Crossref PubMed Scopus (93) Google Scholar) and bacteriorhodopsin (32.Lanyi J.K. Schobert B. Mechanism of proton transport in bacteriorhodopsin from crystallographic structures of the K, L, M1, M2, and M2′ intermediates of the photocycle.J. Mol. Biol. 2003; 328: 439-450Crossref PubMed Scopus (143) Google Scholar, 33.Neutze R. Pebay-Peyroula E. Edman K. Royant A. Navarro J. Landau E.M. Bacteriorhodopsin, a high-resolution structural view of vectorial proton transport.Biochim. Biophys. Acta. 2002; 1565: 144-167Crossref PubMed Scopus (170) Google Scholar, 34.Wolf S. Freier E. Gerwert K. How does a membrane protein achieve a vectorial proton transfer via water molecules?.Chemphyschem. 2008; 9: 2772-2778Crossref PubMed Scopus (24) Google Scholar, 35.Wolf S. Freier E. Potschies M. Hofmann E. Gerwert K. Directional proton transfer in membrane proteins achieved through protonated protein-bound water molecules. A proton diode.Angew. Chem. Int. Ed. Engl. 2010; 49: 6889-6893Crossref PubMed Scopus (49) Google Scholar). It contains a “mobile” carboxylic acid residue (Glu292) accommodated within an otherwise comparatively hydrophobic channel interior. The side chain of Glu292 was seen to adopt distinct conformations down (Fig. 1A) and up (Fig. 1B) the channel in the holoenzyme bound with NAD+ and a ternary complex of PfM2DH with NAD+ and mannitol, respectively. The down → up movement of Glu292 represents the largest (≈5 Å displacement) among a series of small structural rearrangements in and outside of the PfM2DH channel that occur in response to mannitol binding, as shown in Fig. 1 and supplemental Movie S1. Globally, the conformational change of PfM2DH can be characterized as a subtle domain-opening process in which the C-terminal substrate-binding domain rotates away by about ∼2° from the N-terminal NAD+-binding domain, using the loop region of residues 282–287 as the hinge region. At the top of the channel on the PfM2DH surface, two ionizable amino acids (His294, Glu293) are positioned opposite to each other within hydrogen bonding distance (Fig. 1A). The interaction between His294 and Glu293 becomes disrupted as a result of the down → up movement of Glu292 (Fig. 1B). We extrapolate from reported relationships between structure and function of proton channels (36.Wraight C.A. Chance and design, proton transfer in water, channels, and bioenergetic proteins.Biochim. Biophys. Acta. 2006; 1757: 886-912Crossref PubMed Scopus (319) Google Scholar) that the particular arrangement of His294 and Glu293 might serve the role of a local proton reservoir, facilitating uptake and release of protons in each direction through adjustment of trans-channel proton gradient. Two mechanisms of proton transfer via the catalytic base Lys295 (37.Klimacek M. Kavanagh K.L. Wilson D.K. Nidetzky B. On the role of Brønsted catalysis in Pseudomonas fluorescens mannitol 2-dehydrogenase.Biochem. J. 2003; 375: 141-149Crossref PubMed Google Scholar, 38.Klimacek M. Nidetzky B. A catalytic consensus motif for d-mannitol 2-dehydrogenase, a member of a polyol-specific long-chain dehydrogenase family, revealed by kinetic characterization of site-directed mutants of the enzyme from Pseudomonas fluorescens.Biochem. J. 2002; 367: 13-18Crossref PubMed Google Scholar) were considered for PfM2DH (30.Kavanagh K.L. Klimacek M. Nidetzky B. Wilson D.K. Crystal structure of Pseudomonas fluorescens mannitol 2-dehydrogenase binary and ternary complexes. Specificity and catalytic mechanism.J. Biol. Chem. 2002; 277: 43433-43442Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). One involves a shuttling motion of Glu292 (Fig. 1, I) comparable with the mode of proton translocation utilized by known proton pumps (34.Wolf S. Freier E. Gerwert K. How does a membrane protein achieve a vectorial proton transfer via water molecules?.Chemphyschem. 2008; 9: 2772-2778Crossref PubMed Scopus (24) Google Scholar, 35.Wolf S. Freier E. Potschies M. Hofmann E. Gerwert K. Directional proton transfer in membrane proteins achieved through protonated protein-bound water molecules. A proton diode.Angew. Chem. Int. Ed. Engl. 2010; 49: 6889-6893Crossref PubMed Scopus (49) Google Scholar, 39.Garczarek F. Brown L.S. Lanyi J.K. Gerwert K. Proton binding within a membrane protein by a protonated water cluster.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 3633-3638Crossref PubMed Scopus (176) Google Scholar, 40.Belevich I. Gorbikova E. Belevich N.P. Rauhamäki V. Wikström M. Verkhovsky M.I. Initiation of the proton pump of cytochrome c oxidase.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 18469-18474Crossref PubMed Scopus (30) Google Scholar). Of note, assistance from Glu292 to the removal of protons from the active site could provide internal “pull” to oxidation of the bound alcohol substrate. In an alternative scenario, Glu292 acts as a gate that opens as a result of the up-flip of the Glu side chain, thereby establishing a “water wire” along which protons can flow to solvent (Fig. 1C, II). In both mechanisms, proton translocation would be effectively discontinuous, and the essential time switch for the conformational change of Glu292 might be provided by events of the catalytic cycle. In marked contrast to PfM2DH, proton relays of other ADHs are typically built from hydrogen bond networks that seem to operate continuously and do not involve major conformational rearrangements to achieve the proton translocation (11.Klimacek M. Hellmer H. Nidetzky B. Catalytic mechanism of Zn2+-dependent polyol dehydrogenases: kinetic comparison of sheep liver sorbitol dehydrogenase with wild-type and Glu154 → Cys forms of yeast xylitol dehydrogenase.Biochem. J. 2007; 404: 421-429Crossref PubMed Scopus (11) Google Scholar, 16.Kovaleva E.G. Plapp B.V. Deprotonation of the horse liver alcohol dehydrogenase-NAD+ complex controls formation of the ternary complexes.Biochemistry. 2005; 44: 12797-127808Crossref PubMed Scopus (29) Google Scholar). In this work, site-directed mutagenesis, kinetics, and molecular dynamics (MD) simulation were used to interrogate the proposed dynamic function of Glu292 during proton transfer by PfM2DH. Replacement of the Glu by Ala was chosen to disrupt shuttling motions (Fig. 1C, I) or to prevent closing of the proton channel (Fig. 1C, II). The requirement to decrease steric bulk at position 292 in the channel explains the use of a small residue as compared with the approximately isosteric Gln. Catalysis to mannitol oxidation could thus be compared for situations in which protein control over proton translocation to solvent was present (wild-type enzyme) or absent (E292A). A dynamic mechanism of proton transfer is suggested in which, once mannitol has bound to the enzyme-NAD+ complex, a down → up conformational change of Glu292 results in positioning of Lys295 such that it connects the reactive C2 hydroxyl of substrate to a chain of water molecules in the now open proton conduit (Fig. 1C, II). The opposite up → down motion of Glu292 in the step post-catalysis results in release of Lys295 from being bonded with the carbonyl group of fructose, thereby promoting the product dissociation. Materials and chemicals were described elsewhere (37.Klimacek M. Kavanagh K.L. Wilson D.K. Nidetzky B. On the role of Brønsted catalysis in Pseudomonas fluorescens mannitol 2-dehydrogenase.Biochem. J. 2003; 375: 141-149Crossref PubMed Google Scholar, 38.Klimacek M. Nidetzky B. A catalytic consensus motif for d-mannitol 2-dehydrogenase, a member of a polyol-specific long-chain dehydrogenase family, revealed by kinetic characterization of site-directed mutants of the enzyme from Pseudomonas fluorescens.Biochem. J. 2002; 367: 13-18Crossref PubMed Google Scholar, 41.Klimacek M. Nidetzky B. The oxyanion hole of Pseudomonas fluorescens mannitol 2-dehydrogenase. A novel structural motif for electrostatic stabilization in alcohol dehydrogenase active sites.Biochem. J. 2010; 425: 455-463Crossref Scopus (10) Google Scholar, 42.Klimacek M. Nidetzky B. Examining the relative timing of hydrogen abstraction steps during NAD(+)-dependent oxidation of secondary alcohols catalyzed by long-chain d-mannitol dehydrogenase from Pseudomonas fluorescens using pH and kinetic isotope effects.Biochemistry. 2002; 41: 10158-10165Crossref PubMed Scopus (16) Google Scholar). Wild-type PfM2DH was prepared by known procedures (43.Slatner M. Nidetzky B. Kulbe K.D. Kinetic study of the catalytic mechanism of mannitol dehydrogenase from Pseudomonas fluorescens.Biochemistry. 1999; 38: 10489-10498Crossref PubMed Scopus (49) Google Scholar). Mutation Glu292 → Ala was introduced by inverse PCR (38.Klimacek M. Nidetzky B. A catalytic consensus motif for d-mannitol 2-dehydrogenase, a member of a polyol-specific long-chain dehydrogenase family, revealed by kinetic characterization of site-directed mutants of the enzyme from Pseudomonas fluorescens.Biochem. J. 2002; 367: 13-18Crossref PubMed Google Scholar) using a pair of oligonucleotide primers: 5′-GTG ACA CCC TAT GCA GAG ATG AAG-3′ and 5′-GGA CAT CAC GGT AAA CGC-3′. The mismatched bases are underlined. The mutated gene was expressed in Escherichia coli JM109, and the E292A mutant was isolated according to protocol (43.Slatner M. Nidetzky B. Kulbe K.D. Kinetic study of the catalytic mechanism of mannitol dehydrogenase from Pseudomonas fluorescens.Biochemistry. 1999; 38: 10489-10498Crossref PubMed Scopus (49) Google Scholar). CD spectra of wild-type PfM2DH and E292A were recorded as described elsewhere (38.Klimacek M. Nidetzky B. A catalytic consensus motif for d-mannitol 2-dehydrogenase, a member of a polyol-specific long-chain dehydrogenase family, revealed by kinetic characterization of site-directed mutants of the enzyme from Pseudomonas fluorescens.Biochem. J. 2002; 367: 13-18Crossref PubMed Google Scholar). Molar enzyme concentrations were determined from absorbance (280 nm) using an extinction coefficient of 55.4 mm−1 cm−1 for wild-type enzyme and mutant (44.Gasteiger E. Hoogland C. Gattiker A. Duvaud S. Wilkins M.R. Appel R.D. Bairoch A. Walker J.M. The Proteomics Protocols Handbook. Humana Press Inc., Totowa, NJ2005: 571-607Crossref Google Scholar). Initial reaction rates were recorded spectrophotometrically at 25 °C, measuring the change in absorbance of NADH at 340 nm (ϵ340 = 6.22 mm−1 cm−1). A full steady-state kinetic analysis was performed at pH 7.1 (100 mm Tris-HCl) and pH 10.0 (100 mm glycine-NaOH). Kinetic data were collected with the concentrations of substrate and coenzyme being varied against one another, and parameters (kcat, KiC, KC, KS) were obtained by nonlinear regression, as described elsewhere (38.Klimacek M. Nidetzky B. A catalytic consensus motif for d-mannitol 2-dehydrogenase, a member of a polyol-specific long-chain dehydrogenase family, revealed by kinetic characterization of site-directed mutants of the enzyme from Pseudomonas fluorescens.Biochem. J. 2002; 367: 13-18Crossref PubMed Google Scholar, 42.Klimacek M. Nidetzky B. Examining the relative timing of hydrogen abstraction steps during NAD(+)-dependent oxidation of secondary alcohols catalyzed by long-chain d-mannitol dehydrogenase from Pseudomonas fluorescens using pH and kinetic isotope effects.Biochemistry. 2002; 41: 10158-10165Crossref PubMed Scopus (16) Google Scholar, 43.Slatner M. Nidetzky B. Kulbe K.D. Kinetic study of the catalytic mechanism of mannitol dehydrogenase from Pseudomonas fluorescens.Biochemistry. 1999; 38: 10489-10498Crossref PubMed Scopus (49) Google Scholar). kcat is the turnover number, KiC is the dissociation constant of the enzyme-coenzyme complex, KC and KS are Michaelis constants for coenzyme and substrate, respectively. We use subscript O and R on kcat and the transient rate constant kobs (see later) to indicate the direction of mannitol oxidation (kcatO, kobsO) and fructose reduction (kcatR, kobsR), respectively. The pH dependences of kinetic parameters for E292A were determined in the pH range 7.1–10.5 for mannitol oxidation (kcatO, KC, and KS) and 7.1–10.0 for fructose reduction (kcatR, KS) as described previously in studies of other PfM2DH mutants (41.Klimacek M. Nidetzky B. The oxyanion hole of Pseudomonas fluorescens mannitol 2-dehydrogenase. A novel structural motif for electrostatic stabilization in alcohol dehydrogenase active sites.Biochem. J. 2010; 425: 455-463Crossref Scopus (10) Google Scholar). A two-component buffer (Tris, glycine) having pH-independent ionic strength of 0.1 m was used (45.Ellis K.J. Morrison J.F. Buffers of constant ionic strength for studying pH-dependent processes.Methods Enzymol. 1982; 87: 405-426Crossref PubMed Scopus (648) Google Scholar). Kinetic isotope effects (KIE) resulting from deuteration of one of the reactants (2-[2H]mannitol; S-4-[2H]NADH) or solvent were determined using reported procedures (42.Klimacek M. Nidetzky B. Examining the relative timing of hydrogen abstraction steps during NAD(+)-dependent oxidation of secondary alcohols catalyzed by long-chain d-mannitol dehydrogenase from Pseudomonas fluorescens using pH and kinetic isotope effects.Biochemistry. 2002; 41: 10158-10165Crossref PubMed Scopus (16) Google Scholar, 43.Slatner M. Nidetzky B. Kulbe K.D. Kinetic study of the catalytic mechanism of mannitol dehydrogenase from Pseudomonas fluorescens.Biochemistry. 1999; 38: 10489-10498Crossref PubMed Scopus (49) Google Scholar). A nomenclature is used where superscript D describes the primary deuterium KIE and superscript D2O describes the solvent KIE on the respective isotope-sensitive parameter (46.Northrop D.B. Cleland W.W. O'Leary M.H. Northrop D.B. Isotope Effects on Enzyme-catalyzed Reactions. University Park Press, Baltimore, MD1977: 122Google Scholar). DKIEs were obtained in" @default.
• W2071957028 created "2016-06-24" @default.
• W2071957028 creator A5018956751 @default.
• W2071957028 creator A5033525050 @default.
• W2071957028 creator A5051203357 @default.
• W2071957028 date "2012-02-01" @default.
• W2071957028 modified "2023-09-26" @default.
• W2071957028 title "Dynamic Mechanism of Proton Transfer in Mannitol 2-Dehydrogenase from Pseudomonas fluorescens" @default.
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