Choline oxidase (E.C. 1.1.3.17) catalyzes the two-step, four-electron oxidation of choline to glycine betaine with betaine aldehyde as enzyme-associated intermediate and molecular oxygen as final electron acceptor (Scheme 1). The gem-diol, hydrated species of the aldehyde intermediate of the reaction acts as substrate for aldehyde oxidation, suggesting that the enzyme may use similar strategies for the oxidation of the alcohol substrate and aldehyde intermediate. The determination of the chemical mechanism for alcohol oxidation has emerged from biochemical, mechanistic, mutagenetic, and structural studies. As illustrated in the mechanism of Scheme 2, the alcohol substrate is initially activated in the active site of the enzyme by removal of the hydroxyl proton. The resulting alkoxide intermediate is then stabilized in the enzyme-substrate complex via electrostatic interactions with active site amino acid residues. Alcohol oxidation then occurs quantum mechanically via the transfer of the hydride ion from the activated substrate to the N(5) flavin locus. An essential requisite for this mechanism of alcohol oxidation is the high degree of preorganization of the activated enzyme-substrate complex, which is achieved through an internal equilibrium of the Michaelis complex occurring prior to, and independently from, the subsequent hydride transfer reaction. The experimental evidence that support the mechanism for alcohol oxidation shown in Scheme 2 is briefly summarized in the Results and Discussion section

Finnegan, S.

Francis, K.

Gadda, G.

Mijatovic, S.

Nguyen, T.

Orville, A.

Pennati, A.

Quaye, O.

Rungsrisuriyachai, K.

Yuan, H.

UNT Digital Library

   BNL-90632-2009-CP   Hydride Transfer Made Easy in the Oxidation of Alcohols Catalyzed by Choline Oxidase   Giovanni Gadda, Andrea Pennati, Kevin Francis, Osbourne Quaye, Hongling Yuan, Kunchala Rungsrisuriyachai, Steffan Finnegan, Slavica Mijatovic, Tranbao Nguyen  Departments of Chemistry and Biology and The Center for Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30302-4098   Allen M. Orville Biology Department, Brookhaven National Laboratory, Upon, NY 11973-5000    Presented at the 16th International Symposium on Flavins and Flavoproteins Jaca, Spain June 8-13, 2008    January 2008    Biology Department  Brookhaven National Laboratory P.O. Box 5000 Upton, NY 11973-5000 www.bnl.gov  Notice: This manuscript has been authored by employees of Brookhaven Science Associates, LLC under Contract No. DE-AC02-98CH10886 with the U.S. Department of Energy. The publisher by accepting the manuscript for publication acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes.  This preprint is intended for publication in a journal or proceedings.  Since changes may be made before publication, it may not be cited or reproduced without the author’s permission.    DISCLAIMER  This report was prepared as an account of work sponsored by an agency of the United States Government.  Neither the United States Government nor any agency thereof, nor any of their employees, nor any of their contractors, subcontractors, or their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or any third party’s use or the results of such use of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof or its contractors or subcontractors.  The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof.                                    309 d '­e e It n i S' 1 Hydride Transfer Made Easy in the Oxidation of Alcohols Catalyzed by Choline Oxidase Giovanni Gadda, Andrea Pennati, Kevin Francis. Osbourne Quaye. Hongling Yuan, Kunchala Rungsrisuriyachai, Steffan Finnegan, Siavica Mijatovic, Tranbao Nguyen Departments of Chemistry and Biology and The Center for Biotechnology and Drug Design, Georgia State University, Atlanta, Georgia 30302-4098 Allen M. Orville Biology Department, Brookhaven National Laboratory, Upton, NY 11973-5000 Introduction Choline oxidase (E.C. 1.1.3.17) catalyzes the two-step, four-electron oxidation of choline to glycine betaine with betaine aldehyde as enzyme-associated intermediate and molecular oxygen as final electron acceptor (Scheme I) [I]. The gem-diol, hydrated species of the aldehyde intermediate of the reaction acts as substrate for aldehyde oxidation [2], suggesting that the enzyme may use similar strategies tor the oxidation of the alcohol substrate and aldehyde intermediate. 1,/". /H /::~~ -N 1\ FAD FADH' 0 OH FAD FADH1 H20 H20 2 O2 H20 2 O2 Scheme 1: Two-step, j()Ur-elcctron oxidation or choline cataly,(ed by cholinc oxidase The detemlination of the chemical mechanism for alcohol oxidation has emerged trom biochemical [1, 3-6], mechanistic [2, 7-11], mutagenetic [12-14], and structural studies [14]. As illustrated in the mechanism of Scheme 2, the alcohol substrate is initially activated in the active site of the enzyme by removal of the hydroxyl proton [3, 4, TJ. The resulting alkoxide intermediate is then stabilized in the enzyme-substrate complex via electrostatic interactions with active site amino acid residues [\2.14]. Alcohol oxidation then occurs quantum mechanically via the transfer of the hydride ion from the activated substrate to the N(5) flavin locus [8, 9]. An essential requisite tc)r this mechanism of alcohol oxidation is the high degree of preorganization of the activated enzyme-substrate complex, which is achieved through an internal equilibrium of the Michaelis complex occurring prior to, and independently from, the subsequent hydride transfer reaction [I I, 14]. The experimental evidence that support the mechanism lor alcohol oxidation shown in Scheme 2 is briefly summarized in the Results and Discussion section. 310 N E:J1 .l' F3S7 VV:P1 R N N 0 H NH NN -H 0 H N H4Go N - 0 H~ ~, :B N N F1S7 ~~ VV'j)1 I V464 R . N N 0 N NHN , H 0 N ,'" 0 H H BH 1~ NN H99R N R N N 0 N N 0 t H NH NN NH NI I H 0 N E312 o .N 1 ~ HF3S7 N 0 H F357 N W331 H W3J , V4t;4 BH B,. Scheme 2: Chcmical mcchanism f(.Jr the llxidation of choline by choline oxidll,e Results and Discussion Activation o(Thc a/coho! suhsTraTe The pH profiles for the steady state kinetic parameters have been determincd for the wild type enzyme using choline and betaine aldehyde, and a numher of substrate analogues, such as 1,2-[:'114]-cI10Iine, N.N-dimethyletl1anolaminc. ,v. methylethanolamine, and 3,3-dimethyl-butan-1-ol [2-4,7,8. 10]. In all cascs. both the kea/KIll and kelH pH profiles increase to limiting values with increasing pH. suggesting the requirement of a group with pK" value of 7.5 to be unprotonatcd for catalysis. The value of 7.5 is a thermodynamic pK", as suggested by pH dependence studies of enzyme inhibition using glycine betaine [4]. A reasonable role for the active site group that needs to be unprotonated for catalysis is to act as a catalytic base lor the abstraction of the hydroxyl proton of the alcohol substrate (top /ett ponel o( 5;cheme 2). Two histidine residues E312 are suitably located in the active \ FJ51site cavity of the enzyme with the potential to activate choline during turnover of the eni'yme, 11351 , 5101 namely His-351 and His-466 -~ <~~,," \ W::13i (Figure I) [14]. Site-directed replacement of either residue with Figure I: Thc active site residues of cholinealanine results in mutant variants m.idasc (PDB 2jb\). ;\ hypothetical cholineof the enzyme (H351 A, Figure 2: molecule has bcen dockcd in the cCllkr orth~ ac;i\t H466A [12]) with significantly sile ca\ity. :111 lowered kca/K", and kGi' values for choline at pH 10, i.e., the pi I independent region pH profiles. Interestingly. both single mutants show pH profiles far the k,a/Kin and kcal values that are still consistent with the presence of an unprotonated group that is required for cata lysis. with thermodynamic pKl values of R.O and 9.0 tar "':cn 61 .6 H351A (data not shown) and "': • •• • .1 H466A [12]. respectively. These ;4 0~~E 41 co suggest that neither of the • • • • (')~two histidine residues is solely ~_2 2 ~_., • responsible for the activation of .:c (,) •• 1 (fj•the choline suhstrate. Instead. ! 0 .:...0) 0' they may either act together in the o abstraction of the hydroxyl proton -2 -2 of the alcohol substrate, or 5 6 7 8 9 10 substitute far each other when one is replaced by alanine. Studies on pH double mutant variants in which both histidine residues have been Figure 2: pH prolilcs 1'01' the kc"iK", (e). lic ,,' (e). and replaced with other amino acids (_) vallics ,vitll choline as substrate for tile 1135 Ii\ varinnt or cholinc oxidase.art.: currently in progress to address this issue. Slabi!i::Uliol1 ollhe ulkoxide intermediute Substrate and solvent kinetic isotope effects on the k,,/Kill and k,cd val ues with choline at pH 10 havc demonstrated that ckavage of the OH bond or choline occlIrs prior to. and completely decoupkd from. the subsequent hydride transfer reaction thal results in the cleavage 01' the ell bond (Scheme 2) [9]. Mechanistic data for mutant forms of choline oxidase in which G lu-3 12 [14] and II is-466 [12. 13] have bccn replaced by other amino acids strongly suggest that these active site residues play crucial roles for [he stahilization of the alkoxide intermediate that is formed in catalysis. The negatively charged side chain of Glu-312 interacts electrostatically \\ ith the positively charged trimethylammoniul11 moiety of the organic substrate and, by extension. with the intermediates and the product of the enzymatic reaction. Indeed. replacement of Glu-312 ,vith glutamine results in a 500-fold increase in the Kd value for chol ine, as determined using rapid kinetics techniques [14]. This corresponds to an energetic contribution or -15 kJ/mol, in good agreement with the estimate of -13 k.J/mol that was independently determined from mechanistic studies with the choline analogue lacking the positive charge. i.e.. 3.3-dimethyl-butan-l-ol [10]. Mechanistic, spectroscopic, and biochemical studies on the enzyme variant in which Ilis-466 is rcplaeed with alanine strongly support the notion that His-466 is protonatcd during the reductive half-reaction in which choline is oxidized to betaine aldehyde (Scheme 2) [12]. Mechanistic data further suggest that the protonated histidine contributes to the 31:: electrostatic stabilization of the alkoxide intermediate and the transition stale for the oxidation of choline to betaine aldehyde [12]. Qualltum mechanical hydride tral1sfcrji'o/11 the activated slIhsrrate to {hef/arin The effect of oxygen and temperature on the kinetic isotope effeets on the steady state kinetic parameters kcjKm and kca, with I)-(H 4]-ehol ine as substrate as a function of pH have demonstrated that oxygen availability modulates whether the reduced enzyme-betaine aldehyde complex partitions forward to catalysis rather than reverting to the oxidized enzyme-choline alkoxide species [8]. At saturating concentrations of oxygen, i.e., under an irreversible catalytic regime. the value is 10.6 ::± 0.6 and temperature independent [8]. The corresponding isotope elfect on thc Eyring preexponential factors (AH'/ An') is significantly larger than unity tl4 3) [8]. Furthermore, the enthalpies of activation (AN:) for the hydride transfer reaction with choline and 1.2-(H4]-choline are not significantly different from each other, with values or 18 2 and 18 ± 5 kJ/mol, respectively [8]. All taken together, these data are consistent with a mechanism of hydride transkr in which the hydride tunnels quantum mechanically from the a-carbon of the alkoxide species to the N(5) atom of the enzyme-bound Havin within a highly preorganized activated enzyme-substrate complex (Scheme 2) [8]. The effects or pH and temperature on the substrate kinetic isotope effects with 1.2-[CH.d-choline have been studied also at the subsaturating concentration of oxygen of 0.2 111M. to gain insights into the mechanism or hydride transfer under a reversible catalytic regime [11]. The flux or reaction intermediates through the reverse of the hydride transfer step, i.e., the so-called reverse commitment to catalysis, changes with temperature. with the hydride transfer reaction becoming more reversible as the temperature increases from I () (lC to 45 (lC [II]. After correction for the kinetic complexity of the reaction due to the reverse commitment to catalysis. analyses of the kinetic data according to both Arrhenius' and Eyring's formalisms demonstrate that the quantum mechanical nature of the hydride transfer reaction is maintained irrespective of whether the regime of catalysis shi fts from irreversible to reversIble [II]. However, the comparison of the thermodynamic parameters f()r the hydride transfer reaction under reversible and irreversible catalytic regimes unveils an enthalpically unfavourable internal equilibrium of the enzyme-substrate complex that occurs prior to the hydride transfer reaction [11]. Such an internal equilihrium is kinetically undetectable in the wild type enzyme, and is likely required to preorganize the activated enzyme-substrate complex for the efficient quantum mechanical transfer of the hydride from the substrate a-carbon to the N(5) atom of the enzyme-bound navin. Interestingly, replacement of Glu-312 with aspartate results in an enzyme variant in which the conformational change of the enzyme· substrate complex becomes kinetically relevant, as suggested by the etfects of solvent viscosity on the kea;!Kill value with choline and substrate kinetic isotope effects [14]. / 1 t( P b i~ tl o SI N d~ rf Cl: C in at rf a( C T d. cl lr e1 V\­tc F el G rr p C' if \\ o tl A 1 p p 313 PI'('()rguni:;Uliol1 o(thc actimted CI7::I'me-Sllhstrale complex The results of the crystallographic and mechanistic studies on choline oxidase reported thus nlr are consistent vvilh the active site of the enzyme being well-suited 10 ma:\imize several aspects of the oxidation reaction. Choline is bound productively through its trimethylamlllonium group so that alcohol activation can be efliciently achieved by the action of the catalytic base. Once alcohol activation is attained. the resulting negatively charged alkoxide spccies is locked in position through electrostatic interaction with a [lositive charge provided by the side chain of His-466. Flavin movement with respect to the organic substrate is also signiticantly restricted due to the covalent linkage or the FAD CX\1 atom with the \<:2 atom or Hi s-99 [14]. Thus. very Iittle independent movement of the hydride donor and acceptor is allowed in the activated enzyme-substrate eOlll[licx, thereby rendering the quantum mechanical transfer of the hydride ion from the alkoxide u­carbon to the flavin 0.1(5) atom the most hlvoured outcome t()r the reaction. Currently. site-directed replacements of amino acids that do not participate directly in the oxidation of choline to betaine aldehyde in the active site of choline oxidase are under mechanistic investigation in order to establish how the hydride trans leI' reaction is affected by the disruption of [lrcorgani7ation in the enzyme-substrate actilated com [llcx. COf1(-/l!si(JIIS The mechanistic and structural studies reported thus far have contributed to the delineation or the mcchanism for alcohol oxidation in the reaction catalyzed by cholinc oxidase (Scheme 2). The importance of enzymc-substrate preorganization in the active site or the enzyme has heen emerging as an important factor fix the etliciellt oxidation reaction catalyzed by the enzyme. Future mechanistic studies I\ill be aimed at the disnt[ltion or sLich enzyme-substrate preorganization in order '0 estahlish the impact on the hydride transfer reaction catalyzed by the enzyme. Fi11311y. choline oxidase has been grou[led in a sU[lcrfamily of !lavin-dependent en/ymes that oxidize alcohols to the corresponding carbonyl compounds. the Glucose-Mcthanol-Choline Oxidoreduc1ases. The crystal structures of several members of the supcrfamily. including glucose oxidase. cholesterol oxidase, pyranose 2-oxidase. cellobiose dehydrogenase, and choline oxidase itsel( are currently available, showing that the active sites of these enzymes contain similar. if not idcntical. amino acid residues. Therefore. it will be interesting to establish ",hether the icssons we are continuing to learn regarding the mechanism of alcohol oxidation in the reaction catalY7ed hy choline oxidase can he extended to the rest of the tilll1ily members. Ackntm ledgements This \\ork was sup[lOftecl in [larl by a National Science Foundation CAREER A\\an1 (MCB-0545712) and a grant hom the American Chemical Society Petroleum Rcsearch Fund (37351-04) to G.G. 314 References I. 	 Gadda. G. (2003) Kinetic mechanism of choline oxidase from Arrhrohacter glohi/iJrmis, Biochim. Biophys. Acta 1646. 112-118. 2. 	 Fan. F., Germann. M. W'o and Gadda. G. (2006) Mechanistic studies ofcholme oxidase with betaine aldehyde and its isosteric analogue dimethylbutyraldehyde. Biochemistry 45, 1979- I986. 3. 	 Gadda. G. (2003) pH and deuterium kinetic isoiope effects studies on the oxidation of choline to betaine-aldehyde catalyzed by cholinc oxidase, Biochim. Biophys. Acta 1650,4-9. 4. 	 Ghanem. M ... Fan. F .. Francis. K .• and Gadda. G. (2003) Spectroscopic and kinetic properties of recombinant choline oxidase from Arrhrohacter glohi(ormis, Biochemistry 42. 15179-/5188. 5. 	 Fan, F., Ghanem, 0..1., and Gadda, G. (2004) Cloning, sequence analysis. and purification of choline oxidase from Art/1rohacler g/o/Ji/hrmis: a bacterial enzyme involved in osmotic stress tolerance, Arch. BiocI1l.:'111. Biophys. 421. 149-158. 6. 	 Hoang, J. V.. and Gadda, G. (2007) Trapping choline oxidase in a nonfunctional conformation by freezing at low pi-I. Prot;;.:ins 66, 611-620. 7. 	 Gadda. G .. Pow;;.:IL N. L and Menon, P. (2004) The trimethylammoniurn hcadgroup of cholinc is a major determinant for substrate binding and specificity in choline oxidase, Arch. Biochem. Biophys. 430. 264-273. 8. 	 Fan, F., and Gadda. G. (2005) Oxygen- and tcmperature-dcpcndent kinetic isotopl' eflects in choline oxidase: correlating reversible hydride transfer with environmentally enhanced tunneling, J. Am. Chem. Soc. 127. 17954-17961. 9. 	 Fan, F., and Gadda, G. (2005) On lhc catalytic rncchanism of choline oxidase, J. Am. Chem. Soc. 127,2067-2074. 10. 	Gadda, G., Fan. F.. and Hoang, J. V. (2006) 011 the contribution of the positively charged headgroup of choline to substrate binding and catalysis in the rcaction catalyzed by choline oxidase, Arch. Biochem. Biophys. 451, 182· 187. I J. Fan, F.. and Gadda. G. (2007) An internal cquilibrium preorganizes the en7yme-substrate complex t()r hydride tunneling in choline oxidase. Biochemistry 46. 6402-6408. 12. 	Ghanem. M., and Gadda. G. (2005) On th;;.: catalytic role of the conserved a;;.:live site rcsidue His466 of cholinc oxidase. Biochemistry 44. 893-904. J 3. Ghanem, \;1.. and Gadda, G. (2006) Effects of rcversing the protein positive chargc in the proximity of the tlavin N( I) locus of choline oxidase. Biochemistry 45. 3437-3447. 14. 	QuayI..', 0., Lounios, G. T.. Fan, F .. Orville, A. M.. and Gadda. G. (200X) Role of Glu3 J 2 in binding and positioning of the substrate for the hydride transfer reaction in choline oxidase. Biochemistry 47, 243-256. 

Hydride transfer made easy in the oxidation of alcohols catalyzed by choline oxidase

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Hydride transfer made easy in the oxidation of alcohols catalyzed by choline oxidase

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