The Experts below are selected from a list of 294 Experts worldwide ranked by ideXlab platform
Giovanni Gadda - One of the best experts on this subject based on the ideXlab platform.
-
Substitutions of S101 decrease proton and hydride transfers in the oxidation of betaine aldehyde by Choline Oxidase.
Archives of biochemistry and biophysics, 2017Co-Authors: Giovanni Gadda, Hongling YuanAbstract:Choline Oxidase oxidizes Choline to glycine betaine, with two flavin-mediated reactions to convert the alcohol substrate to the carbon acid product. Proton abstraction from Choline or hydrated betaine aldehyde in the wild-type enzyme occurs in the mixing time of the stopped-flow spectrophotometer, thereby precluding a mechanistic investigation. Mutagenesis of S101 rendered the proton transfer reaction amenable to study. Here, we have investigated the aldehyde oxidation reaction catalyzed by the mutant enzymes using steady-state and rapid kinetics with betaine aldehyde. Stopped-flow traces for the reductive half-reaction of the S101T/V/C variants were biphasic, corresponding to the reactions of proton abstraction and hydride transfer. In contrast, the S101A enzyme yielded monophasic traces like wild-type Choline Oxidase. The rate constants for proton transfer in the S101T/C/V variants decreased logarithmically with increasing hydrophobicity of residue 101, indicating a behavior different from that seen previously with Choline for which no correlation was determined. The rate constants for hydride transfer also showed a logarithmic decrease with increasing hydrophobicity at position 101, which was similar to previous results with Choline as a substrate for the enzyme. Thus, the hydrophilic character of S101 is necessary not only for efficient hydride transfer but also for the proton abstraction reaction.
-
Evidence for proton tunneling and a transient covalent flavin-substrate adduct in Choline Oxidase S101A.
Biochimica et biophysica acta. Proteins and proteomics, 2017Co-Authors: Rizvan Uluisik, Elvira Romero, Giovanni GaddaAbstract:The effect of temperature on the reaction of alcohol oxidation catalyzed by Choline Oxidase was investigated with the S101A variant of Choline Oxidase. Anaerobic enzyme reduction in a stopped-flow spectrophotometer was biphasic using either Choline or 1,2-[2H4]-Choline as a substrate. The limiting rate constants klim1 and klim2 at saturating substrate were well separated (klim1/klim2>9), and were >15-fold slower than for wild-type Choline Oxidase. Solvent deuterium kinetic isotope effects (KIEs) ~4 established that klim1 probes the proton transfer from the substrate hydroxyl to a catalytic base. Primary substrate deuterium KIEs ≥7 demonstrated that klim2 reports on hydride transfer from the Choline alkoxide to the flavin. Between 15°C and 39°C the klim1 and klim2 values increased with increasing temperature, allowing for the analyses of H+ and H- transfers using Eyring and Arrhenius formalisms. Temperature-independent KIE on the klim1 value (H2Oklim1/D2Oklim1) suggests that proton transfer occurs within a highly reorganized tunneling-ready-state with a narrow distribution of donor-acceptor distances. Eyring analysis of the klim2 value gave lines with the slope(Choline)>slope(D-Choline), suggesting kinetic complexity. Spectral evidence for the transient occurrence of a covalent flavin-substrate adduct during the first phase of the anaerobic reaction of S101A CHO with Choline is presented, supporting the notion that an important role of amino acid residues in the active site of flavin-dependent enzymes is to eliminate alternative reactions of the versatile enzyme-bound flavin for the reaction that needs to be catalyzed.
-
Role of F357 as an Oxygen Gate in the Oxidative Half-Reaction of Choline Oxidase
Biochemistry, 2016Co-Authors: Francesca Salvi, Isela Rodriguez, Donald Hamelberg, Giovanni GaddaAbstract:Choline Oxidase from Arthrobacter globiformis catalyzes the oxidation of Choline to glycine betaine by using oxygen as an electron acceptor. A partially rate limiting isomerization of the reduced wild-type enzyme during the reaction with oxygen was previously detected using solvent viscosity effects. In this study, we hypothesized that the side chains of M62 and F357, located at the entrance to the active site of Choline Oxidase, may be related to the slow isomerization detected. We engineered a double-variant enzyme M62A/F357A. The kinetic characterization of the double-variant enzyme showed a lack of the isomerization detected in wild-type Choline Oxidase, and a lack of saturation with an oxygen concentration as high as 1 mM, while most other kinetic parameters were similar to those of wild-type Choline Oxidase. The kinetic characterization of the single-variant enzymes established that only the side chain of F357 plays a role in the isomerization of Choline Oxidase in the oxidative half-reaction. Molec...
-
Role of F357 as an Oxygen Gate in the Oxidative Half-Reaction of Choline Oxidase
2016Co-Authors: Francesca Salvi, Isela Rodriguez, Donald Hamelberg, Giovanni GaddaAbstract:Choline Oxidase from Arthrobacter globiformis catalyzes the oxidation of Choline to glycine betaine by using oxygen as an electron acceptor. A partially rate limiting isomerization of the reduced wild-type enzyme during the reaction with oxygen was previously detected using solvent viscosity effects. In this study, we hypothesized that the side chains of M62 and F357, located at the entrance to the active site of Choline Oxidase, may be related to the slow isomerization detected. We engineered a double-variant enzyme M62A/F357A. The kinetic characterization of the double-variant enzyme showed a lack of the isomerization detected in wild-type Choline Oxidase, and a lack of saturation with an oxygen concentration as high as 1 mM, while most other kinetic parameters were similar to those of wild-type Choline Oxidase. The kinetic characterization of the single-variant enzymes established that only the side chain of F357 plays a role in the isomerization of Choline Oxidase in the oxidative half-reaction. Molecular dynamics studies suggest that the slow isomerization related to F357 is possibly due to the participation of the phenyl ring in a newly proposed gating mechanism for a narrow tunnel, assumed to regulate the access of oxygen to the reduced cofactor
-
Identification of the catalytic base for alcohol activation in Choline Oxidase.
Biochemistry, 2014Co-Authors: Crystal Smitherman, Kunchala Rungsrisuriyachai, Markus W. Germann, Giovanni GaddaAbstract:Choline Oxidase catalyzes the oxidation of Choline to glycine betaine through a two-step, four-electron reaction with betaine aldehyde as an intermediate. Oxygen is the final electron acceptor. Alcohol oxidation is initiated by the removal of the substrate hydroxyl proton by an unknown active site residue with a pKa value of ∼7.5. In the crystal structure of the enzyme in complex with glycine betaine, H466 is ≤3.1 A from the carboxylate oxygen of the reaction product, suggesting a possible role in the proton abstraction reaction catalyzed by the enzyme. H466, along with another potential candidate, H351, was previously mutated to alanine, but this failed to establish if either residue was involved in activation of the substrate. In this study, single variants of Choline Oxidase with H466 and H351 substituted with glutamine were prepared, purified, and characterized. The kcat and kcat/Km values of the H351Q enzyme in atmospheric oxygen were 45- and 5000-fold lower than those of the wild-type enzyme, respec...
Norio Murata - One of the best experts on this subject based on the ideXlab platform.
-
transformation of arabidopsis with the coda gene for Choline Oxidase enhances freezing tolerance of plants
Plant Journal, 2000Co-Authors: Atsushi Sakamoto, Roberto Valverde, Tony H H Chen, Norio MurataAbstract:Summary Arabidopsis thaliana was transformed with the codA gene from Arthrobacter globiformis, which encodes Choline Oxidase, the enzyme that synthesizes glycinebetaine from Choline. The transformation enabled the plants to accumulate glycinebetaine in chloroplasts, and significantly enhanced the freezing tolerance of plants. Furthermore, the photosynthetic machinery of transformed plants was more tolerant to freezing stress than that of wild-type plants. Exogenous application of glycinebetaine also increased the freezing tolerance of wild-type plants, suggesting that the presence of glycinebetaine in transformed plants had enhanced their ability to tolerate freezing stress. Northern blotting analysis revealed that the enhancement of freezing tolerance was not related to the expression of four cold-regulated genes. These results suggest that engineering of the biosynthesis of glycinebetaine by transformation with the codA gene might be an effective method for enhancing the freezing tolerance of plants.
-
Transformation of Japanese persimmon (Diospyros kaki Thunb.) with a bacterial gene for Choline Oxidase
Molecular Breeding, 2000Co-Authors: Mei Gao, Norio Murata, Atsushi Sakamoto, Keisuke Miura, Akira Sugiura, Ryutaro TaoAbstract:This report describes the first successful genetic engineering of tolerance to salt in an agriculturally important species of woody plants by Agrobacterium-mediated transformation with the codA gene of Arthrobacter globiformis. This gene encodes Choline Oxidase, which catalyzes the oxidation of Choline to glycinebetaine. The binary plasmid vector pGC95.091, containing a kanamycin-resistance gene (nptII), a gene for β-glucuronidase (gusA) and the codA gene in its T-DNA region, was used with a disarmed strain of Agrobacterium tumefaciens, EHA101, to transform Japanese persimmon (Diospyros kaki Thunb. `Jiro') by the leaf disk transformation method. The pRS95.101 plasmid that included only nptII and gusA in the T-DNA region was used as a control. We selected eight transgenic lines with one or two copies of the T-DNA after transformation with pGC95.091 (PC lines) and three lines after transformation with pRS95.101 (PR lines). The eight PC lines produced Choline Oxidase and glycinebetaine whereas neither was found in untransformed `Jiro' and in the control PR lines. Transgenic plants grew normally, resembling wild-type plants both in vitro and ex vitro. The activity of photosystem II in leaves of the transgenic Japanese persimmon plants under NaCl stress was determined in terms of the ratio of the variable (Fv) to the maximum (Fm) fluorescence of chlorophyll (Fv/Fm). The rate of decline in (Fv/Fm under NaCl stress was lower in the PC lines than in the control PR lines. These results demonstrated that genetic engineering of Japanese persimmon, which allowed it to accumulate glycinebetaine, enhanced the tolerance to salt stress of this plant.
-
Enhanced tolerance to light stress of transgenic Arabidopsis plants that express the codA gene for a bacterial Choline Oxidase
Plant molecular biology, 1999Co-Authors: Alia, Tony H H Chen, Atsushi Sakamoto, Yasuo Kondo, Hideko Nonaka, Hidenori Hayashi, P. Pardha Saradhi, Norio MurataAbstract:Arabidopsis thaliana was transformed with the codA gene from Arthrobacter globiformis. This gene encodes Choline Oxidase, an enzyme that converts Choline to glycinebetaine. The photosynthetic activity, monitored in terms of chlorophyll fluorescence, of transformed plants was more tolerant to light stress than that of wild-type plants. This enhanced tolerance to light stress was caused by acceleration of the recovery of the photosystem II (PS II) complex from the photo-inactivated state. The transformed plants synthesized glycinebetaine, but no changes were detected in the relative levels of membrane lipids or in the relative levels of fatty acids in the various membrane lipids. Transformation with the codA gene increased levels of H2O2, a by-product of the reaction catalyzed by Choline Oxidase, by only 50% to 100% under stress or non-stress conditions. The activity of ascorbate perOxidase and, to a lesser extent, that of catalase in transformed plants were significantly higher than in the wild-type plants. These observations suggest that H2O2 produced by Choline Oxidase in the transformed plants might have stimulated the expression of H2O2 scavenging enzymes, with resultant maintenance of the level of H2O2 within a certain limited range. It appears that glycinebetaine produced in vivo, but not changes in membrane lipids or in the level of H2O2, protected the PS II complex in transformed plants from damage due to light stress.
-
Transformation with a gene for Choline Oxidase enhances the cold tolerance of Arabidopsis during germination and early growth
Plant Cell and Environment, 1998Co-Authors: Alia, Tony H H Chen, Hidenori Hayashi, Norio MurataAbstract:We transformed Arabidopsis thaliana with the codA gene from Arthrobacter globiformis. This gene encodes Choline Oxidase, the enzyme that converts Choline to glycinebetaine. The presence of Choline Oxidase and glycinebetaine in seeds of transformed lines was confirmed by Western blotting and nuclear magnetic resonance (NMR) spectrometry, respectively. The transformation with the codA gene significantly enhanced the tolerance of seeds to low temperatures, such as 0 °C, during imbibition. The transformation accelerated the germination and growth of seedlings at 10 and 15 °C. It appears that the presence of glycinebetaine in transformed plants enhances their ability to tolerate low-temperature stress during the imbibition and germination of seeds and the growth of seedlings.
-
Transformation of Synechococcus with a gene for Choline Oxidase enhances tolerance to salt stress
Plant Molecular Biology, 1995Co-Authors: Patcharaporn Deshnium, Hidenori Hayashi, Dmitry A. Los, Laszlo Mustardy, Norio MurataAbstract:Choline Oxidase, isolated from the soil bacterium Arthrobacter globiformis , converts Choline to glycinebetaine (N-trimethylglycine) without a requirement for any cofactors. The gene for this enzyme, designated codA , was cloned and introduced into the cyanobacterium Synechococcus sp. PCC 7942. The codA gene was experssed under the control of a strong constitutive promoter, and the transformed cells accumulated glycinebetaine at intracellular levels of 60–80 mM. Consequently the cells acquired tolerance to salt stress, as evaluated in terms of growth, accumulation of chlorophyll and photosynthetic activity.
Kunchala Rungsrisuriyachai - One of the best experts on this subject based on the ideXlab platform.
-
Identification of the catalytic base for alcohol activation in Choline Oxidase.
Biochemistry, 2014Co-Authors: Crystal Smitherman, Kunchala Rungsrisuriyachai, Markus W. Germann, Giovanni GaddaAbstract:Choline Oxidase catalyzes the oxidation of Choline to glycine betaine through a two-step, four-electron reaction with betaine aldehyde as an intermediate. Oxygen is the final electron acceptor. Alcohol oxidation is initiated by the removal of the substrate hydroxyl proton by an unknown active site residue with a pKa value of ∼7.5. In the crystal structure of the enzyme in complex with glycine betaine, H466 is ≤3.1 A from the carboxylate oxygen of the reaction product, suggesting a possible role in the proton abstraction reaction catalyzed by the enzyme. H466, along with another potential candidate, H351, was previously mutated to alanine, but this failed to establish if either residue was involved in activation of the substrate. In this study, single variants of Choline Oxidase with H466 and H351 substituted with glutamine were prepared, purified, and characterized. The kcat and kcat/Km values of the H351Q enzyme in atmospheric oxygen were 45- and 5000-fold lower than those of the wild-type enzyme, respec...
-
On the Catalytic Roles of HIS351, ASN510, and HIS466 in Choline Oxidase and the Kinetic Mechanism of Pyranose 2-Oxidase
2010Co-Authors: Kunchala RungsrisuriyachaiAbstract:Choline Oxidase (E.C. 1.1.3.17) from Arthrobacter globiformis catalyzes the fourelectron oxidation of Choline to glycine betaine (N,N,N-trimethylglycine) via two sequential, FAD-dependent reactions in which betaine aldehyde is formed as an enzyme-bound intermediate. In each oxidative half-reaction, molecular oxygen acts as electron acceptor and is converted into hydrogen peroxide. Biochemical, structural, and mechanistic studies on the wildtype and a number of mutant variants of Choline Oxidase have recently been carried out, allowing for the depiction of the mechanism of alcohol oxidation catalyzed by the enzyme. Catalysis by Choline Oxidase is initiated by the removal of the hydroxyl proton of alcohol substrate by a catalytic base in the enzyme-substrate complex, yielding the formation of the alkoxide species. In this dissertation, the roles of His351 and conserved His466 were investigated. The results presented demonstrate that His351 is involved in the stabilization of the transition state for the hydride transfer reaction and contributes to substrate binding. His466 is likely to be a catalytic base in Choline Oxidase due to its dramatic effect on enzymatic activity. Comparison of Choline Oxidase and other enzymes within its superfamily reveals the presence of a conserved His-Asn pair within the active site of enzymes. Therefore, the role of the conserved Asn510 in Choline Oxidase was examined in this study. The results presented here establish the importance of Asn510 in both the reductive and oxidative half-reactions. The lost of ability to form a hydrogen bond interaction between the side chain at position 510 with neighboring residues such as His466 resulted in a change from stepwise to concerted mechanism for the cleavages of OH and CH bonds of Choline, as seen in the Asn510Ala mutant. Finally, the steadystate kinetic mechanism of pyranose 2-Oxidase in the pH range from 5.5 to 8.5 was investigated. It was found that pH exerts significant effects on enzyme mechanism. This study has established the involvement of the residues in the initiation of enzyme catalysis and the stabilization of the alkoxide intermediate in Choline Oxidase. In addition, this work demonstrates the first instance in which the kinetic mechanism of a flavin-dependent Oxidase is governed by pH. INDEX WORDS: Choline Oxidase, Pyranose 2-Oxidase, Flavin, Catalytic base, Hydride ion transfer, Chemical mechanism, Flavoprotein. ON THE CATALYTIC ROLES OF HIS351, ASN510, AND HIS466 IN Choline Oxidase AND THE KINETIC MECHANISM OF PYRANOSE 2-Oxidase
-
Role of Asparagine 510 in the Relative Timing of Substrate Bond Cleavages in the Reaction Catalyzed by Choline Oxidase
Biochemistry, 2010Co-Authors: Kunchala Rungsrisuriyachai, Giovanni GaddaAbstract:The flavoprotein Choline Oxidase catalyzes the oxidation of Choline to glycine betaine with transient formation of an aldehyde intermediate and molecular oxygen as final electron acceptor. The enzyme has been grouped in the glucose−methanol−Choline oxidoreductase enzyme superfamily, which shares a highly conserved His-Asn catalytic pair in the active site. In this study, the conserved asparagine residue at position 510 in Choline Oxidase was replaced with alanine, aspartate, histidine, or leucine by site-directed mutagenesis, and the resulting mutant enzymes were purified and characterized in their biochemical and mechanistic properties. All of the substitutions resulted in low incorporation of FAD into the protein. The Asn510Asp enzyme was not catalytically active with Choline and had 75% of the flavin associated noncovalently. The most notable changes in the catalytic parameters with respect to wild-type Choline Oxidase were seen in the Asn510Ala enzyme, with decreases of 4300-fold in the kcat/KCholine,...
-
On the role of histidine 351 in the reaction of alcohol oxidation catalyzed by Choline Oxidase.
Biochemistry, 2008Co-Authors: Kunchala Rungsrisuriyachai, Giovanni GaddaAbstract:Choline Oxidase catalyzes the four-electron, flavin-linked oxidation of Choline to glycine betaine with transient formation of an enzyme-bound aldehyde intermediate. The recent determination of the crystal structure of Choline Oxidase to a resolution of 1.86 A established the presence of two histidine residues in the active site, which may participate in catalysis. His466 was the subject of a previous study [Ghanem, M., and Gadda, G. (2005) Biochemistry 44, 893−904]. In this study, His351 was replaced with alanine using site-directed mutagenesis, and the resulting mutant enzyme was purified and characterized in its mechanistic properties. The results presented establish that His351 contributes to substrate binding and positioning and stabilizes the transition state for the hydride transfer reaction to the flavin, as suggested by anaerobic substrate reduction stopped-flow data. Furthermore, His351 contributes to the overall polarity of the active site by modulating the pKa of the group that deprotonates ch...
Steffan Finnegan - One of the best experts on this subject based on the ideXlab platform.
-
Structural and kinetic studies on the Ser101Ala variant of Choline Oxidase: catalysis by compromise.
Archives of biochemistry and biophysics, 2010Co-Authors: Steffan Finnegan, Allen M. Orville, Yuan-fang Wang, Irene T Weber, Hongling Yuan, Giovanni GaddaAbstract:The oxidation of Choline catalyzed by Choline Oxidase includes two reductive half-reactions where FAD is reduced by the alcohol substrate and by an aldehyde intermediate transiently formed in the reaction. Each reductive half-reaction is followed by an oxidative half-reaction where the reduced flavin is oxidized by oxygen. Here, we have used mutagenesis to prepare the Ser101Ala mutant of Choline Oxidase and have investigated the impact of this mutation on the structural and kinetic properties of the enzyme. The crystallographic structure of the Ser101Ala enzyme indicates that the only differences between the mutant and wild-type enzymes are the lack of a hydroxyl group on residue 101 and a more planar configuration of the flavin in the mutant enzyme. Kinetics established that replacement of Ser101 with alanine yields a mutant enzyme with increased efficiencies in the oxidative half-reactions and decreased efficiencies in the reductive half-reactions. This is accompanied by a significant decrease in the overall rate of turnover with Choline. Thus, this mutation has revealed the importance of a specific residue for the optimization of the overall turnover of Choline Oxidase, which requires fine-tuning of four consecutive half-reactions for the conversion of an alcohol to a carboxylic acid.
-
role of valine 464 in the flavin oxidation reaction catalyzed by Choline Oxidase
Biochemistry, 2010Co-Authors: Steffan Finnegan, Irene T Weber, Johnson Agniswamy, Giovanni GaddaAbstract:The oxidation of reduced flavin cofactors by oxygen is a very important reaction that is central to the chemical versatility of hundreds of flavoproteins classified as monooxygenases and Oxidases. These enzymes are characterized by bimolecular rate constants ≥105 M−1 s−1 and produce water and hydrogen peroxide, respectively. A hydrophobic cavity close to the reactive flavin C(4a) atom has been previously identified in the 3D structure of monooxygenases but not in flavoprotein Oxidases. In the present study, we have investigated by X-ray crystallography, mutagenesis, steady-state, and rapid reaction approaches the role of Val464, which is <6 A from the flavin C(4a) atom in Choline Oxidase. The 3D structure of the Val464Ala enzyme was essentially identical to that of the wild-type enzyme as shown by X-ray crystallography. Time-resolved anaerobic substrate reduction of the enzymes showed that replacement of Val464 with alanine or threonine did not affect the reductive half-reaction. Steady-state and rapid ki...
-
Crystallographic, Spectroscopic, and Computational Analysis of a Flavin C4a−Oxygen Adduct in Choline Oxidase
Biochemistry, 2009Co-Authors: Allen M. Orville, Giovanni Gadda, George T. Lountos, Steffan Finnegan, Rajeev PrabhakarAbstract:Flavin C4a-OO(H) and C4a-OH adducts are critical intermediates proposed in many flavoenzyme reaction mechanisms, but they are rarely detected even by rapid transient kinetics methods. We observe a trapped flavin C4a-OH or C4a-OO(H) adduct by single-crystal spectroscopic methods and in the 1.86 A resolution X-ray crystal structure of Choline Oxidase. The microspectrophotometry results show that the adduct forms rapidly in situ at 100 K upon exposure to X-rays. Density functional theory calculations establish the electronic structures for the flavin C4a-OH and C4a-OO(H) adducts and estimate the stabilization energy of several active site hydrogen bonds deduced from the crystal structure. We propose that the enzyme-bound FAD is reduced in the X-ray beam. The aerobic crystals then form either a C4a-OH or C4a-OO(H) adduct, but an insufficient proton inventory prevents their decay at cryogenic temperatures.
-
crystallographic spectroscopic and computational analysis of a flavin c4a oxygen adduct in Choline Oxidase
Biochemistry, 2009Co-Authors: Allen M. Orville, Giovanni Gadda, George T. Lountos, Steffan Finnegan, Rajeev PrabhakarAbstract:Flavin C4a-OO(H) and C4a-OH adducts are critical intermediates proposed in many flavoenzyme reaction mechanisms, but they are rarely detected even by rapid transient kinetics methods. We observe a trapped flavin C4a-OH or C4a-OO(H) adduct by single-crystal spectroscopic methods and in the 1.86 A resolution X-ray crystal structure of Choline Oxidase. The microspectrophotometry results show that the adduct forms rapidly in situ at 100 K upon exposure to X-rays. Density functional theory calculations establish the electronic structures for the flavin C4a-OH and C4a-OO(H) adducts and estimate the stabilization energy of several active site hydrogen bonds deduced from the crystal structure. We propose that the enzyme-bound FAD is reduced in the X-ray beam. The aerobic crystals then form either a C4a-OH or C4a-OO(H) adduct, but an insufficient proton inventory prevents their decay at cryogenic temperatures.
-
Substitution of an active site valine uncovers a kinetically slow equilibrium between competent and incompetent forms of Choline Oxidase.
Biochemistry, 2008Co-Authors: Steffan Finnegan, Giovanni GaddaAbstract:The enzymatic oxidation of Choline to glycine betaine is of interest because organisms accumulate glycine betaine intracellularly in response to stress conditions. This is relevant for the genetic engineering of crops with economic interest that do not naturally possess efficient pathways for the synthesis of glycine betaine and for the potential development of drugs that target the glycine betaine biosynthetic pathway in human pathogens. To date, the best characterized Choline-oxidizing enzyme is the flavin-dependent Choline Oxidase from Arthrobacter globiformis, for which structural, mechanistic, and biochemical data are available. Here, we have replaced a hydrophobic residue (Val464) lining the active site cavity close to the N(5) atom of the flavin with threonine or alanine to investigate its role in the reaction of Choline oxidation catalyzed by Choline Oxidase. The reductive half-reactions of the enzyme variants containing Thr464 or Ala464 were investigated using substrate and solvent kinetic isotop...
Osbourne Quaye - One of the best experts on this subject based on the ideXlab platform.
-
Rescuing of the hydride transfer reaction in the Glu312Asp variant of Choline Oxidase by a substrate analogue.
Archives of biochemistry and biophysics, 2010Co-Authors: Osbourne Quaye, Tranbao Nguyen, Swathi Gannavaram, Andrea Pennati, Giovanni GaddaAbstract:In the active site of Choline Oxidase, Glu312 participates in binding the trimethylammonium group of Choline, thereby positioning the alcohol substrate properly for efficient hydride transfer to the enzyme-bound flavin. Previous studies have shown that substitution of Glu312 with aspartate results in a perturbed mechanism of hydride transfer, with a 260-fold decrease in the rate associated with the mutation. Here, the reaction of alcohol oxidation catalyzed by the Glu312Asp enzyme has been investigated with 3-hydroxypropyl-trimethylamine (3-HPTA), a Choline analogue with an extra methylene, as substrate. The results of the kinetic investigation using steady state and rapid reaction approaches showed that the impaired ability of the Glu312Asp enzyme to catalyze a hydride transfer reaction can be effectively, but not completely, rescued in the presence of an extra methylene group on the substrate that compensates for the equivalent shortening of the side chain on residue 312. This observation is consistent with Choline Oxidase having evolved to optimally catalyze the oxidation of Choline.
-
contribution of flavin covalent linkage with histidine 99 to the reaction catalyzed by Choline Oxidase
Journal of Biological Chemistry, 2009Co-Authors: Osbourne Quaye, Sharonda Cowins, Giovanni GaddaAbstract:The FAD-dependent Choline Oxidase has a flavin cofactor covalently attached to the protein via histidine 99 through an 8α-N(3)-histidyl linkage. The enzyme catalyzes the four-electron oxidation of Choline to glycine betaine, forming betaine aldehyde as an enzyme-bound intermediate. The variant form of Choline Oxidase in which the histidine residue has been replaced with asparagine was used to investigate the contribution of the 8α-N(3)-histidyl linkage of FAD to the protein toward the reaction catalyzed by the enzyme. Decreases of 10-fold and 30-fold in the kcat/Km and kcat values were observed as compared with wild-type Choline Oxidase at pH 10 and 25 °C, with no significant effect on kcat/KO using Choline as substrate. Both the kcat/Km and kcat values increased with increasing pH to limiting values at high pH consistent with the participation of an unprotonated group in the reductive half-reaction and the overall turnover of the enzyme. The pH independence of both D(kcat/Km) and Dkcat, with average values of 9.2 ± 3.3 and 7.4 ± 0.5, respectively, is consistent with absence of external forward and reverse commitments to catalysis, and the chemical step of CH bond cleavage being rate-limiting for both the reductive half-reaction and the overall enzyme turnover. The temperature dependence of the Dkred values suggests disruption of the preorganization in the asparagine variant enzyme. Altogether, the data presented in this study are consistent with the FAD-histidyl covalent linkage being important for the optimal positioning of the hydride ion donor and acceptor in the tunneling reaction catalyzed by Choline Oxidase.
-
On the Preorganization of the Active Site of Choline Oxidase for Hydride Transfer and Tunneling Mechanism
2009Co-Authors: Osbourne QuayeAbstract:Choline Oxidase catalyzes the two-step oxidation of Choline to glycine betaine, one of limited osmoprotectants, with the formation of betaine aldehyde as an enzyme bound intermediate. Glycine betaine accumulates in the cytoplasm of plants and bacteria as a defensive mechanism to withstand hyperosmolarity and elevated temperatures. This makes the genetic engineering of relevant plants which lack the property of salt accumulation of economic interest, and the biosynthetic pathway of the osmolyte a potential drug target in microbial infections. The reaction of alcohol oxidation occurs via a hydride ion tunneling transfer from the substrate donor to a flavin acceptor within a highly preorganized active site environment in which Choline and FAD are in a rigidly close proximity. In this dissertation, factors contributing to the enzyme-substrate preorganization which is required for the hydride ion tunneling reaction mechanism in Choline Oxidase have been investigated. Crystallographic studies of wild-type Choline Oxidase revealed a covalent linkage between C8M atom of the FAD isoalloxazine ring and the N(3) atom of the side chain of a histidine at position 99, and a solvent excluded cavity in the substrate binding domain containing glutamic acid at position 312 as the only negatively charged amino acid residue in the active site of the enzyme. The role of the histidine residue and the contribution of the 8α-N(3)-histidyl covalent linkage of the flavin cofactor to the reaction of alcohol oxidation was investigated in a variant form of Choline Oxidase in which the histidine residue was replaced with an asparagine. The role of the glutamate residue and the importance of the spatial location of the negative charge at position 312 was investigated in variant forms of Choline Oxidase in which the negatively charged residue was replaced with glutamine and aspartate. Mechanistic data obtained for the variant enzymes and their comparison to previous data obtained for wild-type Choline Oxidase are consistent with the residues at positions 99 and 312 being important for relative positioning of the hydride ion donor and acceptor. The residues are important for the enzyme-substrate preorganization that is required for the hydride tunneling reaction in Choline Oxidase. INDEX WORDS: Flavoproteins, Flavinylation, Quantum Mechanical Tunneling, Hydride Ion Transfer, Preorganization, Choline Oxidase, Alcohol Oxidizing Enzymes ON THE PREORGANIZATION OF THE ACTIVE SITE OF Choline Oxidase FOR HYDRIDE TRANSFER AND TUNNELING MECHANISM
-
Role of Glu312 in Binding and Positioning of the Substrate for the Hydride Transfer Reaction in Choline Oxidase
Biochemistry, 2007Co-Authors: Osbourne Quaye, George T. Lountos, Allen M. Orville, Fan Fan, Giovanni GaddaAbstract:Choline Oxidase catalyzes the oxidation of Choline to glycine betaine, a compatible solute that accumulates in pathogenic bacteria and plants so they can withstand osmotic and temperature stresses. The crystal structure of Choline Oxidase was determined and refined to a resolution of 1.86 A with data collected at 100 K using synchrotron X-ray radiation. The structure reveals a covalent linkage between His99 Ne2 and FAD C8M atoms, and a 123 A3 solvent-excluded cavity adjacent to the re face of the flavin. A hypothetical model for Choline docked into the cavity suggests that several aromatic residues and Glu312 may orient the cationic substrate for efficient catalysis. The role of the negative charge on Glu312 was investigated by engineering variant enzymes in which Glu312 was replaced with alanine, glutamine, or aspartate. The Glu312Ala enzyme was inactive. The Glu312Gln enzyme exhibited a Kd value for Choline at least 500 times larger than that of the wild-type enzyme. The Glu312Asp enzyme had a kcat/KO2 ...