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Bernd Nidetzky - One of the best experts on this subject based on the ideXlab platform.
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Catalytic mechanism of human UDP-glucose 6-dehydrogenase: in situ proton NMR studies reveal that the C-5 hydrogen of UDP-glucose is not exchanged with bulk water during the enzymatic reaction.
Carbohydrate Research, 2012Co-Authors: Thomas Eixelsberger, Lothar Brecker, Bernd NidetzkyAbstract:Human UDP-glucose 6-dehydrogenase (hUGDH) catalyzes the biosynthetic oxidation of UDP-glucose into UDP-glucuronic acid. The catalytic reaction proceeds in two NAD+-dependent steps via covalent Thiohemiacetal and thioester enzyme intermediates. Formation of the Thiohemiacetal adduct occurs through attack of Cys276 on C-6 of the UDP-gluco-hexodialdose produced in the first oxidation step. Because previous studies of the related enzyme from bovine liver had suggested loss of the C-5 hydrogen from UDP-gluco-hexodialdose due to keto-enol tautomerism, we examined incorporation of solvent deuterium into product(s) of UDP-glucose oxidation by hUGDH. We used wild-type enzyme and a slow-reacting Glu161→Gln mutant that accumulates the thioester adduct at steady state. In situ proton NMR measurements showed that UDP-glucuronic acid was the sole detectable product of both enzymatic transformations. The product contained no deuterium at C-5 within the detection limit (⩽2%). The results are consistent with the proposed mechanistic idea for hUGDH that incipient UDP-gluco-hexodialdose is immediately trapped by Thiohemiacetal adduct formation.
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Structural and Kinetic Evidence That Catalytic Reaction of Human UDP-glucose 6-Dehydrogenase Involves Covalent Thiohemiacetal and Thioester Enzyme Intermediates
Journal of Biological Chemistry, 2011Co-Authors: Sigrid Egger, Udo Oppermann, Apirat Chaikuad, Mario Klimacek, Kathryn L. Kavanagh, Bernd NidetzkyAbstract:Biosynthesis of UDP-glucuronic acid by UDP-glucose 6-dehydrogenase (UGDH) occurs through the four-electron oxidation of the UDP-glucose C6 primary alcohol in two NAD(+)-dependent steps. The catalytic reaction of UGDH is thought to involve a Cys nucleophile that promotes formation of a Thiohemiacetal enzyme intermediate in the course of the first oxidation step. The Thiohemiacetal undergoes further oxidation into a thioester, and hydrolysis of the thioester completes the catalytic cycle. Herein we present crystallographic and kinetic evidence for the human form of UGDH that clarifies participation of covalent catalysis in the enzymatic mechanism. Substitution of the putative catalytic base for water attack on the thioester (Glu(161)) by an incompetent analog (Gln(161)) gave a UGDH variant (E161Q) in which the hydrolysis step had become completely rate-limiting so that a thioester enzyme intermediate accumulated at steady state. By crystallizing E161Q in the presence of 5 mm UDP-glucose and 2 mm NAD(+), we succeeded in trapping a Thiohemiacetal enzyme intermediate and determined its structure at 2.3 A resolution. Cys(276) was covalently modified in the structure, establishing its role as catalytic nucleophile of the reaction. The Thiohemiacetal reactive C6 was in a position suitable to become further oxidized by hydride transfer to NAD(+). The proposed catalytic mechanism of human UGDH involves Lys(220) as general base for UDP-glucose alcohol oxidation and for oxyanion stabilization during formation and breakdown of the Thiohemiacetal and thioester enzyme intermediates. Water coordinated to Asp(280) deprotonates Cys(276) to function as an aldehyde trap and also provides oxyanion stabilization. Glu(161) is the Bronsted base catalytically promoting the thioester hydrolysis.
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Structure and mechanism of human UDP-glucose 6-dehydrogenase
Journal of Biological Chemistry, 2011Co-Authors: Sigrid Egger, K L Kavanagh, Udo Oppermann, Apirat Chaikuad, Bernd NidetzkyAbstract:Elevated production of the matrix glycosaminoglycan hyaluronan is strongly implicated in epithelial tumor progression. Inhibition of synthesis of the hyaluronan precursor UDP-glucuronic acid (UDP-GlcUA) therefore presents an emerging target for cancer therapy. Human UDP-glucose 6-dehydrogenase (hUGDH) catalyzes, in two NAD(+)-dependent steps without release of intermediate aldehyde, the biosynthetic oxidation of UDP-glucose (UDP-Glc) to UDP-GlcUA. Here, we present a structural characterization of the hUGDH reaction coordinate using crystal structures of the apoenzyme and ternary complexes of the enzyme bound with UDP-Glc/NADH and UDP-GlcUA/NAD(+). The quaternary structure of hUGDH is a disc-shaped trimer of homodimers whose subunits consist of two discrete α/β domains with the active site located in the interdomain cleft. Ternary complex formation is accompanied by rigid-body and restrained movement of the N-terminal NAD(+) binding domain, sequestering substrate and coenzyme in their reactive positions through interdomain closure. By alternating between conformations in and out of the active site during domain motion, Tyr(14), Glu(161), and Glu(165) participate in control of coenzyme binding and release during 2-fold oxidation. The proposed mechanism of hUGDH involves formation and breakdown of Thiohemiacetal and thioester intermediates whereby Cys(276) functions as the catalytic nucleophile. Stopped-flow kinetic data capture the essential deprotonation of Cys(276) in the course of the first oxidation step, allowing the thiolate side chain to act as a trap of the incipient aldehyde. Because Thiohemiacetal intermediate accumulates at steady state under physiological reaction conditions, hUGDH inhibition might best explore ligand binding to the NAD(+) binding domain.
Sigrid Egger - One of the best experts on this subject based on the ideXlab platform.
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Structural and Kinetic Evidence That Catalytic Reaction of Human UDP-glucose 6-Dehydrogenase Involves Covalent Thiohemiacetal and Thioester Enzyme Intermediates
Journal of Biological Chemistry, 2011Co-Authors: Sigrid Egger, Udo Oppermann, Apirat Chaikuad, Mario Klimacek, Kathryn L. Kavanagh, Bernd NidetzkyAbstract:Biosynthesis of UDP-glucuronic acid by UDP-glucose 6-dehydrogenase (UGDH) occurs through the four-electron oxidation of the UDP-glucose C6 primary alcohol in two NAD(+)-dependent steps. The catalytic reaction of UGDH is thought to involve a Cys nucleophile that promotes formation of a Thiohemiacetal enzyme intermediate in the course of the first oxidation step. The Thiohemiacetal undergoes further oxidation into a thioester, and hydrolysis of the thioester completes the catalytic cycle. Herein we present crystallographic and kinetic evidence for the human form of UGDH that clarifies participation of covalent catalysis in the enzymatic mechanism. Substitution of the putative catalytic base for water attack on the thioester (Glu(161)) by an incompetent analog (Gln(161)) gave a UGDH variant (E161Q) in which the hydrolysis step had become completely rate-limiting so that a thioester enzyme intermediate accumulated at steady state. By crystallizing E161Q in the presence of 5 mm UDP-glucose and 2 mm NAD(+), we succeeded in trapping a Thiohemiacetal enzyme intermediate and determined its structure at 2.3 A resolution. Cys(276) was covalently modified in the structure, establishing its role as catalytic nucleophile of the reaction. The Thiohemiacetal reactive C6 was in a position suitable to become further oxidized by hydride transfer to NAD(+). The proposed catalytic mechanism of human UGDH involves Lys(220) as general base for UDP-glucose alcohol oxidation and for oxyanion stabilization during formation and breakdown of the Thiohemiacetal and thioester enzyme intermediates. Water coordinated to Asp(280) deprotonates Cys(276) to function as an aldehyde trap and also provides oxyanion stabilization. Glu(161) is the Bronsted base catalytically promoting the thioester hydrolysis.
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Structure and mechanism of human UDP-glucose 6-dehydrogenase
Journal of Biological Chemistry, 2011Co-Authors: Sigrid Egger, K L Kavanagh, Udo Oppermann, Apirat Chaikuad, Bernd NidetzkyAbstract:Elevated production of the matrix glycosaminoglycan hyaluronan is strongly implicated in epithelial tumor progression. Inhibition of synthesis of the hyaluronan precursor UDP-glucuronic acid (UDP-GlcUA) therefore presents an emerging target for cancer therapy. Human UDP-glucose 6-dehydrogenase (hUGDH) catalyzes, in two NAD(+)-dependent steps without release of intermediate aldehyde, the biosynthetic oxidation of UDP-glucose (UDP-Glc) to UDP-GlcUA. Here, we present a structural characterization of the hUGDH reaction coordinate using crystal structures of the apoenzyme and ternary complexes of the enzyme bound with UDP-Glc/NADH and UDP-GlcUA/NAD(+). The quaternary structure of hUGDH is a disc-shaped trimer of homodimers whose subunits consist of two discrete α/β domains with the active site located in the interdomain cleft. Ternary complex formation is accompanied by rigid-body and restrained movement of the N-terminal NAD(+) binding domain, sequestering substrate and coenzyme in their reactive positions through interdomain closure. By alternating between conformations in and out of the active site during domain motion, Tyr(14), Glu(161), and Glu(165) participate in control of coenzyme binding and release during 2-fold oxidation. The proposed mechanism of hUGDH involves formation and breakdown of Thiohemiacetal and thioester intermediates whereby Cys(276) functions as the catalytic nucleophile. Stopped-flow kinetic data capture the essential deprotonation of Cys(276) in the course of the first oxidation step, allowing the thiolate side chain to act as a trap of the incipient aldehyde. Because Thiohemiacetal intermediate accumulates at steady state under physiological reaction conditions, hUGDH inhibition might best explore ligand binding to the NAD(+) binding domain.
Aimin Liu - One of the best experts on this subject based on the ideXlab platform.
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Crystallographic and spectroscopic snapshots reveal a dehydrogenase in action
Nature Communications, 2015Co-Authors: Lu Huo, Ian Davis, Fange Liu, Babak Andi, Shingo Esaki, Hiroaki Iwaki, Yoshie Hasegawa, Allen M. Orville, Aimin LiuAbstract:Aldehydes are common intermediates in enzymatic pathways, but their high reactivity can make them difficult to observe. Here, the authors study the mechanism of aldehyde deactivation in a dehydrogenase, showing a key E/Z isomerization and observing a Thiohemiacetal intermediate by crystal structure analysis. Aldehydes are ubiquitous intermediates in metabolic pathways and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation that can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for ‘disarming’ the final aldehydic intermediate. Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, Thiohemiacetal intermediate, a thioacyl intermediate and an NAD^+-bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E / Z isomerization at the enzyme active site before an sp ^3-to- sp ^2 transition during enzyme-mediated oxidation.
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Crystallographic and spectroscopic snapshots reveal a dehydrogenase in action
Nature Communications, 2015Co-Authors: Lu Huo, Ian Davis, Fange Liu, Babak Andi, Shingo Esaki, Hiroaki Iwaki, Yoshie Hasegawa, Allen M. Orville, Aimin LiuAbstract:Aldehydes are ubiquitous intermediates in metabolic pathways and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation that can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for ‘disarming’ the final aldehydic intermediate. Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, Thiohemiacetal intermediate, a thioacyl intermediate and an NAD þ -bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site before an sp 3 -to-sp 2
Ian Davis - One of the best experts on this subject based on the ideXlab platform.
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a pitcher and catcher mechanism drives endogenous substrate isomerization by a dehydrogenase in kynurenine metabolism
Journal of Biological Chemistry, 2016Co-Authors: Yu Yang, Ian Davis, Uyen Ha, Yifan Wang, Inchul ShinAbstract:Abstract Aldehyde dehydrogenase typically performs oxidation of aldehydes to their corresponding carboxylic acid while reducing NAD(P)+ to NAD(P)H via covalent catalysis mediated by an active-site cysteine residue. One member of this superfamily, the enzyme 2-aminomuconate-6-semialdehyde dehydrogenase (AMSDH), is a component of the kynurenine pathway, which catabolizes tryptophan in mammals and certain bacteria. AMSDH catalyzes the NAD+-dependent oxidation of 2-aminomuconate semialdehyde to 2-aminomuconate. We recently determined the first crystal structure of AMSDH and several catalytic cycle intermediates. A conserved asparagine in the oxyanion hole, Asn-169, is found to be H-bonded to substrate-derived intermediates in the active site of AMSDH during catalysis, including both the covalently bound Thiohemiacetal and thioacyl intermediates. To better interrogate the significance of the hydrogen bond provided by Asn-169 to the reaction mechanism of AMSDH, we created Ala, Ser, Asp, and Gln mutants and studied them using biochemical, kinetic, crystallographic, and computational studies. The in crystallo chemical reaction of the primary substrate with the co-crystalized complex of the N169D mutant and NAD+ led to the successful trapping of a new catalytic intermediate that was not previously seen. The structural and computational data are consistent with a substrate imine/enol tautomer intermediate being formed prior to the formation of the covalent bond between the substrate and the active-site cysteine. Thus, AMSDH surprisingly includes an isomerization process within its known catalytic mechanism. These data establish a hidden intrinsic isomerization activity of the dehydrogenase and allow us to propose a pitcher-catcher type of catalytic mechanism for the isomerization.
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Crystallographic and spectroscopic snapshots reveal a dehydrogenase in action
Nature Communications, 2015Co-Authors: Lu Huo, Ian Davis, Fange Liu, Babak Andi, Shingo Esaki, Hiroaki Iwaki, Yoshie Hasegawa, Allen M. Orville, Aimin LiuAbstract:Aldehydes are common intermediates in enzymatic pathways, but their high reactivity can make them difficult to observe. Here, the authors study the mechanism of aldehyde deactivation in a dehydrogenase, showing a key E/Z isomerization and observing a Thiohemiacetal intermediate by crystal structure analysis. Aldehydes are ubiquitous intermediates in metabolic pathways and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation that can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for ‘disarming’ the final aldehydic intermediate. Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, Thiohemiacetal intermediate, a thioacyl intermediate and an NAD^+-bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E / Z isomerization at the enzyme active site before an sp ^3-to- sp ^2 transition during enzyme-mediated oxidation.
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Crystallographic and spectroscopic snapshots reveal a dehydrogenase in action
Nature Communications, 2015Co-Authors: Lu Huo, Ian Davis, Fange Liu, Babak Andi, Shingo Esaki, Hiroaki Iwaki, Yoshie Hasegawa, Allen M. Orville, Aimin LiuAbstract:Aldehydes are ubiquitous intermediates in metabolic pathways and their innate reactivity can often make them quite unstable. There are several aldehydic intermediates in the metabolic pathway for tryptophan degradation that can decay into neuroactive compounds that have been associated with numerous neurological diseases. An enzyme of this pathway, 2-aminomuconate-6-semialdehyde dehydrogenase, is responsible for ‘disarming’ the final aldehydic intermediate. Here we show the crystal structures of a bacterial analogue enzyme in five catalytically relevant forms: resting state, one binary and two ternary complexes, and a covalent, thioacyl intermediate. We also report the crystal structures of a tetrahedral, Thiohemiacetal intermediate, a thioacyl intermediate and an NAD þ -bound complex from an active site mutant. These covalent intermediates are characterized by single-crystal and solution-state electronic absorption spectroscopy. The crystal structures reveal that the substrate undergoes an E/Z isomerization at the enzyme active site before an sp 3 -to-sp 2
Udo Oppermann - One of the best experts on this subject based on the ideXlab platform.
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Structural and Kinetic Evidence That Catalytic Reaction of Human UDP-glucose 6-Dehydrogenase Involves Covalent Thiohemiacetal and Thioester Enzyme Intermediates
Journal of Biological Chemistry, 2011Co-Authors: Sigrid Egger, Udo Oppermann, Apirat Chaikuad, Mario Klimacek, Kathryn L. Kavanagh, Bernd NidetzkyAbstract:Biosynthesis of UDP-glucuronic acid by UDP-glucose 6-dehydrogenase (UGDH) occurs through the four-electron oxidation of the UDP-glucose C6 primary alcohol in two NAD(+)-dependent steps. The catalytic reaction of UGDH is thought to involve a Cys nucleophile that promotes formation of a Thiohemiacetal enzyme intermediate in the course of the first oxidation step. The Thiohemiacetal undergoes further oxidation into a thioester, and hydrolysis of the thioester completes the catalytic cycle. Herein we present crystallographic and kinetic evidence for the human form of UGDH that clarifies participation of covalent catalysis in the enzymatic mechanism. Substitution of the putative catalytic base for water attack on the thioester (Glu(161)) by an incompetent analog (Gln(161)) gave a UGDH variant (E161Q) in which the hydrolysis step had become completely rate-limiting so that a thioester enzyme intermediate accumulated at steady state. By crystallizing E161Q in the presence of 5 mm UDP-glucose and 2 mm NAD(+), we succeeded in trapping a Thiohemiacetal enzyme intermediate and determined its structure at 2.3 A resolution. Cys(276) was covalently modified in the structure, establishing its role as catalytic nucleophile of the reaction. The Thiohemiacetal reactive C6 was in a position suitable to become further oxidized by hydride transfer to NAD(+). The proposed catalytic mechanism of human UGDH involves Lys(220) as general base for UDP-glucose alcohol oxidation and for oxyanion stabilization during formation and breakdown of the Thiohemiacetal and thioester enzyme intermediates. Water coordinated to Asp(280) deprotonates Cys(276) to function as an aldehyde trap and also provides oxyanion stabilization. Glu(161) is the Bronsted base catalytically promoting the thioester hydrolysis.
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Structure and mechanism of human UDP-glucose 6-dehydrogenase
Journal of Biological Chemistry, 2011Co-Authors: Sigrid Egger, K L Kavanagh, Udo Oppermann, Apirat Chaikuad, Bernd NidetzkyAbstract:Elevated production of the matrix glycosaminoglycan hyaluronan is strongly implicated in epithelial tumor progression. Inhibition of synthesis of the hyaluronan precursor UDP-glucuronic acid (UDP-GlcUA) therefore presents an emerging target for cancer therapy. Human UDP-glucose 6-dehydrogenase (hUGDH) catalyzes, in two NAD(+)-dependent steps without release of intermediate aldehyde, the biosynthetic oxidation of UDP-glucose (UDP-Glc) to UDP-GlcUA. Here, we present a structural characterization of the hUGDH reaction coordinate using crystal structures of the apoenzyme and ternary complexes of the enzyme bound with UDP-Glc/NADH and UDP-GlcUA/NAD(+). The quaternary structure of hUGDH is a disc-shaped trimer of homodimers whose subunits consist of two discrete α/β domains with the active site located in the interdomain cleft. Ternary complex formation is accompanied by rigid-body and restrained movement of the N-terminal NAD(+) binding domain, sequestering substrate and coenzyme in their reactive positions through interdomain closure. By alternating between conformations in and out of the active site during domain motion, Tyr(14), Glu(161), and Glu(165) participate in control of coenzyme binding and release during 2-fold oxidation. The proposed mechanism of hUGDH involves formation and breakdown of Thiohemiacetal and thioester intermediates whereby Cys(276) functions as the catalytic nucleophile. Stopped-flow kinetic data capture the essential deprotonation of Cys(276) in the course of the first oxidation step, allowing the thiolate side chain to act as a trap of the incipient aldehyde. Because Thiohemiacetal intermediate accumulates at steady state under physiological reaction conditions, hUGDH inhibition might best explore ligand binding to the NAD(+) binding domain.