Catalytic mechanism of UDP-glucose dehydrogenase

AbstractUDP-glucose dehydrogenase (UGDH), an oxidoreductase, catalyzes the NAD+-dependent four-electron oxidation of UDP-glucose to UDP-glucuronic acid. The catalytic mechanism of UGDH remains controversial despite extensive investigation and is classified into two types according to whether an aldehyde intermediate is generated in the first oxidation step. The first type, which involves the presence of this putative aldehyde, is inconsistent with some experimental findings. In contrast, the second type, which indicates that the first oxidation step bypasses the aldehyde via an NAD+-dependent bimolecular nucleophilic substitution (SN2) reaction, is consistent with the experimental phenomena, including those that cannot be explained by the first type. This NAD+-dependent SN2 mechanism is thus more reasonable and likely applicable to other oxidoreductases that catalyze four-electron oxidation reactions.

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A DFT study on the catalytic mechanism of UDP-glucose dehydrogenase

Canadian Journal of Chemistry ◽

10.1139/v10-044 ◽

2010 ◽

Vol 88 (8) ◽

pp. 804-814 ◽

Cited By ~ 1

Author(s):

WenJuan Huang ◽

Jorge Llano ◽

James W. Gauld

Keyword(s):

Pathogenic Bacteria ◽

Catalytic Mechanism ◽

Glucuronic Acid ◽

Glucose Dehydrogenase ◽

Polarizable Continuum Model ◽

Uridine Diphosphate ◽

Reaction Field ◽

Enzyme Active Site ◽

Site Model ◽

Diphosphate Glucose

Uridine 5′-diphosphate glucuronic acid (UDPGlcUA) is a key intermediary metabolite in many species, including pathogenic bacteria and humans. It is biosynthesized from UDP-glucose (UDPGlc) by uridine diphosphate glucose dehydrogenase (UDPGlcDH) via a twofold two-electron–one-proton oxidation that successively transforms the 6-hydroxymethyl of glucopyranose into a formyl, and the latter into the final carboxylic function. The catalytic mechanism of UDPGlcDH was investigated using a large enzyme active-site model in combination with the B3LYP method and the polarizable continuum model (IEF-PCM) self-consistent reaction field. The latter was used to correct for the long-range electrostatic effect of the protein environment. The overall mechanism consists of four catalytic steps: (i) NAD+-dependent oxidation of glucose to glucuronaldehyde, (ii) nucleophilic addition of Cys260–SH to glucuronaldehyde to form a 6-thiohemiacetal intermediate, (iii) NAD+-dependent oxidation of the 6-thiohemiacetal to form a 6-thioester intermediate, and finally, (iv) hydrolysis of the 6-thioester to give glucuronic acid. In addition, this study also provides insight into the debated roles of Lys204 and Asp264, and the most likely protonation state of a reactive Michaelis complex of UDPGlcDH.

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The binding of oxidized and reduced nicotinamide–adenine dinucleotides to bovine liver uridine diphosphate glucose dehydrogenase

Biochemical Journal ◽

10.1042/bj1410667 ◽

1974 ◽

Vol 141 (3) ◽

pp. 667-673 ◽

Cited By ~ 14

Author(s):

Paul A. Gainey ◽

Charles F. Phelps

Keyword(s):

Glucuronic Acid ◽

Glucose Dehydrogenase ◽

Gel Filtration ◽

Bovine Liver ◽

Uridine Diphosphate ◽

Protein Fluorescence ◽

Diphosphate Glucose ◽

Ph Dependent ◽

Uridine Diphosphate Glucose Dehydrogenase ◽

Biphasic Profile

The binding of NAD+and NADH to bovine liver UDP-glucose dehydrogenase was studied by using gel-filtration and fluorescence-titration methods. The enzyme bound 0.5mol of NAD+and 2 mol of NADH/mol of subunit at saturating concentrations of both substrate and product. The dissociation constant for NADH was 4.3μm. The binding of NAD+to the enzyme resulted in a small quench of protein fluorescence whereas the binding of NADH resulted in a much larger (60–70%) quench of protein fluorescence. The binding of NADH to the enzyme was pH-dependent. At pH8.1 a biphasic profile was obtained on titrating the enzyme with NADH, whereas at pH8.8 the titration profile was hyperbolic. UDP-xylose, and to a lesser extent UDP-glucuronic acid, lowered the apparent affinity of the enzyme for NADH.

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UDP-glucose dehydrogenase: structure and function of a potential drug target

Biochemical Society Transactions ◽

10.1042/bst0381378 ◽

2010 ◽

Vol 38 (5) ◽

pp. 1378-1385 ◽

Cited By ~ 42

Author(s):

Sigrid Egger ◽

Apirat Chaikuad ◽

Kathryn L. Kavanagh ◽

Udo Oppermann ◽

Bernd Nidetzky

Keyword(s):

Sequence Comparison ◽

Drug Target ◽

Glucuronic Acid ◽

Glucose Dehydrogenase ◽

Enzymatic Reaction ◽

Cysteine Residue ◽

Structure And Function ◽

Eukaryotic Group ◽

And Function ◽

Covalent Catalysis

Biosynthesis of the glycosaminoglycan precursor UDP-α-D-glucuronic acid occurs through a 2-fold oxidation of UDP-α-D-glucose that is catalysed by UGDH (UDP-α-D-glucose 6-dehydrogenase). Structure–function relationships for UGDH and proposals for the enzymatic reaction mechanism are reviewed in the present paper, and structure-based sequence comparison is used for subclassification of UGDH family members. The eukaryotic group of enzymes (UGDH-II) utilize an extended C-terminal domain for the formation of complex homohexameric assemblies. The comparably simpler oligomerization behaviour of the prokaryotic group of enzymes (UGDH-I), in which dimeric forms prevail, is traced back to the lack of relevant intersubunit contacts and trimmings within the C-terminal region. The active site of UGDH contains a highly conserved cysteine residue, which plays a key role in covalent catalysis. Elevated glycosaminoglycan formation is implicated in a variety of human diseases, including the progression of tumours. The inhibition of synthesis of UDP-α-D-glucuronic acid using UGDH antagonists might therefore be a useful strategy for therapy.

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Biosynthesis of glycosaminoglycans in bovine cornea. The effect of uridine diphosphate xylose

Biochemical Journal ◽

10.1042/bj1200719 ◽

1970 ◽

Vol 120 (4) ◽

pp. 719-723 ◽

Cited By ~ 23

Author(s):

C. Balduini ◽

A. Brovelli ◽

A. A. Castellani

Keyword(s):

Glucuronic Acid ◽

Chondroitin Sulphate ◽

Glucose Dehydrogenase ◽

Uridine Diphosphate ◽

Regulatory Effect ◽

Keratan Sulphate ◽

Polysaccharide Chain

1. The role of UDP-xylose in the regulation of corneal glycosaminoglycan biosynthesis was investigated. Bovine corneas were incubated with [U-14C]-glucose in the presence and in the absence of the nucleotide, and the radioactivity of chondroitin, chondroitin sulphate and keratan sulphate, as well as of their monosaccharide constituents, was determined. 2. A decrease in the rate of biosynthesis of chondroitin and chondroitin sulphate and an increase in that of keratan sulphate were observed in the samples incubated with UDP-xylose. 3. The UDP-glucuronic acid isolated after the incubation in the presence of UDP-xylose showed a noticeable decrease in the amount of radioactivity incorporated; this result suggests that UDP-xylose inhibits the UDP-glucose dehydrogenase, causing an accumulation of UDP-glucose and consequently an increase in the formation of UDP-galactose and keratan sulphate. 4. Galactose and galactosamine isolated from the polysaccharides showed variations in the amount of radioactivity incorporated in accordance with those observed for the macromolecules; this fact confirms that in the system we used in vitro a real biosynthesis of the polysaccharide chain took place and that the regulatory effect of UDP-xylose was active at the monosaccharide level.

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Uridine diphosphate glucuronic acid production and utilization in various tissues actively synthesizing glycosaminoglycans

Biochemical Journal ◽

10.1042/bj1280215 ◽

1972 ◽

Vol 128 (2) ◽

pp. 215-227 ◽

Cited By ~ 17

Author(s):

P. A. Gainey ◽

C. F. Phelps

Keyword(s):

Nasal Septum ◽

Glucuronic Acid ◽

Glucose Dehydrogenase ◽

Neonatal Rat ◽

Uridine Diphosphate ◽

Ph Profile ◽

Nasal Cartilage ◽

Space Experiments ◽

Tissue Space ◽

Wet Weight

1. UDP-glucose dehydrogenase has been partially purified from sheep nasal septum cartilage, neonatal rat skin and bovine corneal epithelium. 2. The pH profile, Km values for NAD+ and UDP-glucose, activation energy and molecular weight have been determined for the enzyme from several of the tissues. 3. The sugar nucleotide concentrations in each of the tissues have been related to the spectrum of glycosaminoglycans produced by each tissue. 4. The presence of an allosteric UDP-xylose-binding site distinct from the active site(s) in sheep nasal septum UDP-glucose dehydrogenase has been demonstrated. 5. An active UDP-glucuronic acid nucleotidase has been demonstrated in sheep nasal cartilage. 6. Tissue-space experiments have shown the cell water content of sheep nasal septum cartilage to be 14% of the wet weight. 7. Glucuronic acid 1-phosphate does not occur in measurable amounts in sheep nasal septum cartilage and no UDP-glucuronic acid pyrophosphorylase activity could be detected in this tissue. 8. The inhibition by UDP-xylose with respect to both substrates, UDP-glucose and NAD+, has been examined, and shown to be allosteric.

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UDP-Glucose Dehydrogenases: Identification, Expression, and Function Analyses in Upland Cotton (Gossypium hirsutum)

Frontiers in Genetics ◽

10.3389/fgene.2020.597890 ◽

2021 ◽

Vol 11 ◽

Author(s):

Tingting Jia ◽

Qun Ge ◽

Shuya Zhang ◽

Zhen Zhang ◽

Aiying Liu ◽

...

Keyword(s):

Gene Expression ◽

Upland Cotton ◽

Plant Cell Wall ◽

Glucuronic Acid ◽

Glucose Dehydrogenase ◽

Fiber Development ◽

Protein Domain ◽

Cell Wall Synthesis ◽

Wild Type ◽

And Function

UDP-glucose dehydrogenase (UGD; EC1.1.1.22) is a NAD+-dependent enzyme that catalyzes the two-fold oxidation of UDP-glucose (UDP-Glc) to produce UDP-glucuronic acid and plays an important role in plant cell wall synthesis. A total of 42 UGD genes from four Gossypium genomes including G. hirsutum, G. arboretum, G. barbadense, and G. raimondii were identified and found that the UGD gene family has conservative evolution patterns in gene structure and protein domain. The growth of fibers can be effectively promoted after adding the UDP-Glc to the medium, and the GhUGD gene expression enhanced. In addition, the transgenic Arabidopsis lines over-expressing GH_D12G1806 had longer root lengths and higher gene expression level than the wild-type plants of Columbia-0. These results indicated that UGD may play important roles in cotton fiber development and has a guiding significance for dissecting fiber development mechanism.

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Enzyme Electrochemistry — Biocatalysis on an Electrode

Australian Journal of Chemistry ◽

10.1071/ch05340 ◽

2006 ◽

Vol 59 (4) ◽

pp. 233 ◽

Cited By ~ 54

Author(s):

Paul V. Bernhardt

Keyword(s):

Electron Transfer ◽

Small Molecule ◽

Electrode Surface ◽

Catalytic Mechanism ◽

Organic Substrates ◽

Oxidation Reactions ◽

Accurate Analysis ◽

Practical Applications ◽

Redox Partner ◽

Oxidoreductase Enzymes

Oxidoreductase enzymes catalyze single- or multi-electron reduction/oxidation reactions of small molecule inorganic or organic substrates, and they are integral to a wide variety of biological processes including respiration, energy production, biosynthesis, metabolism, and detoxification. All redox enzymes require a natural redox partner such as an electron-transfer protein (e.g. cytochrome, ferredoxin, flavoprotein) or a small molecule cosubstrate (e.g. NAD(P)H, dioxygen) to sustain catalysis, in effect to balance the substrate/product redox half-reaction. In principle, the natural electron-transfer partner may be replaced by an electrochemical working electrode. One of the great strengths of this approach is that the rate of catalysis (equivalent to the observed electrochemical current) may be probed as a function of applied potential through linear sweep and cyclic voltammetry, and insight to the overall catalytic mechanism may be gained by a systematic electrochemical study coupled with theoretical analysis. In this review, the various approaches to enzyme electrochemistry will be discussed, including direct and indirect (mediated) experiments, and a brief coverage of the theory relevant to these techniques will be presented. The importance of immobilizing enzymes on the electrode surface will be presented and the variety of ways that this may be done will be reviewed. The importance of chemical modification of the electrode surface in ensuring an environment conducive to a stable and active enzyme capable of functioning natively will be illustrated. Fundamental research into electrochemically driven enzyme catalysis has led to some remarkable practical applications. The glucose oxidase enzyme electrode is a spectacularly successful application of enzyme electrochemistry. Biosensors based on this technology are used worldwide by sufferers of diabetes to provide rapid and accurate analysis of blood glucose concentrations. Other applications of enzyme electrochemistry are in the sensing of macromolecular complexation events such as antigen–antibody binding and DNA hybridization. The review will include a selection of enzymes that have been successfully investigated by electrochemistry and, where appropriate, discuss their development towards practical biotechnological applications.

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Contributions of two UDP-glucose dehydrogenases to viability and polymyxin B resistance of Burkholderia cenocepacia

Microbiology ◽

10.1099/mic.0.027607-0 ◽

2009 ◽

Vol 155 (6) ◽

pp. 2029-2039 ◽

Cited By ~ 24

Author(s):

Slade A. Loutet ◽

S. Josefin Bartholdson ◽

John R. W. Govan ◽

Dominic J. Campopiano ◽

Miguel A. Valvano

Keyword(s):

Real Time Pcr ◽

Biochemical Analysis ◽

Glucuronic Acid ◽

Glucose Dehydrogenase ◽

Polymyxin B ◽

Burkholderia Cenocepacia ◽

Pcr Analysis ◽

Increased Sensitivity ◽

In Trans

Burkholderia cenocepacia is highly resistant to antimicrobial peptides and we hypothesized that the conversion of UDP-glucose to UDP-glucuronic acid, a reaction catalysed by the enzyme UDP-glucose dehydrogenase (Ugd) would be important for this resistance. The genome of B. cenocepacia contains three predicted ugd genes: ugdBCAL2946 , ugdBCAM0855 and ugdBCAM2034 , all of which were individually inactivated. Only inactivation of ugdBCAL2946 resulted in increased sensitivity to polymyxin B and this sensitivity could be overcome when either ugdBCAL2946 or ugdBCAM0855 but not ugdBCAM2034 was expressed from plasmids. The growth of a conditional ugdBCAL2946 mutant, created in the ΔugdBCAM0855 background, was significantly impaired under non-permissive conditions. Growth could be rescued by either ugdBCAL2946 or ugdBCAM0855 expressed in trans, but not by ugdBCAM2034 . Biochemical analysis of the purified, recombinant forms of UgdBCAL2946 and UgdBCAM0855 revealed that they are soluble homodimers with similar in vitro Ugd activity and comparable kinetic constants for their substrates UDP-glucose and NAD+. Purified UgdBCAM2034 showed no in vitro Ugd activity. Real-time PCR analysis showed that the expression of ugdBCAL2946 was 5.4- and 135-fold greater than that of ugdBCAM0855 and ugdBCAM2034 , respectively. Together, these data indicate that the combined activity of UgdBCAL2946 and UgdBCAM0855 is essential for the survival of B. cenocepacia but only the most highly expressed ugd gene, ugdBCAL2946 , is required for polymyxin B resistance.

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Theoretical study on the mechanism and enantioselectivity of NHC-catalyzed intramolecular SN2′ nucleophilic substitution: what are the roles of NHC and DBU?

Organic Chemistry Frontiers ◽

10.1039/c8qo00129d ◽

2018 ◽

Vol 5 (9) ◽

pp. 1493-1501 ◽

Cited By ~ 15

Author(s):

Huimin Zhang ◽

Hao Xu ◽

Huining Bai ◽

Donghui Wei ◽

Yanyan Zhu ◽

...

Keyword(s):

Nucleophilic Substitution ◽

Catalytic Mechanism ◽

Theoretical Study ◽

Dft Method

A possible catalytic mechanism was proposed and studied in very detail by using the DFT method for a recently reported enantioselective intramolecular SN2′ substitution of aldehydes with trisubstituted allylic bromides.

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Biosynthesis of heparin/heparan sulphate: mechanism of epimerization of glucuronyl C-5

Biochemical Journal ◽

10.1042/bj3470069 ◽

2000 ◽

Vol 347 (1) ◽

pp. 69-75 ◽

Cited By ~ 15

Author(s):

Åsa HAGNER-MCWHIRTER ◽

Ulf LINDAHL ◽

Jin-ping LI

Keyword(s):

Capsular Polysaccharide ◽

Catalytic Mechanism ◽

Glucuronic Acid ◽

Process Analysis ◽

Heparan Sulphate ◽

Bovine Liver ◽

Base Function ◽

Rate Limiting Step ◽

Hexuronic Acid ◽

Lysine Residues

In the biosynthesis of heparin and heparan sulphate, D-glucuronic acid residues are converted into L-iduronic acid (IdoA) units by C-5 epimerization, at the polymer level. The reaction catalysed by the epimerase occurs by reversible abstraction and readdition of a proton at C-5 of target hexuronic acid residues, through a carbanion intermediate, with or without an inversion of configuration at C-5 [Prihar, Campbell, Feingold, Jacobsson, Jensen, Lindahl and Rodén (1980) Biochemistry 19, 495-500]. Incubation of chemically N-sulphated capsular polysaccharide from Escherichia coli K5 ([4GlcAβ1-4GlcNSO3α1-]n), or of O-desulphated heparin (predominantly [4IdoAα1-4GlcNSO3α1-]n) with purified C-5 epimerase from bovine liver, resulted in the interconversion of glucuronic acid and IdoA residues, which reached equilibrium (30-40% IdoA/total hexuronic acid) after approx. 1 h of incubation. Similar incubations performed in the presence of 3H2O resulted in progressive labelling at C-5 of the target hexuronic acid units of either substrate polysaccharide. Contrary to chemical D-gluco/L-ido equilibrium, established within 1 h of incubation, the accumulation of 3H label continued for at least 6 h. This isotope effect suggests that the second stage of the reaction, i.e. the re-addition of a proton to the carbanion intermediate, is the rate-limiting step of the overall process. Analysis of the 5-3H-labelled polysaccharide products showed that the 3H was approximately equally distributed between glucuronic acid and IdoA units, irrespective of incubation time (from 15 min to 72 h) and of the relative proportions of the two epimers in the substrate. This finding points to a catalytic mechanism in which the abstraction and re-addition of C-5 protons are effected by two polyprotic bases, presumably lysine residues. Previous experiments relating to the biosynthesis of dermatan sulphate were similarly interpreted in terms of a two-base epimerization mechanism but differed from the present findings by implicating one monoprotic and one polyprotic base function [Hannesson, Hagner-McWhirter, Tiedemann, Lindahl and Malmström (1996) Biochem. J. 313, 589-596].

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