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Literature summary extracted from

  • Hall, J.; Reschke, S.; Cao, H.; Leimkuehler, S.; Hille, R.
    The reductive half-reaction of xanthine dehydrogenase from Rhodobacter capsulatus the role of Glu232 in catalysis (2014), J. Biol. Chem., 289, 32121-32130 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.17.1.4 gene XDH1, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli Rhodobacter capsulatus
1.17.3.2 gene xdhA, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain TP 1000 Rhodobacter capsulatus

Protein Variants

EC Number Protein Variants Comment Organism
1.17.1.4 E232A site-directed mutagenesis, the mutant exhibits a 12fold decrease in kred compared to wild-type Rhodobacter capsulatus
1.17.1.4 E232Q site-directed mutagenesis, kred, the limiting rate constant for reduction at high [xanthine], is significantly compromised in the mutant variant E232Q, the mutant exhibits a 12fold decrease in kred, a result that is inconsistent with Glu232 being neutral in the active site of the wild-type enzyme Rhodobacter capsulatus
1.17.3.2 E232A site-directed mutagenesis, the mutant exhibits a 12fold decrease in kred compared to wild-type Rhodobacter capsulatus
1.17.3.2 E232Q site-directed mutagenesis, kred, the limiting rate constant for reduction at high [xanthine], is significantly compromised in the mutant variant E232Q, the mutant exhibits a 12fold decrease in kred, a result that is inconsistent with Glu232 being neutral in the active site of the wild-type enzyme Rhodobacter capsulatus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.17.1.4 additional information
-
 additional information steady-state kinetics and reductive half-reaction, stopped flow kinetics, kinetic analysis of wild-type and mutant xanthine dehydrogenases, overview. kred, the limiting rate constant for reduction at high [xanthine], is significantly compromised in the mutant variant E232Q, a result that is inconsistent with Glu232 being neutral in the active site of the wild-type enzyme. The ionized Glu232 of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of substrate, effectively increasing the pKa of the substrate by two pH units and ensuring that at physiological pH the neutral form of the substrate predominates in the Michaelis complex. The product release is principally rate-limiting in catalysis. The disparity in rate constants for the chemical step of the reaction and product release is not as great in the bacterial enzyme as compared with the vertebrate forms. The faster turnover observed with the bacterial enzyme isdue to a faster rate constant for product release than is seen with the vertebrate enzyme Rhodobacter capsulatus
1.17.3.2 additional information
-
 additional information pH-dependent steady-state kinetics and reductive half-reaction, stopped flow kinetics, kinetic analysis of wild-type and mutant xanthine dehydrogenases, detailed overview. kred, the limiting rate constant for reduction at high [xanthine], is significantly compromised in the mutant variant E232Q, a result that is inconsistent with Glu232 being neutral in the active site of the wild-type enzyme. The ionized Glu232 of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of substrate, effectively increasing the pKa of the substrate by two pH units and ensuring that at physiological pH the neutral form of the substrate predominates in the Michaelis complex. The product release is principally rate-limiting in catalysis. The disparity in rate constants for the chemical step of the reaction and product release is not as great in the bacterial enzyme as compared with the vertebrate forms. The faster turnover observed with the bacterial enzyme is due to a faster rate constant for product release than is seen with the vertebrate enzyme Rhodobacter capsulatus
1.17.3.2 0.114
-
 xanthine pH 8.5, 25°C, recombinant wild-type enzyme Rhodobacter capsulatus
1.17.3.2 0.115
-
 xanthine pH 8.5, 25°c, recombinant mutant E232Q Rhodobacter capsulatus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.17.1.4 xanthine + NAD+ + H2O Rhodobacter capsulatus
-
 urate + NADH + H+
-
 ?
1.17.3.2 xanthine + H2O + O2 Rhodobacter capsulatus
-
 urate + H2O2
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.17.1.4 Rhodobacter capsulatus O54050 AND O54051 small and large subunits encoded by genes xdhA and xdhB
-
1.17.3.2 Rhodobacter capsulatus O54050 small subunit encoded by gene xdhA
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.17.1.4 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli by nickel affinity and anion exchange chromatography, followed by gel filtration Rhodobacter capsulatus
1.17.3.2 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli by nickel affinity and anion exchange chromatography, followed by gel filtration Rhodobacter capsulatus

Reaction

EC Number Reaction Comment Organism Reaction ID
1.17.3.2 xanthine + H2O + O2 = urate + H2O2 mechanism of the reductive half-reaction of xanthine oxidase Rhodobacter capsulatus
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.17.1.4 additional information ionized Glu232 of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of xanthine Rhodobacter capsulatus ?
-
 ?
1.17.1.4 xanthine + NAD+ + H2O
-
 Rhodobacter capsulatus urate + NADH + H+
-
 ?
1.17.1.4 xanthine + NAD+ + H2O product release is principally rate-limiting in catalysis Rhodobacter capsulatus urate + NADH + H+
-
 ?
1.17.3.2 additional information ionized Glu232 of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of xanthine. Proposed orientations of xanthine binding in the active site of xanthine oxidoreductase, using the predominant tautomers of the neutral and monoanionic forms of xanthine, overview Rhodobacter capsulatus ?
-
 ?
1.17.3.2 xanthine + H2O + O2
-
 Rhodobacter capsulatus urate + H2O2
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.17.1.4 XDH1
-
 Rhodobacter capsulatus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.17.1.4 25
-
 assay at Rhodobacter capsulatus
1.17.3.2 25
-
 assay at Rhodobacter capsulatus

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.17.3.2 2.14
-
 xanthine pH 8.5, 25°c, recombinant mutant E232Q Rhodobacter capsulatus
1.17.3.2 135
-
 xanthine pH 8.5, 25°C, recombinant wild-type enzyme Rhodobacter capsulatus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.17.1.4 7.9
-
 assay at Rhodobacter capsulatus
1.17.3.2 8.5
-
 assay at Rhodobacter capsulatus

pH Range

EC Number pH Minimum pH Maximum Comment Organism
1.17.1.4 additional information
-
 comparison of the pH dependence of both kred and kred/Kd from reductive half-reaction experiments between wild-type enzyme and mutant E232Q, overview. The ionized Glu232 of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of substrate, effectively increasing the pKa of the substrate by two pH units and ensuring that at physiological pH the neutral form of the substrate predominates in the Michaelis complex Rhodobacter capsulatus
1.17.3.2 additional information
-
 comparison of the pH dependence of both kred and kred/Kd from reductive half-reaction experiments between wild-type enzyme and mutant E232Q, overview. The ionized Glu232 of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of substrate, effectively increasing the pKa of the substrate by two pH units and ensuring that at physiological pH the neutral form of the substrate predominates in the Michaelis complex Rhodobacter capsulatus

Cofactor

EC Number Cofactor Comment Organism Structure
1.17.1.4 NAD+
-
 Rhodobacter capsulatus

General Information

EC Number General Information Comment Organism
1.17.1.4 additional information XDH active site structure with conserved Glu232 and Arg310 residues. Analysis of crystal structure of xanthine dehydrogenase, PDB ID 2W3S, where an ionized glutamate 802/232 acts as a hydrogen bonding acceptor from the substrate N3 nitrogen Rhodobacter capsulatus
1.17.3.2 additional information enzyme active site structure, overview Rhodobacter capsulatus

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.17.3.2 18.6
-
 xanthine pH 8.5, 25°c, recombinant mutant E232Q Rhodobacter capsulatus
1.17.3.2 1184.2
-
 xanthine pH 8.5, 25°C, recombinant wild-type enzyme Rhodobacter capsulatus