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Literature summary extracted from

  • Venskutonyte, R.; Koh, A.; Stenstroem, O.; Khan, M.T.; Lundqvist, A.; Akke, M.; Baeckhed, F.; Lindkvist-Petersson, K.
    Structural characterization of the microbial enzyme urocanate reductase mediating imidazole propionate production (2021), Nat. Commun., 12, 1347 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.3.99.33 gene urdA, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli Shewanella oneidensis

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.3.99.33 two-domain construct of UrdA (UrdA') at four specific states: an ADP-bound structure without substrate, and three FAD-bound structures in complex with either the substrate urocanate or the product ImP, or without any ligand bound. For the urocanate and FAD-bound structure crystals: mixing of 30 mg/ml protein in 3.4 mM FAD and 3.0 mM urocanic acid with a reservoir solution consisting of 27% PEG 8000, 0.3 M (NH4)2SO4, 0.1 M HEPES, pH 7.0. Crystals in complex with imidazole propionate by mixing of 26 mg/ml protein in 20 mM HEPES, pH 7.0, 150 mM NaCl, 3.2 mM FAD, and 7 mM imidazole propionate with a reservoir solution containing 20% PEG 8000, 0.2 M NH4Cl, and 0.1 M HEPES, pH 7.0. Crystals for apo-FAD-bound structure by mixing of 0.001 ml of 30 mg/ml protein in 20 mM HEPES, pH 7.0, 150 mM NaCl, and 3.2 mM FAD with 0.001 ml of reservoir solution containing 0.1 M Tris, pH 8.5, 2 M (NH4)2SO4, and 200 nl of 30% xylitol from the additive screen, X-ray diffraction structure determination and analysis at 1.1-2.56 A resolution Shewanella oneidensis

Protein Variants

EC Number Protein Variants Comment Organism
1.3.99.33 D388A site-directed mutagenesis of wild-type enzyme, inactive mutant Shewanella oneidensis
1.3.99.33 R411A site-directed mutagenesis of wild-type enzyme, inactive mutant Shewanella oneidensis
1.3.99.33 R560A site-directed mutagenesis of wild-type enzyme, inactive mutant Shewanella oneidensis
1.3.99.33 Y373H site-directed mutagenesis of two-domain enzyme mutant enzyme, the mutant shows low fumarate reductase activity Shewanella oneidensis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.3.99.33 additional information
-
 additional information ligand binding thermodynamcis, overview Shewanella oneidensis
1.3.99.33 0.22
-
 Urocanate recombinant two-domain construct UrdA, pH 7.0, 22°C Shewanella oneidensis
1.3.99.33 0.43
-
 Urocanate recombinant wild-type UrdA, pH 7.0, 22°C Shewanella oneidensis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.3.99.33 urocanate + FADH2 Shewanella oneidensis
-
 dihydrourocanate + FAD
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.3.99.29 Shewanella oneidensis
-
-
-
1.3.99.33 Shewanella oneidensis Q8CVD0
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.3.99.33 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli by nickel affinity chromatography and gel filtration Shewanella oneidensis

Reaction

EC Number Reaction Comment Organism Reaction ID
1.3.99.33 dihydrourocanate + acceptor = urocanate + reduced acceptor reaction mechanism, overview. Apo-ADP enzyme represents an inactive state, while the binding of FAD induces movement of the clamp domain that enables binding of the substrate urocanate to form an active complex with a solvent-accessible channel for water-mediated proton delivery to Arg411, and subsequent protonation of the substrate. Once urocanate is reduced to imidazole propionate, Arg411 turns away accompanied by conformational changes, resulting in closure of the solvent-accessible channel by the hydrophobic lid and release of imidazole propionate, allowing for a new substrate molecule to enter the active site. Binding is entropically driven for both urocanate and imidazole propionate Shewanella oneidensis
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.3.99.33 urocanate + FADH2
-
 Shewanella oneidensis dihydrourocanate + FAD
-
 ?
1.3.99.33 urocanate + FADH2 3-(1H-imidazol-4-yl)prop-2-enoate or imidazole propionate (ImP) Shewanella oneidensis dihydrourocanate + FAD
-
 ?
1.3.99.33 urocanate + reduced methyl viologen
-
 Shewanella oneidensis dihydrourocanate + methyl viologen
-
 ?

Subunits

EC Number Subunits Comment Organism
1.3.99.33 More enzyme UrdA is organized as a three-domain protein, with an N-terminal domain harboring a covalently bound FMN, an FAD-bound domain, and a mobile capping domain, the latter two forming a substrate-binding site, structural organization and active site interactions, structure-function analysis, overview Shewanella oneidensis

Synonyms

EC Number Synonyms Comment Organism
1.3.99.33 SO_4620
-
 Shewanella oneidensis
1.3.99.33 urdA
-
 Shewanella oneidensis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.3.99.33 22
-
 assay at room temperature Shewanella oneidensis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.3.99.33 1.43
-
 Urocanate recombinant two-domain construct UrdA, pH 7.0, 22°C Shewanella oneidensis
1.3.99.33 5.62
-
 Urocanate recombinant wild-type UrdA, pH 7.0, 22°C Shewanella oneidensis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.3.99.33 7
-
 assay at Shewanella oneidensis

Cofactor

EC Number Cofactor Comment Organism Structure
1.3.99.33 FAD binding structure Shewanella oneidensis
1.3.99.33 FADH2 binding structure Shewanella oneidensis
1.3.99.33 methyl viologen artificial Shewanella oneidensis

General Information

EC Number General Information Comment Organism
1.3.99.33 metabolism enzyme UrdA provides an alternative pathway to metabolize the amino acid histidine by unidirectional reduction of urocanate to imidazole propionate Shewanella oneidensis
1.3.99.33 additional information ligand-binding domains of UrdA, structure-function analysis, and mechanism of action of UrdA, overview. Both full-length UrdA and two-domain construct UrdA' show specific activity toward urocanate but not to fumarate, suggesting that the functional integrity and substrate specificity are conserved in the two-domain construct. Specific residues, in combination with larger conformational changes, regulate substrate access, and product release. Residue Tyr373 contributes to substrate specificity; residues R560 D388 and R411 are important for catalysis. A large conformational change is observed upon ligand binding Shewanella oneidensis

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.3.99.33 6.5
-
 Urocanate recombinant two-domain construct UrdA, pH 7.0, 22°C Shewanella oneidensis
1.3.99.33 13.07
-
 Urocanate recombinant wild-type UrdA, pH 7.0, 22°C Shewanella oneidensis