Literature summary for 1.3.99.33 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 .

Search for more literature for 1.3.99.33

Cloned(Commentary)

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

Crystallization (Commentary)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

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

KM Value [mM]

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

Natural Substrates/ Products (Substrates)

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

Organism

Organism	UniProt	Comment	Textmining
Shewanella oneidensis	Q8CVD0	-	-

Purification (Commentary)

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

Reaction

Reaction	Comment	Organism	Reaction ID
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)

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

Subunits

Subunits	Comment	Organism
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

Synonyms	Comment	Organism
SO_4620	-	Shewanella oneidensis
urdA	-	Shewanella oneidensis

Temperature Optimum [°C]

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

Turnover Number [1/s]

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

pH Optimum

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

Cofactor

Cofactor	Comment	Organism
FAD	binding structure	Shewanella oneidensis
FADH2	binding structure	Shewanella oneidensis
methyl viologen	artificial	Shewanella oneidensis

General Information

General Information	Comment	Organism
metabolism	enzyme UrdA provides an alternative pathway to metabolize the amino acid histidine by unidirectional reduction of urocanate to imidazole propionate	Shewanella oneidensis
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]

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