Literature summary for 3.5.2.3 extracted from

Lipowska, J.; Miks, C.D.; Kwon, K.; Shuvalova, L.; Zheng, H.; Lewinski, K.; Cooper, D.R.; Shabalin, I.G.; Minor, W.
Pyrimidine biosynthesis in pathogens - structures and analysis of dihydroorotases from Yersinia pestis and Vibrio cholerae (2019), Int. J. Biol. Macromol., 136, 1176-1187 .

Application

Application	Comment	Organism
drug development	the substantial difference between bacterial and mammalian DHOs makes the bacterial enzyme a promising drug target for disrupting bacterial growth and thus an important candidate to evaluate as a response to antimicrobial resistance on a molecular level	Yersinia pestis
drug development	the substantial difference between bacterial and mammalian DHOs makes the bacterial enzyme a promising drug target for disrupting bacterial growth and thus an important candidate to evaluate as a response to antimicrobial resistance on a molecular level	Vibrio cholerae serotype O1

Cloned(Commentary)

Cloned (Comment)	Organism
gene pyrC, sequence comparisons, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)	Yersinia pestis
gene pyrC, sequence comparisons, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)	Vibrio cholerae serotype O1

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant His-tagged enzyme, enzyme proteolysis before crystallization, sitting drop vapor diffusion technique, mixing of 0.001 ml of 14 mg/ml protein in 20 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% sodium azide, 0.5 mM TCEP, and and 20 mM L-citrulline, with 0.0015 ml of crystallization solution containing 133 mM sodium acetate, 67 mM sodium cacodylate-HCl, pH 6.5, 20% w/v PEG 8000, and 2.3% v/v 1-butanol, and equilibration against 0.034 ml of reservoir solution of 1.5 M NaCl, 4-7 days, 16°C, X-ray diffraction structure determination and analysis at 1.95 A resolution, molecular replacement, modelling	Vibrio cholerae serotype O1
purified recombinant His-tagged enzyme, sitting drop vapor diffusion technique, mixing of 400 nl of 10 mg/ml protein in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% sodium azide, and 0.5 mM TCEP, with 400 nl of crystallization solution containing 0.15 M DL-malic acid, pH 7.0, and 20% w/v PEG 3350, equilibration against 0.034 ml of reservoir solution of 1.3 M NaCl, 4-7 days, 16°C, X-ray diffraction structure determination and analysis at 2.4 A resolution, molecular replacement, modelling	Yersinia pestis

Crystallization (Comment)

Organism

purified recombinant His-tagged enzyme, enzyme proteolysis before crystallization, sitting drop vapor diffusion technique, mixing of 0.001 ml of 14 mg/ml protein in 20 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% sodium azide, 0.5 mM TCEP, and and 20 mM L-citrulline, with 0.0015 ml of crystallization solution containing 133 mM sodium acetate, 67 mM sodium cacodylate-HCl, pH 6.5, 20% w/v PEG 8000, and 2.3% v/v 1-butanol, and equilibration against 0.034 ml of reservoir solution of 1.5 M NaCl, 4-7 days, 16°C, X-ray diffraction structure determination and analysis at 1.95 A resolution, molecular replacement, modelling

Vibrio cholerae serotype O1

purified recombinant His-tagged enzyme, sitting drop vapor diffusion technique, mixing of 400 nl of 10 mg/ml protein in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% sodium azide, and 0.5 mM TCEP, with 400 nl of crystallization solution containing 0.15 M DL-malic acid, pH 7.0, and 20% w/v PEG 3350, equilibration against 0.034 ml of reservoir solution of 1.3 M NaCl, 4-7 days, 16°C, X-ray diffraction structure determination and analysis at 2.4 A resolution, molecular replacement, modelling

Yersinia pestis

Inhibitors

Inhibitors	Comment	Organism
2-oxo-1,2,3,6-tetrahydropyrimidine4,6-dicarboxylate	-	Vibrio cholerae serotype O1
2-oxo-1,2,3,6-tetrahydropyrimidine4,6-dicarboxylate	-	Yersinia pestis
L-6-thiodihydroorotate	-	Vibrio cholerae serotype O1
L-6-thiodihydroorotate	-	Yersinia pestis
additional information	malate can bind to the active site	Vibrio cholerae serotype O1
additional information	malate can bind to the active site	Yersinia pestis

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Zn2+	the active site of VcDHO contains two zinc ions: Znalpha in trigonal bipyramidal coordination and Znbeta in tetrahedral coordination. Each zinc ion is coordinated by two histidine residues, His13 and His15 for Znalpha, and His135 and His173 for Znbeta in VcDHO, structure analysis, overview	Vibrio cholerae serotype O1
Zn2+	the active site of YpDHO contains two zinc ions: Znalpha in trigonal bipyramidal coordination and Znbeta in tetrahedral coordination. Each zinc ion is coordinated by two histidine residues, His17 and His19 for Znalpha, and His140 and His178 for Znbeta in YpDHO, structure analysis, overview	Yersinia pestis

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
(S)-dihydroorotate + H2O	Yersinia pestis	-	N-carbamoyl-L-aspartate	-	r
(S)-dihydroorotate + H2O	Vibrio cholerae serotype O1	-	N-carbamoyl-L-aspartate	-	r
(S)-dihydroorotate + H2O	Vibrio cholerae serotype O1 El Tor Inaba N16961	-	N-carbamoyl-L-aspartate	-	r
(S)-dihydroorotate + H2O	Vibrio cholerae serotype O1 ATCC 39315	-	N-carbamoyl-L-aspartate	-	r
additional information	Yersinia pestis	N-carbamoyl-L-aspartate is preferred as the physiological substrate	?	-	-
additional information	Vibrio cholerae serotype O1	N-carbamoyl-L-aspartate is preferred as the physiological substrate	?	-	-
additional information	Vibrio cholerae serotype O1 El Tor Inaba N16961	N-carbamoyl-L-aspartate is preferred as the physiological substrate	?	-	-
additional information	Vibrio cholerae serotype O1 ATCC 39315	N-carbamoyl-L-aspartate is preferred as the physiological substrate	?	-	-
N-carbamoyl-L-aspartate	Yersinia pestis	-	(S)-dihydroorotate + H2O	-	r
N-carbamoyl-L-aspartate	Vibrio cholerae serotype O1	-	(S)-dihydroorotate + H2O	-	r
N-carbamoyl-L-aspartate	Vibrio cholerae serotype O1 El Tor Inaba N16961	-	(S)-dihydroorotate + H2O	-	r
N-carbamoyl-L-aspartate	Vibrio cholerae serotype O1 ATCC 39315	-	(S)-dihydroorotate + H2O	-	r

Organism

Organism	UniProt	Comment	Textmining
Vibrio cholerae serotype O1	Q9KL24	-	-
Vibrio cholerae serotype O1 ATCC 39315	Q9KL24	-	-
Vibrio cholerae serotype O1 El Tor Inaba N16961	Q9KL24	-	-
Yersinia pestis	Q8ZFU4	-	-

Posttranslational Modification

Posttranslational Modification	Comment	Organism
additional information	VcDHO contains a carboxylated lysine (Kcx) residue in position 96 and two S-oxy cysteine (Csx) residues in positions 259 and 261	Vibrio cholerae serotype O1

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, desalting gel filtration, and ultrafiltration	Yersinia pestis
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, desalting gel filtration, and ultrafiltration	Vibrio cholerae serotype O1

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
(S)-dihydroorotate + H2O	-	Yersinia pestis	N-carbamoyl-L-aspartate	-	r
(S)-dihydroorotate + H2O	-	Vibrio cholerae serotype O1	N-carbamoyl-L-aspartate	-	r
(S)-dihydroorotate + H2O	-	Vibrio cholerae serotype O1 El Tor Inaba N16961	N-carbamoyl-L-aspartate	-	r
(S)-dihydroorotate + H2O	-	Vibrio cholerae serotype O1 ATCC 39315	N-carbamoyl-L-aspartate	-	r
additional information	N-carbamoyl-L-aspartate is preferred as the physiological substrate	Yersinia pestis	?	-	-
additional information	N-carbamoyl-L-aspartate is preferred as the physiological substrate	Vibrio cholerae serotype O1	?	-	-
additional information	malate can bind to the active site	Yersinia pestis	?	-	-
additional information	malate can bind to the active site	Vibrio cholerae serotype O1	?	-	-
additional information	N-carbamoyl-L-aspartate is preferred as the physiological substrate	Vibrio cholerae serotype O1 El Tor Inaba N16961	?	-	-
additional information	malate can bind to the active site	Vibrio cholerae serotype O1 El Tor Inaba N16961	?	-	-
additional information	N-carbamoyl-L-aspartate is preferred as the physiological substrate	Vibrio cholerae serotype O1 ATCC 39315	?	-	-
additional information	malate can bind to the active site	Vibrio cholerae serotype O1 ATCC 39315	?	-	-
N-carbamoyl-L-aspartate	-	Yersinia pestis	(S)-dihydroorotate + H2O	-	r
N-carbamoyl-L-aspartate	-	Vibrio cholerae serotype O1	(S)-dihydroorotate + H2O	-	r
N-carbamoyl-L-aspartate	-	Vibrio cholerae serotype O1 El Tor Inaba N16961	(S)-dihydroorotate + H2O	-	r
N-carbamoyl-L-aspartate	-	Vibrio cholerae serotype O1 ATCC 39315	(S)-dihydroorotate + H2O	-	r

Subunits

Subunits	Comment	Organism
More	three-dimensional structure of DHO from Vibrio cholerae, structure analysis and comparison, overview	Vibrio cholerae serotype O1
More	three-dimensional structure of DHO from Yersinia pestis, structure analysis and comparison, overview. Each subunit has a structure based on the (beta/alpha)8-barrel (TIM-barrel) fold, a characteristic of the amidohydrolase superfamily	Yersinia pestis

Synonyms

Synonyms	Comment	Organism
DHO	-	Yersinia pestis
DHO	-	Vibrio cholerae serotype O1
pyrC	-	Yersinia pestis
pyrC	-	Vibrio cholerae serotype O1
type II DHO	-	Yersinia pestis
VcDHO	-	Vibrio cholerae serotype O1
YpDHO	-	Yersinia pestis

General Information

General Information	Comment	Organism
evolution	the enzyme belongs to the amidohydrolase superfamily	Vibrio cholerae serotype O1
evolution	the structure of YpDHO has essentially the same conformation as the structure of DHO from Escherichia coli (EcDHO), the most thoroughly studied bacterial type II DHO. The enzyme belongs to the amidohydrolase superfamily	Yersinia pestis
metabolism	the de novo pyrimidine biosynthesis pathway is essential for the proliferation of many pathogens. One of the pathway enzymes, dihydroorotase (DHO), catalyzes the reversible interconversion of N-carbamoyl-L-aspartate to 4,5-dihydroorotate. De novo pyrimidine biosynthesis pathway, overview	Yersinia pestis
metabolism	the de novo pyrimidine biosynthesis pathway is essential for the proliferation of many pathogens. One of the pathway enzymes, dihydroorotase (DHO), catalyzes the reversible interconversion of N-carbamoyl-L-aspartate to 4,5-dihydroorotate. De novo pyrimidine biosynthesis pathway, overview	Vibrio cholerae serotype O1
additional information	structure homology modelling using the Plasmodium falciparum enzyme structure as model (PfDHO, UniProtKB ID Q8IKA9)	Yersinia pestis
additional information	structure homology modelling using the Plasmodium falciparum enzyme structure as model (PfDHO,UniProtKB ID Q8IKA9)	Vibrio cholerae serotype O1