Literature summary for 3.5.4.10 extracted from

Kang, Y.N.; Tran, A.; White, R.H.; Ealick, S.E.
A novel function for the N-terminal nucleophile hydrolase fold demonstrated by the structure of an archaeal inosine monophosphate cyclohydrolase (2007), Biochemistry, 46, 5050-5062.

Cloned(Commentary)

Cloned (Comment)	Organism
overexpression of His-tagged enzyme in Escherichia coli strain BL21	Methanothermobacter thermautotrophicus
overexpression of wild-type and mutant enzymes in Escherichia coli strain BL21	Methanothermobacter thermautotrophicus

Crystallization (Commentary)

Crystallization (Comment)	Organism
MthPurO in complex with either IMP or 5-aminoimidazole-4-carboxamide ribonucleotide, crystals from 30 mg/ml protein in 25-30% MPD, 0.2 M ammonium acetate, and 0.1 M sodium citrate buffer, pH 5.6, 0.001 ml protein solution is mixed with 0.001 ml of reservoir solution, equilibration against 0.5 mL reservoir solution, 24°C, od-shaped crystals, 3-4 weeks, X-ray diffraction structure determination and analysis at 2.0 A and 2.6 A resolution, respectively, method optimization, molecular replacement and modelling	Methanothermobacter thermautotrophicus
MthPurO in complex with either IMP or 5-aminoimidazole-4-carboxamide ribonucleotide, X-ray diffraction structure determination and analysis at 2.0 A and 2.6 A resolution, respectively	Methanothermobacter thermautotrophicus

Crystallization (Comment)

Organism

MthPurO in complex with either IMP or 5-aminoimidazole-4-carboxamide ribonucleotide, crystals from 30 mg/ml protein in 25-30% MPD, 0.2 M ammonium acetate, and 0.1 M sodium citrate buffer, pH 5.6, 0.001 ml protein solution is mixed with 0.001 ml of reservoir solution, equilibration against 0.5 mL reservoir solution, 24°C, od-shaped crystals, 3-4 weeks, X-ray diffraction structure determination and analysis at 2.0 A and 2.6 A resolution, respectively, method optimization, molecular replacement and modelling

Methanothermobacter thermautotrophicus

MthPurO in complex with either IMP or 5-aminoimidazole-4-carboxamide ribonucleotide, X-ray diffraction structure determination and analysis at 2.0 A and 2.6 A resolution, respectively

Methanothermobacter thermautotrophicus

Protein Variants

Protein Variants	Comment	Organism
C61A	site-directed mutagenesis, the mutant shows 8fold increased activity compared to the wild-type enzyme	Methanothermobacter thermautotrophicus
E102Q	site-directed mutagenesis, inactive mutant	Methanothermobacter thermautotrophicus
R31K	site-directed mutagenesis, the mutant shows 76% reduced activity compared to the wild-type enzyme	Methanothermobacter thermautotrophicus
Y59F	site-directed mutagenesis, the mutant shows 34% reduced activity compared to the wild-type enzyme	Methanothermobacter thermautotrophicus

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	-	Methanothermobacter thermautotrophicus

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
22000	-	x * 22000, recombinant enzyme, SDS-PAGE	Methanothermobacter thermautotrophicus

Organism

Organism	UniProt	Comment	Textmining
Methanothermobacter thermautotrophicus	-	gene MJ0626	-
Methanothermobacter thermautotrophicus	O27089	gene MTH1020	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged enzyme from Escherichia coli strain BL21 by cobalt affinity chromatography, elution with imidazole, to homogeneity	Methanothermobacter thermautotrophicus
soluble recombinant wild-type and mutant enzymes from Escherichia coli strain BL21 by heat treatment for 15 min at 70°C, and anion exchange chromatography, to homogeneity	Methanothermobacter thermautotrophicus

Reaction

Reaction	Comment	Organism	Reaction ID
IMP + H2O = 5-formamido-1-(5-phospho-D-ribosyl)imidazole-4-carboxamide	catalytic mechanism, Glu104 is essential for catalysis	Methanothermobacter thermautotrophicus	a
IMP + H2O = 5-formamido-1-(5-phospho-D-ribosyl)imidazole-4-carboxamide	two possible catalytic mechanisms for cyclization, Glu102 is essential for catalysis	Methanothermobacter thermautotrophicus	a

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
0.44	-	purified recombinant mutant R31K	Methanothermobacter thermautotrophicus
1.2	-	purified recombinant mutant Y59F	Methanothermobacter thermautotrophicus
1.8	-	purified recombinant wild-type enzyme	Methanothermobacter thermautotrophicus
32	-	purified recombinant mutant C61A	Methanothermobacter thermautotrophicus

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
5-formaminoimidazole-4-carboxamide ribonucleotide	the enzyme catalyzes the cyclization of 5-formaminoimidazole-4-carboxamide ribonucleotide, FAICAR, to IMP in the final step of de novo purine biosynthesis	Methanothermobacter thermautotrophicus	IMP + H2O	-	?

Subunits

Subunits	Comment	Organism
?	x * 22000, recombinant enzyme, SDS-PAGE	Methanothermobacter thermautotrophicus
More	MthPurO has a four-layered alphabetabetaalpha core structure, showing an N-terminal nucleophile hydrolase fold. The active site is located at the deep pocket between two central beta-sheets and contains residues strictly conserved within PurOs	Methanothermobacter thermautotrophicus
tetramer	MthPurO has a four-layered alphabetabetaalpha core structure, showing an N-terminal nucleophile hydrolase fold. The active site is located at the deep pocket between two central beta-sheets and contains residues strictly conserved within PurOs, structure analysis, modelling, detailed overview	Methanothermobacter thermautotrophicus

Synonyms

Synonyms	Comment	Organism
inosine 5'-monophosphate cyclohydrolase	-	Methanothermobacter thermautotrophicus
MjPurO	-	Methanothermobacter thermautotrophicus
MthPurO	-	Methanothermobacter thermautotrophicus
PurO	-	Methanothermobacter thermautotrophicus

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
60	-	assay at	Methanothermobacter thermautotrophicus

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.2	-	assay at	Methanothermobacter thermautotrophicus