Information on EC 3.5.4.27 - methenyltetrahydromethanopterin cyclohydrolase

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The expected taxonomic range for this enzyme is: Archaea, Bacteria

EC NUMBER
COMMENTARY hide
3.5.4.27
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RECOMMENDED NAME
GeneOntology No.
methenyltetrahydromethanopterin cyclohydrolase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
5,10-methenyl-5,6,7,8-tetrahydromethanopterin + H2O = 5-formyl-5,6,7,8-tetrahydromethanopterin
show the reaction diagram
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of cyclic amidines
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-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
formaldehyde oxidation V (H4MPT pathway)
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Metabolic pathways
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Methane metabolism
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methanogenesis from H2 and CO2
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Microbial metabolism in diverse environments
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reductive acetyl coenzyme A pathway II (autotrophic methanogens)
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methanogenesis from CO2
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SYSTEMATIC NAME
IUBMB Comments
5,10-methenyltetrahydromethanopterin 10-hydrolase (decyclizing)
Methanopterin is a pterin analogue. The enzyme is involved in the formation of methane from CO2 in Methanobacterium thermoautotrophicum.
CAS REGISTRY NUMBER
COMMENTARY hide
99533-50-3
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
chemolithoautotrophic
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Manually annotated by BRENDA team
chemolithoautotrophic
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Manually annotated by BRENDA team
DSM7471, originally isolated from the solar saltern of Figueria da Foz, Portugal
UniProt
Manually annotated by BRENDA team
DSM7471, originally isolated from the solar saltern of Figueria da Foz, Portugal
UniProt
Manually annotated by BRENDA team
strain KT
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-
Manually annotated by BRENDA team
Bath
Uniprot
Manually annotated by BRENDA team
15sh
Uniprot
Manually annotated by BRENDA team
15sh
Uniprot
Manually annotated by BRENDA team
strain AS1
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-
Manually annotated by BRENDA team
strain OB3b
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Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
strain H4-14
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Manually annotated by BRENDA team
strain H4-14
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
5,10-methenyl-5,6,7,8-tetrahydromethanopterin + H2O
5-formyl-5,6,7,8-tetrahydromethanopterin
show the reaction diagram
5,10-methenyl-5,6,7,8-tetrahydromethanopterin + H2O
?
show the reaction diagram
5,10-methenyl-5,6,7,8-tetrahydromethanopterin + H2O
N5-formyl-5,6,7,8-tetrahydromethanopterin
show the reaction diagram
formyl-tetrahydromethanopterin
methenyl-tetrahydromethanopterin + H2O
show the reaction diagram
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
5,10-methenyl-5,6,7,8-tetrahydromethanopterin + H2O
5-formyl-5,6,7,8-tetrahydromethanopterin
show the reaction diagram
5,10-methenyl-5,6,7,8-tetrahydromethanopterin + H2O
?
show the reaction diagram
formyl-tetrahydromethanopterin
methenyl-tetrahydromethanopterin + H2O
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
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the enzyme contains no prothetic group
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
(NH4)2SO4
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1.5 M, stimulates
ammonium sulfate
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1.5 M, maximal 100fold stimulation
K+
activity in cell extract is dependent on the presence of high concentrations of potassium phosphate. The activity in 1.0 M potassium phosphate buffer pH 7.6 is eightfold higher than that in 0.12 M potassium phosphate buffer pH 7.6
K2HPO4
K2SO4
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0.8 M, maximal 100fold stimulation; 0.8 M stimulates
KCl
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2.5 M, maximal 100fold stimulation; 2.5 M stimulates
Na2HPO4
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maximal activation by 1.5 M K2HPO4, 1 M Na2HPO4 or 1 M Na2SO4
Na2SO4
NH4Cl
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optimal concentration is 0.3 M
Sodium phosphate
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1 M, 200fold stimulation
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
10-Formyltetrahydromethanopterin
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2,3-bisphosphoglycerate
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the potassium salts of 2,3-bisphosphoglycerate stimulates cyclohydrolase activity
2,3-diphosphoglycerate
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the activity increases with increasing 2,3-diphosphoglycerate concentrations from 30 U/mg up to a specific activity of 7,500 U/mg. The maximal activity is reached at a 2,3-diphosphoglycerate concentration of 0.75 M. At higher concentrations, the activity decreases
phosphate
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the potassium salts of phosphateglycerate stimulates cyclohydrolase activity
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.025 - 0.077
5,10-methenyl-5,6,7,8-tetrahydromethanopterin
0.03 - 0.22
formyl-tetrahydromethanopterin
0.03 - 2
N5,N10-methenyl-5,6,7,8-tetrahydromethanopterin
additional information
additional information
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effect of salts on Km
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.029
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pH 7.5, 65C, cell extract
1.6
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pH 7.5, 65C, cell extract
10
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pH 7.5, 65C
5000
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above, purified native enzyme
additional information
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 70
30C: about 75% of maximal activity, 70C: about 55% of maximal activity
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3.8
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amino acid sequence calculation
4.2
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amino acid sequence calculation
4.8
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amino acid sequence calculation
5.5
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amino acid sequence calculation
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Archaeoglobus fulgidus (strain ATCC 49558 / VC-16 / DSM 4304 / JCM 9628 / NBRC 100126)
Archaeoglobus fulgidus (strain ATCC 49558 / VC-16 / DSM 4304 / JCM 9628 / NBRC 100126)
Archaeoglobus fulgidus (strain ATCC 49558 / VC-16 / DSM 4304 / JCM 9628 / NBRC 100126)
Methanobrevibacter ruminantium (strain ATCC 35063 / DSM 1093 / JCM 13430 / OCM 146 / M1)
Methanopyrus kandleri (strain AV19 / DSM 6324 / JCM 9639 / NBRC 100938)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
33282
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2 * 33282, sequence calculation, 2 * 33000, SDS-PAGE
33972
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3 * 33972, sequence calculation, 3 * 41500, SDS-PAGE
34246
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2 * 34246, sequence calculation, 2 * 41000, SDS-PAGE
34851
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2 * 34851, sequence calculation, 2 * 39000, SDS-PAGE
34889
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2 * 34889, sequence calculation, 2 * 41000, SDS-PAGE
34899
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x * 34899, sequence calculation
34982
x * 34982, calculated from sequence
42000
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non-denaturing PAGE
67000
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non-denaturing PAGE
80000
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non-denaturing PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
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1 * 41500, SDS-PAGE
trimer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging drop vapor diffusion method in an anaerobic tent with an atmosphere of 95% N2/5% H2 under low-intensity red light. X-ray structures of the substrate-free E186Q mutant enzyme, the enzyme:5,10-methenyl-5,6,7,8-tetrahydromethanopterin complex, and the E186Q mutant enzyme:5-formyl-5,6,7,8-tetrahydromethanopterin complex
crystal structure of apoenzyme is solved at 1.37 A resolution
purified recombinant enzyme, X-ray diffraction structure determination and analysis at 2.0 A resolution
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8
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in 0.75 M 2,3-diphosphoglycerate, the cyclohydrolase is completely stable at pH 8.0 and 90C
726828
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
addition of K2HPO4 or other salts prevents inactivation, relatively low concentrations of the salt, below 0.1 mM, are required for the stabilization, potassium salts being more effective than ammonium and sodium salts, at concentrations above 0.5 M, which are required for maximal activity the thermostability decreases again
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at cyclic 2,3-diphosphoglycerate concentrations prevailing in the cells of Methanopyrus kandleri the enzyme is completely thermostable. At molar concentrations also the potassium salts of phosphate and of 2,3-bisphosphoglycerate, the biosynthetic precursor of cyclic 2,3-diphosphoglycerate confer thermostability to the enzymes
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detergents are essential for stability
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OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
stable to oxygen
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246739
stable under oxic conditions
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726828
the enzyme is slowly inactivated under oxic conditions
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486097
the purified enzyme is more resistant to inactivation by O2 than the enzyme in cell extract
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, purified native enzyme, 1.32 M KCl, 50 mM Tris-HCl, pH 7.0, under N2 atmosphere, several months without significaant loss of activity
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-20C, stable for several weeks
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-20C, under N2, 50 mM Tris/HCl buffer, pH 7, containing 1.3 M KCl, no loss of activity for several months
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-20C, under N2, in 50 mM Tris/HCl, pH 7, containing 1.3 M KCl, stable for several months
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0C, both under anaeobic and aerobic conditions, the enzyme also loses very little activity within 24 h
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0C, both under anaerobic and anaerobic conditions, stable for 24 h
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme from Escherichia coli strain BL21(DE3) by ammonium sulfate fractionation and ion exchange chromatography, native enzyme over 600fold from cell supernatant by ammonium sulfate fractionation and two steps of ion exchange chromatography, purification requires anaerobic conditions, the enzyme is completely soluble at 80% saturated ammonium sulfate
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA and amino acid sequence determination and analysis, sequence comparisons to other enzymes of the methanogenesis pathway
expression in Escherichia coli
gene mch, DNA and amino acid sequence determination, overexpression in Escherichia coli strain BL21(DE3)
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overexpression in Escherichia coli
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
NaCl downregulates methenyl-H4MPT cyclohydrolase expression
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E186A
Vmax is 0.035% of wild-type activity, Km for 5,10-methenyl-5,6,7,8-tetrahydromethanopterin is 2.1fold lower compared to wild-type value
E186D
Vmax is 0.56% of wild-type activity, Km for 5,10-methenyl-5,6,7,8-tetrahydromethanopterin is 1.1fold higher compared to wild-type value
E186N
Vmax is 0.1% of wild-type activity, Km for 5,10-methenyl-5,6,7,8-tetrahydromethanopterin is 1.1fold lower compared to wild-type value
E186Q
nearly inactive mutant enhzyme
K94E
Vmax is 86% of wild-type activity, Km for 5,10-methenyl-5,6,7,8-tetrahydromethanopterin is 1.4fold lower compared to wild-type value
K94V
Vmax is 41% of wild-type activity, Km for 5,10-methenyl-5,6,7,8-tetrahydromethanopterin is 1.2fold lower compared to wild-type value
R183a
Vmax is 0.07% of wild-type activity, Km for 5,10-methenyl-5,6,7,8-tetrahydromethanopterin is 1.3fold lower compared to wild-type value
R183E
Vmax is 0.01% of wild-type activity, Km for 5,10-methenyl-5,6,7,8-tetrahydromethanopterin is 2.2fold lower compared to wild-type value
R183E/E186Q
no activity
R183K
Vmax is 15% of wild-type activity, Km for 5,10-methenyl-5,6,7,8-tetrahydromethanopterin is 1.3fold lower compared to wild-type value
R183Q
Vmax is 0.05% of wild-type activity, Km for 5,10-methenyl-5,6,7,8-tetrahydromethanopterin is 1.3fold lower compared to wild-type value
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