Information on EC 2.8.1.12 - molybdopterin synthase

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

EC NUMBER
COMMENTARY hide
2.8.1.12
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RECOMMENDED NAME
GeneOntology No.
molybdopterin synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
cyclic pyranopterin phosphate + 2 [molybdopterin-synthase sulfur-carrier protein]-Gly-NH-CH2-C(O)SH + H2O = molybdopterin + 2 molybdopterin-synthase sulfur-carrier protein
show the reaction diagram
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
molybdenum cofactor biosynthesis
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protein SAMPylation and SAMP-mediated thiolation
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molybdenum cofactor biosynthesis
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Folate biosynthesis
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Metabolic pathways
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SYSTEMATIC NAME
IUBMB Comments
thiocarboxylated molybdopterin synthase:cyclic pyranopterin monophosphate sulfurtransferase
Catalyses the synthesis of molybdopterin from cyclic pyranopterin monophosphate. Two sulfur atoms are transferred to cyclic pyranopterin monophosphate in order to form the characteristic ene-dithiol group found in the molybdenum cofactor. Molybdopterin synthase consists of two large subunits forming a central dimer and two small subunits (molybdopterin-synthase sulfur-carrier proteins) that are thiocarboxylated at the C-terminus by EC 2.8.1.11, molybdopterin synthase sulfurtransferase. The reaction occurs in prokaryotes and eukaryotes.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
carrier subunit MoaX
UniProt
Manually annotated by BRENDA team
no activity in Saccharomyces cerevisiae
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Manually annotated by BRENDA team
Thermus thermophilus HB8 / ATCC 27634 / DSM 579
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Manually annotated by BRENDA team
seed mutant viviparous15 (vp15) isolated from the UniformMu transposon-tagging population, the subunits are encoded by CNX genes
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
metabolism
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
cyclic pyranopterin phosphate + 2 [molybdopterin-synthase sulfur-carrier protein]-Gly-NH-CH2-C(O)SH + H2O
molybdopterin + 2 molybdopterin-synthase sulfur-carrier protein
show the reaction diagram
cyclic pyranopterin phosphate + [molybdopterin-synthase sulfur-carrier protein]-Gly-NH-CH2-C(O)SH + H2O
molybdopterin + molybdopterin-synthase sulfur-carrier protein
show the reaction diagram
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?
additional information
?
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in vitro generation of carboxylated and thiocarboxylated MoaD, overview
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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
cyclic pyranopterin phosphate + 2 [molybdopterin-synthase sulfur-carrier protein]-Gly-NH-CH2-C(O)SH + H2O
molybdopterin + 2 molybdopterin-synthase sulfur-carrier protein
show the reaction diagram
cyclic pyranopterin phosphate + [molybdopterin-synthase sulfur-carrier protein]-Gly-NH-CH2-C(O)SH + H2O
molybdopterin + molybdopterin-synthase sulfur-carrier protein
show the reaction diagram
Q6MWY3
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?
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
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assay at
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6900
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2 * 6900, MoaD, + 2 * 16500, MoaE, SDS-PAGE, composed of two small MoaD and two large subunits MoaE. Both forms of MoaD, carboxylated and thiocarboxylated, are monomeric and are able to form a heterotetrameric complex after coincubation in equimolar ratios with MoaE, but only the thiocarboxylated MPT synthase complex is able to convert precursor Z in vitro to MPT
9600
x * 9600, small subunit CnxG, SDS-PAGE
9700
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2 * 9700, subunit MOCO1-A, + 2 * 20900, subunit MOCO1-B, SDS-PAGE
9800
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2 * 9800, subunit MOCS2A, + 2 * 20800, subunit MCS2B, SDS-PAGE
16500
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2 * 6900, MoaD, + 2 * 16500, MoaE, SDS-PAGE, composed of two small MoaD and two large subunits MoaE. Both forms of MoaD, carboxylated and thiocarboxylated, are monomeric and are able to form a heterotetrameric complex after coincubation in equimolar ratios with MoaE, but only the thiocarboxylated MPT synthase complex is able to convert precursor Z in vitro to MPT
20800
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2 * 9800, subunit MOCS2A, + 2 * 20800, subunit MCS2B, SDS-PAGE
20900
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2 * 9700, subunit MOCO1-A, + 2 * 20900, subunit MOCO1-B, SDS-PAGE
21600
x * 21600, large sbunit CnxH, SDS-PAGE
46100
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carboxylated MPT synthase, gel filtration
52800
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thiocarboxylated MPT synthase, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterotetramer
tetramer
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dimer of dimers containing the MoaD and MoaE proteins, the enzyme is an alpha2beta2 heterotetramer of the smaller MoaD and larger MoaE proteins
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant apo form molybdopterin synthase and molybdopterin synthase-precursor Z complex using wild-type and mutant K126A MoeE, X-ray diffraction structure determination and analysis, structure modeling with the enzyme from Staphylococcu aureus, overview
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purified recombinant MoaE, MoaD, and mutant MoeE E141DELTA, the MoaD-MoeB complex is crystallized using 1.1 M (NH4)2SO4 and 0.1 M HEPES, pH 7.5, as precipitant at a protein concentration of 15 mg/ml within 4-8 months, MoeE mutant E141DELTA at a protein concentration of 20 mg/ml is crystallized from a solution containing 600 mM sodium formate, 15% polyethylene glycol 4000, 10% isopropyl alcohol, and 100 mM Tris, pH 7.5, within 2-3 days, X-ray diffraction structure determination and analysis at 1.9-2.4 A resolution
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purified recombinant enzyme, vapor diffusion, protein in M NaCl, 0.1 M HEPES, pH 7.5, is mixed with mother liquor containing 20% v/v glycerol, X-ray diffraction structure determination and analysis at 1.45 A resolution
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purified recombinant His-tagged apo form molybdopterin synthase and molybdopterin synthase-precursor Z complex using wild-type and mutant K123A MoeE, 10 mg/ml protein in a buffer containing 50 mM Tris-HCl, pH 8.0, and 50 mM NaCl, mixed with a precipitant consisting of 2.0 M sodium formate and 0.1 M sodium acetate, pH 5.3, at 22°C, 2 days, for the enzyme complex with precursor Z, 18% w/v PEG 8000, 0.1% polyvinylpyrrollidone K15, and 0.1 M Tris-HCl, pH 8.0, at 4°C is used, X-ray diffraction structure determination and analysis at 2.0 A resolution for the apoenzyme and at 2.5 A resolution for the enzyme complex, molecular replacement with MOLREP, modeling using the cyrstal structure of the Escherichia coli MPT synthase, overview
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purified MoaB, sitting drop vapour diffusion method, 0.001 ml of 11 mg/ml protein in 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, are mixed with 0.001 ml of well solution containing 20% w/v PEG 3350, and 0.2 M tripotassium citrate monohydrate, 20°C, 2 weeks, X-ray diffraction structure determination and analysis at 1.64 A resolution
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinaant MoaB from Escherichia coli strain BL21(DE3) by ultracentrifugation, hydrophobic interaction chromatography, gel filtration, anion and cation exchange chromatography, an hydroxyapatite chromatography
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recombinant enzyme from Escherichia coli strain BL21(DE3) by ammonium sulfate fractionation, dialysis, and gel filtration
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recombinant fully activated MoaD, MoaE, and the E141DELTA variant of MoaE
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recombinant His-tagged wild-type and mutant protein from Escherichia coli strain M15 by nickel affinity chromatography and gel filtration. Elution of carboxylated or thiocarboxylated protein is induced by using a cleavage buffer containing 20 mM Tris/HCl, 500 mM NaCl, 0.1 mM EDTA, pH 8.0, with either 30 mM dithiothreitol or 30 mM ammonium sulfide, respectively. The cleavage reaction is performed at 4°C for at least 24 h
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recombinant His6-tagged MoaE and MoaD from Escherichia coli strain Rosetta (DE3) by nickel affinity chromatography and gel filtration
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recombinant human subunits MOCS2A and MOCS2B from Escherichia coli strain BL21(DE3) by ammonium sulfate fractionation and gel filtration. The separately purified subunits readily assemble into a functional MPT synthase tetramer
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recombinant intein-fusion wild-type and mutant MoaDs and MoaEs from Escherichia coli strain BL21(DE3) by ammonium sulfate fractionation, gel filtration, and chitin affinity chromatography, intein cleavage, followed by another step of gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA and amino acid sequence determination and analysis, cDNA clones ATCC 960768 from adult uterus and ATCC 331184 from fetal liver and spleen, the MOCO1 locus resides on human chromosome 5 encoding subunits MOCO1-A and MOCO1-B
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expressed in Escherichia coli BL21(DE3) cells
expression of MoaB in Escherichia coli strain BL21(DE3)
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genes moaD and moaE, expression of intein-fusion thiocarboxylated MoaD, expression of wild-type and mutant MoaDs and MoaEs in Escherichia coli strain BL21(DE3)
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genes moaD and moaE,expression of His-tagged wild-type and mutant protein in Escherichia coli strain M15
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genes moaE and moaD, expression of His6-tagged MoaE and MoaD in Escherichia coli strain Rosetta (DE3)
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MOCS2A and MOCS2B genotyping, recombinant expression of human subunits MOCS2A and MOCS2B in Escherichia coli strain BL21(DE3), functional complementation of Escherichia coli moaD and moaE mutants. In vitro translation and mutagenesis experiments of MOCS2A and MOCS2B
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recombinant expression in Escherichia coli strain BL21(DE3)
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vp15 mutant, DNA and amino acid sequence determination and analysis, using a high-throughput strategy for analysis of high-copy Mu lines, i.e. MuTAIL PCR to extract genomic sequences flanking the Mu transposons in the vp15 line. the vp15 maps to chromosome 5L. Two allelic mutations confirm that Vp15 encodes a plant MPT synthase small subunit, ZmCNX7
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E141DELTA
F34A
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site-directed mutagenesis, mutant of MoaE, the mutant shows reduced activity compared to the wild-type enzyme
G81DELTA
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deletion of the MoaD C-terminal glycine (G81DELTA MoaD-SH) completely abolishes MPT synthase activity
K119A
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site-directed mutagenesis, mutant of MoaE, the mutant shows loss of activity, probably due to complete failure to form the heterotetramer
M115A
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site-directed mutagenesis, mutant of MoaE, the mutant shows reduced activity compared to the wild-type enzyme
R140A
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site-directed mutagenesis, mutant of MoaE, the mutant shows reduced activity compared to the wild-type enzyme
R39A
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site-directed mutagenesis, mutant of MoaE, the mutant shows reduced activity compared to the wild-type enzyme
E168K
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naturally occuring substitution in MOCS2B, the E168K mutation, identified in a severely affected patient, attenuates binding of precursor Z, the MPT synthase tetramer is readily formed in mixtures of MOCS2B-E168K with equimolar amounts of MOCS2A
V7F
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naturally occuring substitution in MOCS2A, identified in a patient with an unusual mild form of the disease, the mutation weakens the interaction between MOCS2A and MOCS2B, the MOCS2A-V7F variant does not form a complex with MOCS2B in either its carboxylated or thiocarboxylated form, and the mutant MPT synthase shows 90% reduced activity compared to the wild-type enzyme
E125K
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inactive mutant
K123A
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site-directed mutagenesis, mutant of MoaE
additional information
Show AA Sequence (1581 entries)
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