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Information on EC 2.8.1.12 - molybdopterin synthase and Organism(s) Escherichia coli and UniProt Accession P30749

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EC Tree
     2 Transferases
         2.8 Transferring sulfur-containing groups
             2.8.1 Sulfurtransferases
                2.8.1.12 molybdopterin synthase
IUBMB Comments
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.
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This record set is specific for:
Escherichia coli
UNIPROT: P30749
Word Map
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria
The taxonomic range for the selected organisms is: Escherichia coli
Synonyms
CnxG, CnxH, MoaB, MoaD, MoaE, MoaX, MPT synthase, Rv3323c, TTHA0341, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
MPT 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
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
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.
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 + 2 [molybdopterin-synthase sulfur-carrier protein]-Gly-NH-CH2-C(O)SH + H2O
molybdopterin + 2 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 SUBSTRATE
NATURAL 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 + 2 [molybdopterin-synthase sulfur-carrier protein]-Gly-NH-CH2-C(O)SH + H2O
molybdopterin + 2 molybdopterin-synthase sulfur-carrier protein
show the reaction diagram
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?
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.2
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assay at
7.2
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assay at
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22
-
assay at room temperature
22
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assay at room temperature
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
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MPT synthase catalyzes the second step of molybdenum cafactor biosynthesis
physiological function
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conversion of the sulfur- and metal-free precursor Z to mylobdopterin by MPT synthase involving the transfer of sulfur atoms from a C-terminal MoaD thiocarboxylate to the C-1' and C-2' positions of precursor Z. In the complex, precursor Z is bound by strictly conserved residues in a pocket at the MoaE dimer interface in close proximity of the C-terminal glycine of MoaD, conformational changes in a loop that participates in interactions with precursor Z
metabolism
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molybdopterin (MPT) synthase catalyzes the final step in the biosynthesis of MPT, the metal-binding organic portion of the molybdenum cofactor, Moco
physiological function
additional information
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
Sequence
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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
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
46100
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carboxylated MPT synthase, gel filtration
52800
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thiocarboxylated MPT synthase, gel filtration
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterotetramer
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the MPT synthase protein consists of two large (MoaE) and two small (MoaD) subunits with the MoaD subunits located at opposite ends of a central MoaE dimer
?
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x * 8757.94, recombinant carboxylated MoaD, sequence calculation, x * 8774.03, recombinant thiocarboxylated MoaD, sequence calculation, x * 8757.72, recombinant carboxylated MoaD, mass spectrometry, x * 8774.00, recombinant thiocarboxylated MoaD, mass spectrometry
heterotetramer
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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
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
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MoaD is capable of forming two different stable, yet reversible, heterotetrameric complexes that perform biochemically distinct reactions involving the C terminus of MoaD
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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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E128K
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inactive mutant
E141DELTA
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme, formate binding site structure in the E141DELTA MoaE variant, overview
K126A
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site-directed mutagenesis, mutant of MoaE
E128K
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site-directed mutagenesis, mutant of MoaE, the MPT synthase containing E128K MoaE is 16.6times slower than the wild-type protein
E141DELTA
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme, 12.3times slower than the wild-type protein, truncation of the C-terminal helix might be predicted to disrupt interaction with MoaD-SH
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
K126A
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site-directed mutagenesis, mutant of MoaE, the mutant shows reduced activity compared to the wild-type enzyme. The intermediate, rather than MPT, is the major product of the K126A synthase reaction
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
PURIFICATION/commentary
ORGANISM
UNIPROT
LITERATURE
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 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
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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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Daniels, J.N.; Wuebbens, M.M.; Rajagopalan, K.V.; Schindelin, H.
Crystal structure of a molybdopterin synthase-precursor Z complex: insight into its sulfur transfer mechanism and its role in molybdenum cofactor deficiency
Biochemistry
47
615-626
2008
Escherichia coli (P30749), Staphylococcus aureus
Manually annotated by BRENDA team
Gutzke, G.; Fischer, B.; Mendel, R.R.; Schwarz, G.
Thiocarboxylation of molybdopterin synthase provides evidence for the mechanism of dithiolene formation in metal-binding pterins
J. Biol. Chem.
276
36268-36274
2001
Escherichia coli
Manually annotated by BRENDA team
Rudolph, M.J.; Wuebbens, M.M.; Turque, O.; Rajagopalan, K.V.; Schindelin, H.
Structural studies of molybdopterin synthase provide insights into its catalytic mechanism
J. Biol. Chem.
278
14514-14522
2003
Escherichia coli (P30749)
Manually annotated by BRENDA team
Wuebbens, M.M.; Rajagopalan, K.V.
Mechanistic and mutational studies of Escherichia coli molybdopterin synthase clarify the final step of molybdopterin biosynthesis
J. Biol. Chem.
278
14523-14532
2003
Escherichia coli
Manually annotated by BRENDA team
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