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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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2.8.1.12 molybdopterin synthaseIUBMB 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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The taxonomic range for the selected organisms is: Escherichia coli
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Reaction Schemes
Synonyms
mpt synthase, molybdopterin synthase, moae2, moad-moae, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
REACTION
REACTION DIAGRAM
COMMENTARY
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
cyclic pyranopterin phosphate + 2 [molybdopterin-synthase sulfur-carrier protein]-Gly-NH-CH2-C(O)SH + H2O = molybdopterin + 2 molybdopterin-synthase sulfur-carrier protein
cyclic pyranopterin phosphate + 2 [molybdopterin-synthase sulfur-carrier protein]-Gly-NH-CH2-C(O)SH + H2O = molybdopterin + 2 molybdopterin-synthase sulfur-carrier protein
catalytic mechanism, structure function-relationship, overview
cyclic pyranopterin phosphate + 2 [molybdopterin-synthase sulfur-carrier protein]-Gly-NH-CH2-C(O)SH + H2O = molybdopterin + 2 molybdopterin-synthase sulfur-carrier protein
catalytic mechanism, structure-function relationshipp analysis, overview
cyclic pyranopterin phosphate + 2 [molybdopterin-synthase sulfur-carrier protein]-Gly-NH-CH2-C(O)SH + H2O = molybdopterin + 2 molybdopterin-synthase sulfur-carrier protein
reaction mechanism via an intermediate, that remains tightly associated with the protein, overview. Stoichiometry of 2 molecules of thiocarboxylated MoaD per conversion of a single precursor Z molecule to molybdopterin, protein-bound intermediate show a monosulfurated structure that contains a terminal phosphate group similar to that present in molybdopterin
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cyclic pyranopterin phosphate + 2 [molybdopterin-synthase sulfur-carrier protein]-Gly-NH-CH2-C(O)SH + H2O = molybdopterin + 2 molybdopterin-synthase sulfur-carrier protein
two-step reaction of MPT synthesis where the dithiolene is generated by two thiocarboxylates derived from a single tetrameric MPT synthase, hypothetical model for the conversion of precursor Z to molybdopterin in the MPT synthase reaction, overview
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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.
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
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
cyclic pyranopterin phosphate + 2 [molybdopterin-synthase sulfur-carrier protein]-Gly-NH-CH2-C(O)SH + H2O
molybdopterin + 2 molybdopterin-synthase sulfur-carrier protein
-
-
-
-
?
additional information
?
-
-
in vitro generation of carboxylated and thiocarboxylated MoaD, overview
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-
?
cyclic pyranopterin phosphate + 2 [molybdopterin-synthase sulfur-carrier protein]-Gly-NH-CH2-C(O)SH + H2O
molybdopterin + 2 molybdopterin-synthase sulfur-carrier protein
-
-
-
?
cyclic pyranopterin phosphate + 2 [molybdopterin-synthase sulfur-carrier protein]-Gly-NH-CH2-C(O)SH + H2O
molybdopterin + 2 molybdopterin-synthase sulfur-carrier protein
via reaction intermediate, overview
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-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
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
-
-
-
?
cyclic pyranopterin phosphate + 2 [molybdopterin-synthase sulfur-carrier protein]-Gly-NH-CH2-C(O)SH + H2O
molybdopterin + 2 molybdopterin-synthase sulfur-carrier protein
-
-
-
-
?
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
metabolism
MPT synthase catalyzes the second step of molybdenum cafactor biosynthesis
physiological function
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
-
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
physiological function
-
MPT is formed by incorporation of two sulfur atoms into precursor Z, which is catalyzed by MPT synthase. The thiocarboxylation of the C-terminus of MoaD serves as the source of sulfur that is transferred to precursor Z
physiological function
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the enzyme is involved in the biosynthesis of the molybdenum cofactor. In the cofactor, the metal is complexed to the unique cis-dithiolene moiety located on the pyran ring of molybdopterin, molybdopterin synthase is responsible for adding the dithiolene to a desulfo precursor termed precursor Z. The sulfur used for dithiolene formation is carried in the form of a thiocarboxylate at the MoaD C terminus. The unique cis-dithiolene moiety of MPT chelates molybdenum or tungsten within Moco. The MoeB protein is essential for the formation of the MoaD thiocarboxylate, serving in the transfer of the dithiolene sulfur atoms, is essential for MPT synthase activity
additional information
active site struture and substrate-binding and catalytically important residues, overview
additional information
molybdopterin synthase contains a binding pocket for the terminal phosphate of molybdopterin, structure of the MoaD thiocarboxylate, overview. The MoaE homodimer shows conformational changes accompanying binding of the MoaD subunit, overview. Position of the thiocarboxylate sulfur at the C-terminus of MoaD
additional information
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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
additional information
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Lys-119 is a residue essential for activity and Arg-39 and Lys-126 are also residues critical for the reaction
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
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
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
heterotetramer
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
-
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
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
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
E141DELTA
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme, formate binding site structure in the E141DELTA MoaE variant, overview
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
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
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
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)
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