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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
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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
catalytic mechanism, structure function-relationship, 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
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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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 + [molybdopterin-synthase sulfur-carrier protein]-Gly-NH-CH2-C(O)SH + H2O
molybdopterin + molybdopterin-synthase sulfur-carrier protein
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additional information
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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
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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
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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
via reaction intermediate, 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
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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
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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
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via reaction intermediate, overview
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evolution
homologues of both molybdopterin synthase subunits are evident in diverse eukaryotic sources such as worm, rat, mouse, rice, and fruit fly as well as humans. In contrast, molybdopterin synthase homologues are absent in the yeast Saccharomyces cerevisiae
evolution
molybdenum cofactor (Moco) biosynthesis is an evolutionarily conserved pathway present in eubacteria, archaea and eukaryotes, including humans. The strong structural similarity between the small subunit of MPT synthase and ubiquitin provides evidence for the evolutionary antecedence of the Moco biosynthetic pathway to the ubiquitin dependent protein degradation pathway
malfunction
characterization of mutants identified in group B patients of molybdenum cofactor deficiency, molecular mechanism leading to human molybdenum cofactor deficiency, overview
malfunction
chlorate-sensitive mutants, all the result of amino acid substitutions, produce low levels of molybdopterin and may have low levels of molybdoenzymes. In contrast, chlorate-resistant cnx strains have undetectable levels of molybdopterin, lack the ability to utilize nitrate or hypoxanthine as sole nitrogen sources, and are probably null mutations. Two independent alterations at residue Gly-148 in the large subunit, CnxH, result in temperature sensitivity, not the thermolabile nitrate reductase
malfunction
chlorate-sensitive mutants, all the result of amino acid substitutions, produce low levels of molybdopterin and may have low levels of molybdoenzymes. In contrast, chlorate-resistant cnx strains have undetectable levels of molybdopterin, lack the ability to utilize nitrate or hypoxanthine as sole nitrogen sources, and are probably null mutations. Two independent alterations at residue Gly-148 in the large subunit, CnxH, result in temperature sensitivity, not the thermolabile nitrate reductase. Molybdopterin is undetectable in the chlorate-resistant mutants, cnxG4 and cnxH3, whereas up to 2% and 10% of the wild-type level is found in the chlorate-sensitive strains cnxG2 and cnxH1, respectively
malfunction
genetic deficiencies of enzymes involved in Moco biosynthesis in humans lead to a severe and usually fatal disease
malfunction
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the viviparous seed mutant, viviparous15 (vp15), isolated from the Uniform Mu transposon-tagging population, shows an early seedling lethal phenotype, it also shows reduced activities of several enzymes that require molybdenum cofactor (MoCo) in vp15 mutant seedlings, overview. Because MoCo is required for abscisic acid biosynthesis, the viviparous phenotype is probably caused by abscisic acid deficiency
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
metabolism
molybdopterin synthase is involved in the conversion of precursor Z to molybdopterin in the molybdenum cofactor biosynthetic pathway
metabolism
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MPT synthase catalyzes the second step of molybdenum cafactor biosynthesis
metabolism
MPT synthase catalyzes the second step of molybdenum cafactor biosynthesis
metabolism
fused MPT synthase protein from Deinococcus radiodurans in which MoaD- and MoaE-like domains are located on a single peptide can be cleaved the JAMM/MPN+ domain containing metalloprotease DR0402 (JAMMDR). Cleavage generates the MoaD having a C-terminal di-Gly motif. JAMMDR can also cleave off the MoaD from MoaD-eGFP fusion protein
metabolism
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fused MPT synthase protein from Deinococcus radiodurans in which MoaD- and MoaE-like domains are located on a single peptide can be cleaved the JAMM/MPN+ domain containing metalloprotease DR0402 (JAMMDR). Cleavage generates the MoaD having a C-terminal di-Gly motif. JAMMDR can also cleave off the MoaD from MoaD-eGFP fusion protein
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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
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
physiological function
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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
physiological function
the molybdenum cofactor contains a tricyclic pyranopterin, termed molybdopterin (MPT), that bears the cis-dithiolene group responsible for molybdenum ligation. The dithiolene group of MPT is generated by MPT synthase
physiological function
the small and large subunits in humans, MOCO1-A and MOCO1-B, respectively, together form molybdopterin synthase which adds sulphur to precursor Z
additional information
active site struture and substrate-binding and catalytically important residues, overview
additional information
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active site struture and substrate-binding and catalytically important residues, overview. Interaction between Glu125 and precursor Z in the enzyme-precursor Z cocrystals
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
in the activated form of the enzyme this C-terminus is present as a thiocarboxylate. In the structure of a covalent complex of MPT synthase, an isopeptide bond is present between the C-terminus of the small subunit and a Lys side chain in the large subunit. The highly conserved active site residues are Arg39, Lys119, Lys126 and Arg140 in the large subunit
additional information
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in the activated form of the enzyme this C-terminus is present as a thiocarboxylate. In the structure of a covalent complex of MPT synthase, an isopeptide bond is present between the C-terminus of the small subunit and a Lys side chain in the large subunit. The highly conserved active site residues are Arg39, Lys119, Lys126 and Arg140 in the large subunit
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
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
the C-terminal Gly of the small subunit CnxG, is crucial for the sulfur transfer process during the formation of molybdopterin, residues essential for function, overview
additional information
the C-terminal Gly of the small subunit CnxG, is crucial for the sulfur transfer process during the formation of molybdopterin, residues essential for function, overview
additional information
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the C-terminal Gly of the small subunit CnxG, is crucial for the sulfur transfer process during the formation of molybdopterin, residues essential for function, overview
additional information
the rate of conversion of precursor Z to MPT by the human enzyme is slower than that of the eubacterial homologue from Escherichia coli
additional information
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the rate of conversion of precursor Z to MPT by the human enzyme is slower than that of the eubacterial homologue from Escherichia coli
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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
2 * 9800, subunit MOCS2A, + 2 * 20800, subunit MCS2B, SDS-PAGE
20900
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
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
2 * 9700, subunit MOCO1-A, + 2 * 20900, subunit MOCO1-B, SDS-PAGE
9800
2 * 9800, subunit MOCS2A, + 2 * 20800, subunit MCS2B, SDS-PAGE
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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
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x * 21600, large sbunit CnxH, SDS-PAGE
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x * 9600, small subunit CnxG, SDS-PAGE
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x * 11000, His-tagged subunit MoaD, plus x * 18000, FLAG-tagged subunit MoaE, SDS-PAGE
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x * 29000, FLAG-tagged Moad-MoaE precursor protein, SDS-PAGE
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x * 11000, His-tagged subunit MoaD, plus x * 18000, FLAG-tagged subunit MoaE, SDS-PAGE
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x * 29000, FLAG-tagged Moad-MoaE precursor protein, SDS-PAGE
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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
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
heterotetramer
2 * 9700, subunit MOCO1-A, + 2 * 20900, subunit MOCO1-B, SDS-PAGE
heterotetramer
2 * 9800, subunit MOCS2A, + 2 * 20800, subunit MCS2B, SDS-PAGE
heterotetramer
the crystal structure of MPT synthase reveals a heterotetrameric protein in which the C-terminus of each small subunit is inserted into a large subunit to form the active site
heterotetramer
2 * 17000 + 2 * 10000, SDS-PAGE
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
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
additional information
comparison of structures and sequences of MPT synthases from different species, overview. The side chains of the aromatic residues Phe D7, Phe D75, Tyr E55 and Trp E125 are forming the hydrophobic core of the heterodimer interface, the highly conserved active site residues are Arg39, Lys119, Lys126 and Arg140 in the large subunit
additional information
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comparison of structures and sequences of MPT synthases from different species, overview. The side chains of the aromatic residues Phe D7, Phe D75, Tyr E55 and Trp E125 are forming the hydrophobic core of the heterodimer interface, the highly conserved active site residues are Arg39, Lys119, Lys126 and Arg140 in the large subunit
additional information
human MPT synthase contains the subunits MOCS2A and MOCS2B
additional information
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human MPT synthase contains the subunits MOCS2A and MOCS2B
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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
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
structure at 2.1 A resolution. The overall structure is hetero-tetrameric, consisting of a MoaE2 dimer flanked on either side by single MoaD2 subunits. The carboxyl-terminal domain of MoaD2 inserted into MoaE2, forming the active pocket
structure at 2.6 A resolution. The overall structure is hetero-tetrameric, consisting of a MoaE2 dimer flanked on either side by single MoaD2 subunits. The carboxyl-terminal domain of MoaD2 inserted into MoaE2, forming the active pocket
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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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
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
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
K123A
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site-directed mutagenesis, mutant of MoaE
E128K
inactive mutant
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
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
K126A
site-directed mutagenesis, mutant of MoaE
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
additional information
isolation of cnxG and cnxH mutants, i.e. mutations cnxG10, cnxG24, cnxG100, cnxG166, cnxH43, cnxH86, cnxH89, cnxH255, and cnxH604, overview. Precursor Z and molybdopterin levels in chlorate-resistant and chlorate-sensitive cnxG and cnxH mutant strains, i.e. strains cnxG4 and cnxH3, and strains cnxG2 and cnxH1, overview
additional information
isolation of cnxG and cnxH mutants, i.e. mutations cnxG10, cnxG24, cnxG100, cnxG166, cnxH43, cnxH86, cnxH89, cnxH255, and cnxH604, overview. Precursor Z and molybdopterin levels in chlorate-resistant and chlorate-sensitive cnxG and cnxH mutant strains, i.e. strains cnxG4 and cnxH3, and strains cnxG2 and cnxH1, overview
additional information
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isolation of cnxG and cnxH mutants, i.e. mutations cnxG10, cnxG24, cnxG100, cnxG166, cnxH43, cnxH86, cnxH89, cnxH255, and cnxH604, overview. Precursor Z and molybdopterin levels in chlorate-resistant and chlorate-sensitive cnxG and cnxH mutant strains, i.e. strains cnxG4 and cnxH3, and strains cnxG2 and cnxH1, overview
additional information
isolation of cnxG and cnxH mutants, i.e. mutations cnxG10, cnxG24, cnxG100, cnxG166, cnxH43, cnxH86, cnxH89, cnxH255, and cnxH604, overview. Precursor Z and molybdopterin levels in chlorate-resistant and chlorate-sensitive cnxG and cnxH mutant strains, overview. Molybdopterin is undetectable in the chlorate-resistant mutants, cnxG4 and cnxH3, whereas up to 2% and 10% of the wild-type level is found in the chlorate-sensitive strains cnxG2 and cnxH1, respectively. The chlorate-sensitive A. nidulans strain, cnxH1, contains an alteration of the translational stop codon such that it is translated to give the amino acid residue Cys, now residue 196, followed by 31 novel amino acids before the next observed stop codon
additional information
isolation of cnxG and cnxH mutants, i.e. mutations cnxG10, cnxG24, cnxG100, cnxG166, cnxH43, cnxH86, cnxH89, cnxH255, and cnxH604, overview. Precursor Z and molybdopterin levels in chlorate-resistant and chlorate-sensitive cnxG and cnxH mutant strains, overview. Molybdopterin is undetectable in the chlorate-resistant mutants, cnxG4 and cnxH3, whereas up to 2% and 10% of the wild-type level is found in the chlorate-sensitive strains cnxG2 and cnxH1, respectively. The chlorate-sensitive A. nidulans strain, cnxH1, contains an alteration of the translational stop codon such that it is translated to give the amino acid residue Cys, now residue 196, followed by 31 novel amino acids before the next observed stop codon
additional information
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isolation of cnxG and cnxH mutants, i.e. mutations cnxG10, cnxG24, cnxG100, cnxG166, cnxH43, cnxH86, cnxH89, cnxH255, and cnxH604, overview. Precursor Z and molybdopterin levels in chlorate-resistant and chlorate-sensitive cnxG and cnxH mutant strains, overview. Molybdopterin is undetectable in the chlorate-resistant mutants, cnxG4 and cnxH3, whereas up to 2% and 10% of the wild-type level is found in the chlorate-sensitive strains cnxG2 and cnxH1, respectively. The chlorate-sensitive A. nidulans strain, cnxH1, contains an alteration of the translational stop codon such that it is translated to give the amino acid residue Cys, now residue 196, followed by 31 novel amino acids before the next observed stop codon
additional information
a deletion of Ala150 is introduced into MOCS2B, attempts to purify MOCS2B-A150DELTA fails because the protein forms inclusion bodies upon expression in the Escherichia coli cells. Construction of a MOCS2B variant lacking the first 43 amino acid residues, the DELTA1-43 variant of MOCS2B is unstable because the majority of the protein was rapidly degraded during or after purification
additional information
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a deletion of Ala150 is introduced into MOCS2B, attempts to purify MOCS2B-A150DELTA fails because the protein forms inclusion bodies upon expression in the Escherichia coli cells. Construction of a MOCS2B variant lacking the first 43 amino acid residues, the DELTA1-43 variant of MOCS2B is unstable because the majority of the protein was rapidly degraded during or after purification
additional information
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the viviparous mutant vp15 shows a Mu insertion in a gene encoding the small subunit of molybdopterin synthase. Mutant seedlings rescued in soil or agar media are typically smaller than the wild-type, with spindly leaves, MoCo-dependent enzyme activities are reduced in the vp15 mutants, phenotype, overview
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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
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 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
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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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
expressed in Escherichia coli BL21(DE3) cells
expression in Escherichia coli
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
recombinant expression in Escherichia coli strain BL21(DE3)
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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Kanaujia, S.P.; Ranjani, C.V.; Jeyakanthan, J.; Ohmori, M.; Agari, K.; Kitamura, Y.; Baba, S.; Ebihara, A.; Shinkai, A.; Kuramitsu, S.; Shiro, Y.; Sekar, K.; Yokoyama, S.
Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of molybdopterin synthase from Thermus thermophilus HB8
Acta Crystallogr. Sect. F
63
324-326
2007
Thermus thermophilus, Thermus thermophilus HB8 / ATCC 27634 / DSM 579
brenda
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
Staphylococcus aureus, Escherichia coli (P30749)
brenda
Unkles, S.E.; Heck, I.S.; Appleyard, M.V.; Kinghorn, J.R.
Eukaryotic molybdopterin synthase. Biochemical and molecular studies of Aspergillus nidulans cnxG and cnxH mutants
J. Biol. Chem.
274
19286-19293
1999
no activity in Saccharomyces cerevisiae, Aspergillus nidulans (Q9Y8C1), Aspergillus nidulans (Q9Y8C2), Aspergillus nidulans
brenda
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
brenda
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)
brenda
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
brenda
Leimkuhler, S.; Freuer, A.; Araujo, J.A.; Rajagopalan, K.V.; Mendel, R.R.
Mechanistic studies of human molybdopterin synthase reaction and characterization of mutants identified in group B patients of molybdenum cofactor deficiency
J. Biol. Chem.
278
26127-26134
2003
Homo sapiens (O96007), Homo sapiens
brenda
Rudolph, M.J.; Wuebbens, M.M.; Rajagopalan, K.V.; Schindelin, H.
Crystal structure of molybdopterin synthase and its evolutionary relationship to ubiquitin activation
Nat. Struct. Biol.
8
42-46
2001
Homo sapiens (O96007), Homo sapiens
brenda
Sloan, J.; Kinghorn, J.R.; Unkles, S.E.
The two subunits of human molybdopterin synthase: evidence for a bicistronic messenger RNA with overlapping reading frames
Nucleic Acids Res.
27
854-858
1999
Homo sapiens (O96007), Homo sapiens
brenda
Suzuki, M.; Settles, A.M.; Tseung, C.W.; Li, Q.B.; Latshaw, S.; Wu, S.; Porch, T.G.; Schmelz, E.A.; James, M.G.; McCarty, D.R.
The maize viviparous15 locus encodes the molybdopterin synthase small subunit
Plant J.
45
264-274
2006
Zea mays
brenda
Narrandes, N.C.; Machowski, E.E.; Mizrahi, V.; Kana, B.D.
Cleavage of the moaX-encoded fused molybdopterin synthase from Mycobacterium tuberculosis is necessary for activity
BMC Microbiol.
15
22
2015
Mycobacterium tuberculosis (Q6MWY3), Mycobacterium tuberculosis
brenda
Yang, Y.M.; Won, Y.B.; Ji, C.J.; Kim, J.H.; Ryu, S.H.; Ok, Y.H.; Lee, J.W.
Cleavage of molybdopterin synthase MoaD-MoaE linear fusion by JAMM/MPN+ domain containing metalloprotease DR0402 from Deinococcus radiodurans
Biochem. Biophys. Res. Commun.
502
48-54
2018
Deinococcus radiodurans (Q9RR88), Deinococcus radiodurans, Deinococcus radiodurans DSM 20539 (Q9RR88)
brenda
Wang, H.; Chen, X.; Zhang, W.; Zhou, W.; Liu, X.; Rao, Z.
Structural analysis of molybdopterin synthases from two mycobacterial pathogens
Biochem. Biophys. Res. Commun.
511
21-27
2019
Mycobacterium tuberculosis (A0A045HUW8), Mycolicibacterium smegmatis (I7FL16), Mycolicibacterium smegmatis ATCC 700084 (I7FL16)
brenda