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hercynine + gamma-L-glutamyl-L-cysteine + O2 = gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
hercynine + gamma-L-glutamyl-L-cysteine + O2 = gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
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hercynine + gamma-L-glutamyl-L-cysteine + O2 = gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
catalytic mechanism in which C-S bond formation is initiated by an iron(III)-complexed thiyl radical attacking the imidazole ring of N-alpha-trimethyl histidine, proposed mechanism for EgtB-catalyzed C-S bond formation and sulfoxidation, overview
hercynine + gamma-L-glutamyl-L-cysteine + O2 = gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
EgtB contains a conserved tyrosine residue that reacts via proton-coupled electron transfer with the iron(III)-superoxo species and creates an iron(III)-hydroperoxo intermediate, thereby preventing the possible thiolate dioxygenation side reaction. The nucleophilic C-S bond-formation step happens subsequently concomitant to relay of the proton of the iron(II)-hydroperoxo back to Tyr377. This is the rate-determining step in the reaction cycle and is followed by hydrogen-atom transfer from the CE1-H group of trimethyl histidine substrate to iron(II)-superoxo. In the final step, a quick and almost barrierless sulfoxidation leads to the sulfoxide product complexes. Quantum mechanics/molecular mechanics study of the mechanism of sulfoxide synthase enzymes as compared to cysteine dioxygenase enzymes and present pathways for both reaction channels in EgtB, reaction mechanism, overview. The active site contains the unusual Tyr157-Cys93 cross-link with a covalent bond between the two amino acid residues. It is believed this cross-link has a steric effect on the overall reaction mechanism. The fast CDO-type side reaction is prevented through a proton-coupled electron transfer from Tyr377 to iron(III)-superoxo, which enables the slower C-S bond formation to take place and blocks the sulfur dioxygenation side reaction
hercynine + gamma-L-glutamyl-L-cysteine + O2 = gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
two different mechanisms are analyzed, depending on whether the sulfoxidation or the S-C bond formation takes place first. The calculations suggest that the S-O bond formation occurs first between the thiolate and the ferric superoxide, followed by homolytic O-O bond cleavage. Subsequently, proton transfer from a second-shell residue Tyr377 to the newly generated iron-oxo moiety takes place, which is followed by proton transfer from the N-alpha-trimethyl histidine imidazole to Tyr377, facilitated by two crystallographically observed water molecules. Next, the S-C bond is formed between gamma-glutamyl cysteine and N-alpha-trimethyl histidine, followed by proton transfer from the imidazole CH moiety to Tyr377, which is calculated to be the rate-limiting step for the whole reaction, with a barrier of 17.9 kcal/mol in the quintet state. Optimized structures for the second, third, and fourth steps. Detailed overview
hercynine + gamma-L-glutamyl-L-cysteine + O2 = gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
EgtB contains a conserved tyrosine residue that reacts via proton-coupled electron transfer with the iron(III)-superoxo species and creates an iron(III)-hydroperoxo intermediate, thereby preventing the possible thiolate dioxygenation side reaction. The nucleophilic C-S bond-formation step happens subsequently concomitant to relay of the proton of the iron(II)-hydroperoxo back to Tyr377. This is the rate-determining step in the reaction cycle and is followed by hydrogen-atom transfer from the CE1-H group of trimethyl histidine substrate to iron(II)-superoxo. In the final step, a quick and almost barrierless sulfoxidation leads to the sulfoxide product complexes. Quantum mechanics/molecular mechanics study of the mechanism of sulfoxide synthase enzymes as compared to cysteine dioxygenase enzymes and present pathways for both reaction channels in EgtB, reaction mechanism, overview. The active site contains the unusual Tyr157-Cys93 cross-link with a covalent bond between the two amino acid residues. It is believed this cross-link has a steric effect on the overall reaction mechanism. The fast CDO-type side reaction is prevented through a proton-coupled electron transfer from Tyr377 to iron(III)-superoxo, which enables the slower C-S bond formation to take place and blocks the sulfur dioxygenation side reaction
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hercynine + gamma-L-glutamyl-L-cysteine + O2 = gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
two different mechanisms are analyzed, depending on whether the sulfoxidation or the S-C bond formation takes place first. The calculations suggest that the S-O bond formation occurs first between the thiolate and the ferric superoxide, followed by homolytic O-O bond cleavage. Subsequently, proton transfer from a second-shell residue Tyr377 to the newly generated iron-oxo moiety takes place, which is followed by proton transfer from the N-alpha-trimethyl histidine imidazole to Tyr377, facilitated by two crystallographically observed water molecules. Next, the S-C bond is formed between gamma-glutamyl cysteine and N-alpha-trimethyl histidine, followed by proton transfer from the imidazole CH moiety to Tyr377, which is calculated to be the rate-limiting step for the whole reaction, with a barrier of 17.9 kcal/mol in the quintet state. Optimized structures for the second, third, and fourth steps. Detailed overview
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hercynine + gamma-L-glutamyl-L-cysteine + O2
gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + gamma-L-Glu-L-Cys sulfinic acid + H2O
Substrates: -
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hercynine + gamma-L-glutamyl-L-cysteine + O2
gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
hercynine + N-glutaryl cysteine + O2
N-glutaryl-(hercyn-2-yl)-L-cysteine S-oxide + H2O
Substrates: N-glutaryl cysteine is a 100fold less efficient sulfur donor for wild type EgtBthermo but a 10fold better substrate for mutant EgtBD416N than gamma-L-glutamyl-L-cysteine
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additional information
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hercynine + gamma-L-glutamyl-L-cysteine + O2
gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
Substrates: -
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hercynine + gamma-L-glutamyl-L-cysteine + O2
gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
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Substrates: -
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hercynine + gamma-L-glutamyl-L-cysteine + O2
gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
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Substrates: sulfoxide incorporation at C2 by EgtB
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hercynine + gamma-L-glutamyl-L-cysteine + O2
gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
Substrates: the enzyme is part of the biosynthesis pathway of ergothioneine
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hercynine + gamma-L-glutamyl-L-cysteine + O2
gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
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Substrates: the enzyme is part of the biosynthesis pathway of ergothioneine
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hercynine + gamma-L-glutamyl-L-cysteine + O2
gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
Substrates: hercynine i.e. Nalpha,Nalpha,Nalpha-trimethyl-L-histidine. The enzyme is specific for both hercynine and gamma-L-glutamyl-L-cysteine. No activity with cysteine, N-acetylcysteine, or glutathione
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hercynine + gamma-L-glutamyl-L-cysteine + O2
gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
Substrates: -
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hercynine + gamma-L-glutamyl-L-cysteine + O2
gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
Substrates: -
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hercynine + gamma-L-glutamyl-L-cysteine + O2
gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
Substrates: i.e. N-alpha-trimethyl histidine
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hercynine + gamma-L-glutamyl-L-cysteine + O2
gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
Substrates: i.e. N-alpha-trimethyl histidine. Substrate binding mode, detailed overview
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hercynine + gamma-L-glutamyl-L-cysteine + O2
gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
Substrates: -
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hercynine + gamma-L-glutamyl-L-cysteine + O2
gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
Substrates: -
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additional information
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Substrates: enzyme additionally accepts L-cysteine, i.e. reaction of EC 1.21.3.10
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additional information
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Substrates: enzyme additionally accepts L-cysteine, i.e. reaction of EC 1.21.3.10
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additional information
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Substrates: enzyme exhibits both EgtB- and Egt1-type activities, and accepts both Cys and Glu-Cys as the sulfur donor, i.e. catalyzes reactions of EC 1.14.99.50 and 1.14.99.51, respectively. The C-S bond is formed at the imidazole epsilon-position
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Substrates: enzyme exhibits both EgtB- and Egt1-type activities, and accepts both Cys and Glu-Cys as the sulfur donor, i.e. catalyzes reactions of EC 1.14.99.50 and 1.14.99.51, respectively. The C-S bond is formed at the imidazole epsilon-position
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Substrates: no activity with L-cysteine and L-histdine
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additional information
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Substrates: enzyme EgtB is extremely specific in terms of substrate specificity. Enzyme activity determination using : a 1H NMR assay of chemical shift of the imidazole hydrogen atoms
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additional information
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Substrates: enzyme EgtB is extremely specific in terms of substrate specificity. Enzyme activity determination using : a 1H NMR assay of chemical shift of the imidazole hydrogen atoms
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additional information
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Substrates: product formation is monitored by cation exchange HPLC using 20 mM phosphoric acid at pH 2 as a mobile phase, and EgtB substrates and products are quantified by 1H NMR, overview
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Substrates: product formation is monitored by cation exchange HPLC using 20 mM phosphoric acid at pH 2 as a mobile phase, and EgtB substrates and products are quantified by 1H NMR, overview
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hercynine + gamma-L-glutamyl-L-cysteine + O2
gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
hercynine + gamma-L-glutamyl-L-cysteine + O2
gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
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Substrates: -
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hercynine + gamma-L-glutamyl-L-cysteine + O2
gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
Substrates: the enzyme is part of the biosynthesis pathway of ergothioneine
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hercynine + gamma-L-glutamyl-L-cysteine + O2
gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
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Substrates: the enzyme is part of the biosynthesis pathway of ergothioneine
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hercynine + gamma-L-glutamyl-L-cysteine + O2
gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
Substrates: -
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hercynine + gamma-L-glutamyl-L-cysteine + O2
gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
Substrates: -
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hercynine + gamma-L-glutamyl-L-cysteine + O2
gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
Substrates: i.e. N-alpha-trimethyl histidine
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hercynine + gamma-L-glutamyl-L-cysteine + O2
gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
Substrates: -
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hercynine + gamma-L-glutamyl-L-cysteine + O2
gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
Substrates: -
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Mn2+
binding structure, overview
Fe2+
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non-heme iron, required for catalysis, the active site iron is coordinated by a 3-His facial triad rather than a 2-His-1-Glu ligand set
Fe2+
requires Fe2+ for activity
Fe2+
non-heme iron, required for catalysis
Fe2+
a non-heme iron enzyme, the two substrates and three histidine residues serve as ligands in an octahedral iron binding site, Glu140 rather than His51 is the metal ligand
Fe2+
the enzyme is a non-heme iron-dependent sulfoxide synthase
Fe2+
an iron-dependent sulfoxide synthase
Fe2+
non-heme iron, requied for catalysis
Iron
protein conatains 0.92 iron per monomer. A mononuclear nonheme iron is coordinated by His62, His153, His157 and three water molecules in an octahedral arrangement
Iron
Tyr93 serves as a proton donor and Tyr94 serves as a hydrogen-bond donor, both facilitating the reduction of the initial iron(III) superoxide species
Iron
protein contains 0.92 iron per monomer
Iron
in a computational model, the abstraction of the proton from the imidazole group in TMH by the Fe(IV)-oxo species is the prerequisite for C-S bond formation, which is the rate-limiting step. The resulting iron(III)-oxo species can easily abstract a proton from Tyr377 to generate a phenolic hydroxyl anion
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3.1 - 5.9
gamma-L-glutamyl-L-cysteine
additional information
additional information
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3.1
gamma-L-glutamyl-L-cysteine
mutant A420Y, pH not specified in the publication, temperature not specified in the publication
3.9
gamma-L-glutamyl-L-cysteine
mutant D52L, pH not specified in the publication, temperature not specified in the publication
4.2
gamma-L-glutamyl-L-cysteine
mutant D52L/A420Y, pH not specified in the publication, temperature not specified in the publication
5.9
gamma-L-glutamyl-L-cysteine
wild-type, pH not specified in the publication, temperature not specified in the publication
0.012
hercynine
pH 8.0, 26°C, recombinant enzyme mutant Y377F
0.013
hercynine
mutant A420Y, pH not specified in the publication, temperature not specified in the publication
0.0132
hercynine
mutant A420Y, pH not specified in the publication, temperature not specified in the publication
0.015
hercynine
mutant D52L/A420Y, pH not specified in the publication, temperature not specified in the publication
0.0151
hercynine
mutant D52L/A420Y, pH not specified in the publication, temperature not specified in the publication
0.0196
hercynine
mutant D52L, pH not specified in the publication, temperature not specified in the publication
0.02
hercynine
mutant D52L, pH not specified in the publication, temperature not specified in the publication
0.022
hercynine
pH 6.0, 26°C, recombinant enzyme mutant Y377F
0.041
hercynine
pH not specified in the publication, temperature not specified in the publication
0.0414
hercynine
wild-type, pH not specified in the publication, temperature not specified in the publication
additional information
additional information
Michaelis-Menten kinetics
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additional information
additional information
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Michaelis-Menten kinetics
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additional information
additional information
Michaelis-Menten kinetics
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additional information
additional information
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Michaelis-Menten kinetics
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0.05 - 0.093
gamma-L-glutamyl-L-cysteine
0.05
gamma-L-glutamyl-L-cysteine
wild-type, pH not specified in the publication, temperature not specified in the publication
0.055
gamma-L-glutamyl-L-cysteine
mutant D52L, pH not specified in the publication, temperature not specified in the publication
0.07
gamma-L-glutamyl-L-cysteine
mutant D52L/A420Y, pH not specified in the publication, temperature not specified in the publication
0.073
gamma-L-glutamyl-L-cysteine
mutant D52L/A420Y, pH not specified in the publication, temperature not specified in the publication
0.09
gamma-L-glutamyl-L-cysteine
mutant A420Y, pH not specified in the publication, temperature not specified in the publication
0.093
gamma-L-glutamyl-L-cysteine
mutant A420Y, pH not specified in the publication, temperature not specified in the publication
7
hercynine
pH not specified in the publication, temperature not specified in the publication
7
hercynine
wild-type, pH not specified in the publication, temperature not specified in the publication
11
hercynine
mutant D52L, pH not specified in the publication, temperature not specified in the publication
20
hercynine
mutant D52L/A420Y, pH not specified in the publication, temperature not specified in the publication
21.7
hercynine
mutant A420Y, pH not specified in the publication, temperature not specified in the publication
57
hercynine
pH 6.0, 26°C, recombinant enzyme mutant Y377F
74
hercynine
pH 8.0, 26°C, recombinant enzyme mutant Y377F
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malfunction
a single point mutation Y377F converts this enzyme into a gamma-glutamyl cysteine dioxygenase with an efficiency that rivals naturally evolved thiol dioxygenases
evolution
enzyme EgtB represents a distinct enzyme class (sulfoxide synthases) with no relation to sulfur oxidizing or C-S bond-forming iron enzymes such as cysteine dioxygenase or isopenicillin synthase
evolution
Some cyanobacteria recruited and adapted a sulfoxide synthase from a different biosynthetic pathway to make ergothioneine. Evolutionary malleability of the thiohistidine biosynthetic machinery. The sulfoxide synthase EgtB catalyzes the sulfurization of N-alpha-trimethylhistidine at the imidazole 2-position and subsequent oxidation to the S-sulfoxide. The homologous sulfoxide synthases OvoA, EC 1.14.99.52, catalyze the formation of 5-histidylcysteine sulfoxide. The stereochemistry of this sulfoxide is unknown, and cyanobacterial OvoA homologues (Egt-B(ovo)) have evolved to catalyze an EgtB-type reaction by convergent evolution. Prokaryotic EgtBs are usually monofunctional, fungal EgtBs are fused to EgtD
evolution
enzyme EgtB belongs to the mononuclear nonheme iron dioxygenase family
evolution
OvoA, EC 1.14.99.52, and EgtB are related in sequence, while they are biochemically distinct
evolution
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EgtB contains a strongly conserved HX3HXE motif, implying that it is a member of the facial triad enzyme family with the Fe(II) site ligated by 2-His-1-Glu
evolution
comparison of EgtB from Mycobacterium thermophilum, EC 1.14.99.50, and EgtB from Chloracidobacterium thermophilum, EC 1.14.99.51, structures enables a classification of all ergothioneine biosynthetic EgtBs into five subtypes, each characterized by unique active-site features
evolution
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OvoA, EC 1.14.99.52, and EgtB are related in sequence, while they are biochemically distinct
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evolution
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enzyme EgtB belongs to the mononuclear nonheme iron dioxygenase family
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metabolism
the enzyme is part of the biosynthesis pathway of ergothioneine
metabolism
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the enzyme is part of the biosynthesis pathway of ergothioneine
metabolism
enzyme EgtB catalyzes O2-dependent C-S bond formation between gamma-glutamyl cysteine and N-alpha-trimethyl histidine as the central step in ergothioneine biosynthesis
metabolism
biosynthesis of N-alpha-trimethyl-2-thiohistidine (ergothioneine) is a frequent trait in cyanobacteria. This sulfur compound may provide essential relief from oxidative stress related to oxygenic photosynthesis. The central steps in ergothioneine biosynthesis are catalyzed by a histidine methyltransferase and the iron-dependent sulfoxide synthase. Ergothioneine biosynthesis starts by trimethylation of the alpha-amino group of histidine. The resulting N-alpha-trimethylhistidine (TMH) is fused to either gamma-glutamylcysteine (in actinomycetes, EC 1.14.99.50) or cysteine (in fungi, EC 1.14.99.51). The sulfoxide product is converted into ergothoneine by removal of the glutamyl and cysteinyl moieties
metabolism
the enzyme catalyzes the key step in the biosynthesis of ergothioneine
metabolism
EgtB is a nonheme iron enzyme catalyzing the C-S bond formation between gamma-glutamyl cysteine and N-alpha-trimethyl histidine in the ergothioneine biosynthesis
metabolism
the mononuclear non-heme iron enzyme EgtB catalyzes an oxidative C-S bond formation in the ergothioneine biosynthesis. One of the EgtB substrates is gamma-Glu-Cys, which is part of the glutathione biosynthesis, resulting in competition between ergothioneine and glutathione biosyntheses
metabolism
the sulfoxidation is prior to the formation of the C-S bond, and the reaction mainly occurs on the quintet state surface. Due to electron transfer from the gamma-glutamyl cysteine to the ferric superoxide, the sulfur atom of gamma-glutamyl cysteine has partial radical characteristics, which facilitates the attack of the distal oxygen atom on the sulfur radical of gamma-glutamyl cysteine to form the sulfoxide. The formation of N-alpha-trimethyl histidine C2 anion, i.e., the abstraction of the proton from the imidazole group by the Fe(IV)-oxo species, is the prerequisite for C-S bond formation, which is the rate-limiting step with an energy barrier of 21.7 kcal/mol. Tyr377 is not directly involved in the sulfoxidation and C-S bond formation
metabolism
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the mononuclear non-heme iron enzyme EgtB catalyzes an oxidative C-S bond formation in the ergothioneine biosynthesis. One of the EgtB substrates is gamma-Glu-Cys, which is part of the glutathione biosynthesis, resulting in competition between ergothioneine and glutathione biosyntheses
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metabolism
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the enzyme catalyzes the key step in the biosynthesis of ergothioneine
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metabolism
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EgtB is a nonheme iron enzyme catalyzing the C-S bond formation between gamma-glutamyl cysteine and N-alpha-trimethyl histidine in the ergothioneine biosynthesis
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metabolism
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the sulfoxidation is prior to the formation of the C-S bond, and the reaction mainly occurs on the quintet state surface. Due to electron transfer from the gamma-glutamyl cysteine to the ferric superoxide, the sulfur atom of gamma-glutamyl cysteine has partial radical characteristics, which facilitates the attack of the distal oxygen atom on the sulfur radical of gamma-glutamyl cysteine to form the sulfoxide. The formation of N-alpha-trimethyl histidine C2 anion, i.e., the abstraction of the proton from the imidazole group by the Fe(IV)-oxo species, is the prerequisite for C-S bond formation, which is the rate-limiting step with an energy barrier of 21.7 kcal/mol. Tyr377 is not directly involved in the sulfoxidation and C-S bond formation
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physiological function
enzyme EgtB catalyzes O2-dependent C-S bond formation between gamma-glutamyl cysteine and N-alpha-trimethyl histidine as the central step in ergothioneine biosynthesis
physiological function
EgtB from Mycobacterium thermoresistibile catalyzes O2-dependent sulfur-carbon bond formation between the side chains of Nalpha-trimethyl histidine and gamma-glutamyl cysteine as a central step in ergothioneine biosynthesis
physiological function
sulfoxide synthase EgtB represents is a non-heme iron enzyme that catalyzes the formation of a C-S bond between N-alpha-trimethyl histidine and gamma-glutamyl cysteine, which is the key step in the biosynthesis of ergothioneine, an important amino acid related to aging
physiological function
EgtB of Candidatus Chloracidobacterium thermophilum has both EgtB- and Egt1-type of activities, i.e. reactions of EC 1.14.99.50 and EC 1.21.3.10
physiological function
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sulfoxide synthase EgtB represents is a non-heme iron enzyme that catalyzes the formation of a C-S bond between N-alpha-trimethyl histidine and gamma-glutamyl cysteine, which is the key step in the biosynthesis of ergothioneine, an important amino acid related to aging
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additional information
the two substrates and three histidine residues serve as ligands in an octahedral iron binding active site, enzyme structure analysis, detailed overview
additional information
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the two substrates and three histidine residues serve as ligands in an octahedral iron binding active site, enzyme structure analysis, detailed overview
additional information
in a competitive reaction containing 1 mM of each histidine, N-alpha-trimethylhistidine, and cysteine, OvoAErwin produces only S-(L-histidin-5-yl)-L-cysteine S-oxide, whereas OvoAErw-NW and EgtB(ovo) produce exclusively gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide
additional information
density functional theory modeling of active-site models of EgtB in a polarized continuum model propose a reaction mechanism starting with sulfoxidation (OAT) of gammaGC followed by C-S bond formation and deprotonation (PT) to form products. Optimized QM geometry of the S-O bond formation transition state for the reaction of iron(III)-superoxo with cysteine in EgtB, overview
additional information
in the active site, the metal is hexacoordinated and ligated by three histidines (His51, His134, and His138), the two substrates (via a sulfide of gamma-glutamyl cysteine and an imidazole nitrogen of N-alpha-trimethyl histidine), and a water molecule. Second-shell residue, Tyr377 forms a hydrogen bond with the water molecule. In addition, two positively charged residues, Arg90 and Arg87, form hydrogen bonds with the substrate gamma-glutamyl cysteine. Several additional water molecules form a hydrogen bonding network interacting with the two substrates
additional information
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density functional theory modeling of active-site models of EgtB in a polarized continuum model propose a reaction mechanism starting with sulfoxidation (OAT) of gammaGC followed by C-S bond formation and deprotonation (PT) to form products. Optimized QM geometry of the S-O bond formation transition state for the reaction of iron(III)-superoxo with cysteine in EgtB, overview
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additional information
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in the active site, the metal is hexacoordinated and ligated by three histidines (His51, His134, and His138), the two substrates (via a sulfide of gamma-glutamyl cysteine and an imidazole nitrogen of N-alpha-trimethyl histidine), and a water molecule. Second-shell residue, Tyr377 forms a hydrogen bond with the water molecule. In addition, two positively charged residues, Arg90 and Arg87, form hydrogen bonds with the substrate gamma-glutamyl cysteine. Several additional water molecules form a hydrogen bonding network interacting with the two substrates
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apo-protein crystallizes in a cubic form with a space group of P21 and diffracted to 2.5 A
comparison with EgtB from Mycobacterium thermophilum, Ec 1.14.99.50 reveals a completely different configuration of active site residues that are involved in oxygen binding and activation. EgtB from Chloracidobacterium thermophilum forms a stable tetramer. In complex with N-alpha-trimethyl histidine, the imidazole rings of His62, His153, and His157 form an iron-binding three histidine facial triad, with the Ntau of the N-alpha-trimethyl histidine imidazole ring joining as the fourth
to 2.5 A resolution. Enzyme is a tetramer. The active site is located at the interface between the N- and C-terminal domains for each monomer where a mononuclear nonheme iron is coordinated by His62, His153, His157 and three water molecules in an octahedral arrangement
analysis of crystal structure of EgtB, PDB ID 4X8D, in complex with iron(II) and N-alpha-trimethylhistidine
analysis of the X-ray crystal structure of EgtB from Mycobacterium thermoresistibile, solved in three different forms, namely, the apo form, in complex with iron and the N-alpha-trimethyl histidine substrate, and in complex with manganese and the two natural substrates gamma-glutamyl cysteine and alpha-trimethyl histidine at 1.98 A resolution, with the active site for the third one. X-ray structure of the active site of EgtB complex with gamma-glutamyl cysteine and N-alpha-trimethyl histidine, coordinates taken from PDB ID 4X8D. Mn is replaced by Fe in the active site
purified recombinant enzyme, as iron-bound holoenzyme, in complex with substrate gamma-glutamyl cysteine, N-alpha-dimethyl histidine and Mn2+ or with substrate N-alpha-trimethyl histidine and Fe2+, X-ray diffraction structure determination and analysis
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Y93F
essential catalytic residue. Mutation significantly reduces sulfoxide synthase activity. Tyr93 serves as a proton donor and Tyr94 serves as a hydrogen-bond donor
Y94F
essential catalytic residue. Mutation significantly reduces sulfoxide synthase activity, and affects the enzyme's ability to consume cysteine. Tyr93 serves as a proton donor and Tyr94 serves as a hydrogen-bond donor
Y377F
-
site-directed mutagenesis, the single point mutation in EgtB completely uncouples substrate consumption from sulfoxide synthase activity with the native substrates hercynine and gamma-glutamyl cysteine, with EgtB exclusively oxidizing gamma-glutamyl cysteine to the sulfinic acid. Tyr377 is hydrogen bonded to a water molecule that coordinates to the iron
D416N
site-directed mutagenesis, the mutation increases KM for gamma-glutamyl cysteine by 200fold but does not significantly change KM for N-alpha-trimethyl histidine or kcat compared to the wild-type enzyme
Y377F
site-directed mutagenesis, the mutation results in conversion of the non-heme iron-dependent sulfoxide synthase into a thiol dioxygenase, purified enzyme mutant EgtBY377F contains 0.64% equiv. of iron as inferred by a ferrozine-based colorimetric assay, and shows reduced activity compared to the wild-type enzyme
A420Y
position is involved in controlling the reaction selectiviy. Mutant leads to an increased amount of side-reaction (cysteine dioxygenase activity)
A420Y
mutation introduced to improve reaction of Egt1-type, i.e. EC 1.14.99.51-type, 10fold decrease in Km-value for L-cysteine
D52L
mutation may disrupt the hydrogen bond between Asp52 and glutamyl group of substrate gamma-Glu-Cys, which in turn alters the substrate selectivity
D52L
D52 may be involved in controlling the partition between Egt1- and EgtB-type of activities
D52L/A420Y
mutation tunes EgtB activity toward Egt1-type, i.e. reaction of EC 1.21.3.10
D52L/A420Y
mutation tunes EgtB activity toward Egt1-type
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Seebeck, F.P.
In vitro reconstitution of Mycobacterial ergothioneine biosynthesis
J. Am. Chem. Soc.
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6632-6633
2010
Mycolicibacterium smegmatis (A0R5N0)
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Goncharenko, K.V.; Vit, A.; Blankenfeldt, W.; Seebeck, F.P.
Structure of the sulfoxide synthase EgtB from the ergothioneine biosynthetic pathway
Angew. Chem. Int. Ed. Engl.
54
2821-2824
2015
Mycolicibacterium thermoresistibile (G7CFI3), Mycolicibacterium thermoresistibile
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Faponle, A.S.; Seebeck, F.P.; de Visser, S.P.
Sulfoxide synthase versus cysteine dioxygenase reactivity in a nonheme iron enzyme
J. Am. Chem. Soc.
139
9259-9270
2017
Mycolicibacterium thermoresistibile (G7CFI3), Mycolicibacterium thermoresistibile ATCC 19527 (G7CFI3)
brenda
Hu, W.; Song, H.; Sae Her, A.; Bak, D.W.; Naowarojna, N.; Elliott, S.J.; Qin, L.; Chen, X.; Liu, P.
Bioinformatic and biochemical characterizations of C-S bond formation and cleavage enzymes in the fungus Neurospora crassa ergothioneine biosynthetic pathway
Org. Lett.
16
5382-5385
2014
Mycolicibacterium smegmatis (A0R5N0), Mycolicibacterium smegmatis ATCC 700084 (A0R5N0)
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Pluskal, T.; Ueno, M.; Yanagida, M.
Genetic and metabolomic dissection of the ergothioneine and selenoneine biosynthetic pathway in the fission yeast, S. pombe, and construction of an overproduction system
PLoS ONE
9
e97774
2014
Mycolicibacterium smegmatis, no activity in Neurospora crassa
brenda
Goncharenko, K.V.; Seebeck, F.P.
Conversion of a non-heme iron-dependent sulfoxide synthase into a thiol dioxygenase by a single point mutation
Chem. Commun. (Camb.)
52
1945-1948
2016
Mycolicibacterium thermoresistibile (G7CFI3), Mycolicibacterium thermoresistibile
brenda
Liao, C.; Seebeck, F.P.
Convergent evolution of ergothioneine biosynthesis in cyanobacteria
ChemBioChem
18
2115-2118
2017
Mycolicibacterium thermoresistibile (G7CFI3)
brenda
Wei, W.; Siegbahn, P.; Liao, R.
Theoretical study of the mechanism of the nonheme iron enzyme EgtB
Inorg. Chem.
56
3589-3599
2017
Mycolicibacterium thermoresistibile (G7CFI3), Mycolicibacterium thermoresistibile ATCC 19527 (G7CFI3)
brenda
Peck, S.C.; van der Donk, W.A.
Go it alone four-electron oxidations by mononuclear non-heme iron enzymes
J. Biol. Inorg. Chem.
22
381-394
2017
Mycobacterium avium
brenda
Naowarojna, N.; Irani, S.; Hu, W.; Cheng, R.; Zhang, L.; Li, X.; Chen, J.; Zhang, Y.J.; Liu, P.
Crystal structure of the ergothioneine sulfoxide synthase from Candidatus Chloracidobacterium thermophilum and structure-guided engineering to modulate its substrate selectivity
ACS Catal.
9
6955-6961
2019
Chloracidobacterium thermophilum (G2LET6), Chloracidobacterium thermophilum
brenda
Tian, G.; Su, H.; Liu, Y.
Mechanism of sulfoxidation and C-S bond formation involved in the biosynthesis of ergothioneine catalyzed by ergothioneine synthase (EgtB)
ACS Catal.
8
5875-5889
2018
Mycolicibacterium thermoresistibile (G7CFI3), Mycolicibacterium thermoresistibile ATCC 19527 (G7CFI3)
-
brenda
Stampfli, A.R.; Goncharenko, K.V.; Meury, M.; Dubey, B.N.; Schirmer, T.; Seebeck, F.P.
An alternative active site architecture for O2 activation in the ergothioneine biosynthetic EgtB from Chloracidobacterium thermophilum
J. Am. Chem. Soc.
141
5275-5285
2019
Chloracidobacterium thermophilum (G2LET6)
brenda
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