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  • 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 (2014), Org. Lett., 16, 5382-5385 .
    View publication on PubMedView publication on EuropePMC

Application

EC Number Application Comment Organism
1.14.99.50 synthesis the ergothioneine biosynthetic pathway (EgtA-EgtE catalysis) provides an opportunity for ergothioneine production through metabolic engineering Mycolicibacterium smegmatis
1.14.99.52 synthesis the ergothioneine biosynthetic pathway (EgtA-EgtE catalysis) provides an opportunity for ergothioneine production through metabolic engineering, but the kinetic properties and the presence of side-reaction render OvoA not suitable for use in ergothioneine production through metabolic engineering Erwinia tasmaniensis
1.21.3.10 synthesis the ergothioneine biosynthetic pathway (EgtA-EgtE catalysis) provides an opportunity for ergothioneine production through metabolic engineering Neurospora crassa
4.4.1.36 synthesis the ergothioneine biosynthetic pathway (EgtA-EgtE catalysis) provides an opportunity for ergothioneine production through metabolic engineering Neurospora crassa

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.14.99.50 contruction of an EgtB homologue sequence similarity network using a BLAST e value cutoff Mycolicibacterium smegmatis
1.21.3.10 gene egt1, Egt1 recombinant overexpression from vector pASK-IBA3+ in Escherichia coli Neurospora crassa
4.4.1.36 expression in Escherichia coli Neurospora crassa
4.4.1.36 gene NCU11365, genome mining, cloning from a sequence determined by screening of an EgtB homologue sequence similarity network from Mycobacterum smegmatis EgtB, recombinant expression of enzyme EgtE in Escherichia coli strain BL21(DE3) Neurospora crassa

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.21.3.10 0.39
-
 hercynine pH and temperature not specified in the publication, with gamma-L-glutamyl-L-cysteine Neurospora crassa
1.21.3.10 0.436
-
 hercynine pH and temperature not specified in the publication, with L-Cys Neurospora crassa
1.21.3.10 0.603
-
 L-cysteine pH and temperature not specified in the publication Neurospora crassa
1.21.3.10 7.68
-
 gamma-L-glutamyl-L-cysteine pH and temperature not specified in the publication Neurospora crassa
4.4.1.36 0.1947
-
 S-(hercyn-2-yl)-L-cysteine S-oxide pH and temperature not specified in the publication Neurospora crassa
4.4.1.36 0.195
-
 S-(hercyn-2-yl)-L-cysteine S-oxide pH not specified in the publication, temperature not specified in the publication Neurospora crassa

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
1.14.99.50 Fe2+ non-heme iron, required for catalysis Mycolicibacterium smegmatis
1.14.99.52 Fe2+ non-heme iron, required for catalysis Erwinia tasmaniensis
1.21.3.10 Fe2+ required for catalysis, the enzyme contains about 0.90 Fe per Egt1 monomer Neurospora crassa

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.14.99.50 hercynine + gamma-L-glutamyl-L-cysteine + O2 Mycolicibacterium smegmatis
-
 gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
-
 ?
1.14.99.50 hercynine + gamma-L-glutamyl-L-cysteine + O2 Mycolicibacterium smegmatis ATCC 700084
-
 gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
-
 ?
1.14.99.52 hercynine + L-cysteine + O2 Erwinia tasmaniensis reaction of EC 1.14.99.51 S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
-
 ?
1.14.99.52 L-histidine + L-cysteine + O2 Erwinia tasmaniensis
-
 S-(L-histidin-5-yl)-L-cysteine S-oxide + H2O
-
 ?
1.21.3.10 hercynine + gamma-L-glutamyl-L-cysteine + O2 Neurospora crassa reaction of EC 1.14.99.50, low activity gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
-
 ?
1.21.3.10 hercynine + gamma-L-glutamyl-L-cysteine + O2 Neurospora crassa ATCC 24698 reaction of EC 1.14.99.50, low activity gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
-
 ?
1.21.3.10 hercynine + L-cysteine + O2 Neurospora crassa
-
 S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
-
 ?
1.21.3.10 hercynine + L-cysteine + O2 Neurospora crassa ATCC 24698
-
 S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
-
 ?
4.4.1.36 2-(hydroxysulfanyl)hercynine + reduced acceptor Neurospora crassa
-
 ergothioneine + acceptor + H2O
-
 ?
4.4.1.36 2-(hydroxysulfanyl)hercynine + reduced acceptor Neurospora crassa DSM 1257
-
 ergothioneine + acceptor + H2O
-
 ?
4.4.1.36 2-(hydroxysulfanyl)hercynine + reduced acceptor Neurospora crassa ATCC 24698
-
 ergothioneine + acceptor + H2O
-
 ?
4.4.1.36 S-(hercyn-2-yl)-L-cysteine S-oxide + H2O Neurospora crassa
-
 2-(hydroxysulfanyl)hercynine + pyruvate + ammonia
-
 ?
4.4.1.36 S-(hercyn-2-yl)-L-cysteine S-oxide + H2O Neurospora crassa DSM 1257
-
 2-(hydroxysulfanyl)hercynine + pyruvate + ammonia
-
 ?
4.4.1.36 S-(hercyn-2-yl)-L-cysteine S-oxide + H2O Neurospora crassa ATCC 24698
-
 2-(hydroxysulfanyl)hercynine + pyruvate + ammonia
-
 ?
4.4.1.36 S-(hercyn-2-yl)-L-cysteine S-oxide + reduced acceptor Neurospora crassa
-
 ergothioneine + pyruvate + ammonia + acceptor
-
 ?
4.4.1.36 S-(hercyn-2-yl)-L-cysteine S-oxide + reduced acceptor Neurospora crassa overall reaction ergothioneine + pyruvate + ammonia + acceptor
-
 ?
4.4.1.36 S-(hercyn-2-yl)-L-cysteine S-oxide + reduced acceptor Neurospora crassa DSM 1257
-
 ergothioneine + pyruvate + ammonia + acceptor
-
 ?
4.4.1.36 S-(hercyn-2-yl)-L-cysteine S-oxide + reduced acceptor Neurospora crassa DSM 1257 overall reaction ergothioneine + pyruvate + ammonia + acceptor
-
 ?
4.4.1.36 S-(hercyn-2-yl)-L-cysteine S-oxide + reduced acceptor Neurospora crassa ATCC 24698
-
 ergothioneine + pyruvate + ammonia + acceptor
-
 ?
4.4.1.36 S-(hercyn-2-yl)-L-cysteine S-oxide + reduced acceptor Neurospora crassa ATCC 24698 overall reaction ergothioneine + pyruvate + ammonia + acceptor
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.14.99.50 Mycolicibacterium smegmatis A0R5N0
-
-
1.14.99.50 Mycolicibacterium smegmatis ATCC 700084 A0R5N0
-
-
1.14.99.52 Erwinia tasmaniensis
-
-
-
1.21.3.10 Neurospora crassa Q7RX33
-
-
1.21.3.10 Neurospora crassa ATCC 24698 Q7RX33
-
-
4.4.1.36 Neurospora crassa A7UX13
-
-
4.4.1.36 Neurospora crassa ATCC 24698 A7UX13
-
-
4.4.1.36 Neurospora crassa DSM 1257 A7UX13
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
4.4.1.36 recombinant enzyme from Escherichia coli strain BL21(DE3) Neurospora crassa

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.14.99.50 hercynine + gamma-L-glutamyl-L-cysteine + O2
-
 Mycolicibacterium smegmatis gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
-
 ?
1.14.99.50 hercynine + gamma-L-glutamyl-L-cysteine + O2
-
 Mycolicibacterium smegmatis ATCC 700084 gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
-
 ?
1.14.99.50 additional information 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 Mycolicibacterium smegmatis ?
-
 ?
1.14.99.50 additional information 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 Mycolicibacterium smegmatis ATCC 700084 ?
-
 ?
1.14.99.52 hercynine + L-cysteine + O2 reaction of EC 1.14.99.51 Erwinia tasmaniensis S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
-
 ?
1.14.99.52 L-histidine + L-cysteine + O2
-
 Erwinia tasmaniensis S-(L-histidin-5-yl)-L-cysteine S-oxide + H2O
-
 ?
1.14.99.52 additional information when OvoA's native substrate histidine is replaced by hercynine, OvoA can catalyze a one-step transformation reaction of hercynylcysteine S-oxide synthase, EC 1.14.99.51, resulting in kobs changes by 2fold for hercynine, while Km for cysteine increases from 0.3 mM to 3.19 mM. OvoA preferentially uses histidine and cysteine. OvoA also catalyzes the oxidation of cysteine to cysteine sulfinic acid, EC 1.13.11.20. Enzyme activity determination using : a 1H NMR assay of chemical shift of the imidazole hydrogen atoms Erwinia tasmaniensis ?
-
 ?
1.21.3.10 hercynine + gamma-L-glutamyl-L-cysteine + O2 reaction of EC 1.14.99.50, low activity Neurospora crassa gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
-
 ?
1.21.3.10 hercynine + gamma-L-glutamyl-L-cysteine + O2 reaction of EC 1.14.99.50, low activity Neurospora crassa ATCC 24698 gamma-L-glutamyl-S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
-
 ?
1.21.3.10 hercynine + L-cysteine + O2
-
 Neurospora crassa S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
-
 ?
1.21.3.10 hercynine + L-cysteine + O2
-
 Neurospora crassa ATCC 24698 S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
-
 ?
1.21.3.10 additional information three different assay methods for enzyme activity determination: a 1H NMR assay of chemical shift of the imidazole hydrogen atoms, an oxygen consumption assay, and a 13C NMR assay to monitor the presence of other cysteine related oxygen consumption. Egt1 from Neurospora crassa prefers cysteine as substrate compared to gamma-L-glutamyl-L-cysteine, Egt1 has a 62fold greater specificity (kobs/Km) for L-cysteine relative to gamma-Glu-Cys Neurospora crassa ?
-
 ?
1.21.3.10 additional information three different assay methods for enzyme activity determination: a 1H NMR assay of chemical shift of the imidazole hydrogen atoms, an oxygen consumption assay, and a 13C NMR assay to monitor the presence of other cysteine related oxygen consumption. Egt1 from Neurospora crassa prefers cysteine as substrate compared to gamma-L-glutamyl-L-cysteine, Egt1 has a 62fold greater specificity (kobs/Km) for L-cysteine relative to gamma-Glu-Cys Neurospora crassa ATCC 24698 ?
-
 ?
4.4.1.36 2-(hydroxysulfanyl)hercynine + reduced acceptor
-
 Neurospora crassa ergothioneine + acceptor + H2O
-
 ?
4.4.1.36 2-(hydroxysulfanyl)hercynine + reduced acceptor
-
 Neurospora crassa DSM 1257 ergothioneine + acceptor + H2O
-
 ?
4.4.1.36 2-(hydroxysulfanyl)hercynine + reduced acceptor
-
 Neurospora crassa ATCC 24698 ergothioneine + acceptor + H2O
-
 ?
4.4.1.36 additional information enzyme activity determination by a 1H NMR assay of chemical shift of the imidazole hydrogen atoms and a colorimetric assay by coupling the C-S lyase reaction with lactate dehydrogenas, product determination by high-resolution mass spectrometry and NMR Neurospora crassa ?
-
 ?
4.4.1.36 additional information enzyme activity determination by a 1H NMR assay of chemical shift of the imidazole hydrogen atoms and a colorimetric assay by coupling the C-S lyase reaction with lactate dehydrogenas, product determination by high-resolution mass spectrometry and NMR Neurospora crassa DSM 1257 ?
-
 ?
4.4.1.36 additional information enzyme activity determination by a 1H NMR assay of chemical shift of the imidazole hydrogen atoms and a colorimetric assay by coupling the C-S lyase reaction with lactate dehydrogenas, product determination by high-resolution mass spectrometry and NMR Neurospora crassa ATCC 24698 ?
-
 ?
4.4.1.36 S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
-
 Neurospora crassa 2-(hydroxysulfanyl)hercynine + pyruvate + ammonia
-
 ?
4.4.1.36 S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
-
 Neurospora crassa DSM 1257 2-(hydroxysulfanyl)hercynine + pyruvate + ammonia
-
 ?
4.4.1.36 S-(hercyn-2-yl)-L-cysteine S-oxide + H2O
-
 Neurospora crassa ATCC 24698 2-(hydroxysulfanyl)hercynine + pyruvate + ammonia
-
 ?
4.4.1.36 S-(hercyn-2-yl)-L-cysteine S-oxide + reduced acceptor
-
 Neurospora crassa ergothioneine + pyruvate + ammonia + acceptor
-
 ?
4.4.1.36 S-(hercyn-2-yl)-L-cysteine S-oxide + reduced acceptor overall reaction Neurospora crassa ergothioneine + pyruvate + ammonia + acceptor
-
 ?
4.4.1.36 S-(hercyn-2-yl)-L-cysteine S-oxide + reduced acceptor
-
 Neurospora crassa DSM 1257 ergothioneine + pyruvate + ammonia + acceptor
-
 ?
4.4.1.36 S-(hercyn-2-yl)-L-cysteine S-oxide + reduced acceptor overall reaction Neurospora crassa DSM 1257 ergothioneine + pyruvate + ammonia + acceptor
-
 ?
4.4.1.36 S-(hercyn-2-yl)-L-cysteine S-oxide + reduced acceptor
-
 Neurospora crassa ATCC 24698 ergothioneine + pyruvate + ammonia + acceptor
-
 ?
4.4.1.36 S-(hercyn-2-yl)-L-cysteine S-oxide + reduced acceptor overall reaction Neurospora crassa ATCC 24698 ergothioneine + pyruvate + ammonia + acceptor
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.14.99.50 EgtB
-
 Mycolicibacterium smegmatis
1.14.99.50 hercynine oxygenase UniProt Mycolicibacterium smegmatis
1.14.99.52 OvoA
-
 Erwinia tasmaniensis
1.21.3.10 Egt1
-
 Neurospora crassa
4.4.1.36 Egt2
-
 Neurospora crassa
4.4.1.36 egtE
-
 Neurospora crassa
4.4.1.36 hercynylcysteine sulfoxide lyase
-
 Neurospora crassa
4.4.1.36 hercynylcysteine sulfoxide lyase UniProt Neurospora crassa
4.4.1.36 NCU01256 protein
-
 Neurospora crassa
4.4.1.36 NCU11365
-
 Neurospora crassa
4.4.1.36 PLP-dependent C-S lyase
-
 Neurospora crassa

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.21.3.10 0.461
-
 gamma-L-glutamyl-L-cysteine pH and temperature not specified in the publication Neurospora crassa
1.21.3.10 0.461
-
 hercynine pH and temperature not specified in the publication, with gamma-L-glutamyl-L-cysteine Neurospora crassa
1.21.3.10 2.267
-
 L-cysteine pH and temperature not specified in the publication Neurospora crassa
1.21.3.10 2.267
-
 hercynine pH and temperature not specified in the publication, with L-Cys Neurospora crassa

Cofactor

EC Number Cofactor Comment Organism Structure
4.4.1.36 pyridoxal 5'-phosphate
-
 Neurospora crassa

General Information

EC Number General Information Comment Organism
1.14.99.50 evolution OvoA, EC 1.14.99.52, and EgtB are related in sequence, while they are biochemically distinct Mycolicibacterium smegmatis
1.14.99.50 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 Mycolicibacterium smegmatis
1.14.99.52 evolution OvoA and EgtB, EC 1.14.99.50, are related in sequence, while they are biochemically distinct Erwinia tasmaniensis
1.14.99.52 metabolism enzyme OvoA is responsible for the oxidative C-S bond formation in ovothiol biosynthesis. OvoA in ovothiol biosynthesis has a relaxed substrate specificity Erwinia tasmaniensis
1.21.3.10 malfunction Egt1 knockout in Neurospora crassa makes the mutant much more sensitive to oxidative stress compared to wild-type Neurospora crassa
1.21.3.10 metabolism the fungus Neurospora crassa has a concise ergothioneine biosynthetic pathway, its nonheme iron enzyme Egt1 makes use of cysteine instead of gamma-Glu-Cys (like EgtB, EC 1.14.99.50) as the substrate. Such a change of substrate preference eliminates the competition between ergothioneine and glutathione biosyntheses. In Neuropsora crassa ergothioneine biosynthesis, there are reactions distinct from the pathway via EgtB: the oxidative C-S bond formation and the subsequent C-S bond cleavage reaction, which result in a net transfer of the sulfur atom form cysteine to histidine imidazole side-chain Neurospora crassa
4.4.1.36 metabolism the enzyme catalyzes a pyridoxal 5'-phosphate-mediated C-S lyase reaction in the ergothioneine biosynthesis Neurospora crassa