EC Number | Crystallization (Comment) | Organism |
---|---|---|
4.2.1.20 | wild-type in complex with L-threonine, to 1.54 A. L-threonine binds non-covalently, no formation of an electrophilic amino-acrylate intermediate. With mutant I16V/E17G/I68V/F95L/F274S/T292S/T321A/V384A, the species is formed | Pyrococcus furiosus |
EC Number | Protein Variants | Comment | Organism |
---|---|---|---|
4.2.1.20 | I16V/E17G/I68V/F95L/F274S/T292S/T321A/V384A | significant increase in activity with L-threonine | Pyrococcus furiosus |
4.2.1.122 | I16V/E17G/Q89L/F95L/T292S/V384A | site-directed mutagenesis, the mutant PfTrpB2B9 exhibits a 6000fold higher activity with L-Thr compared to wild-type | Pyrococcus furiosus |
4.2.1.122 | I16V/Q89L | site-directed mutagenesis | Pyrococcus furiosus |
4.2.1.122 | additional information | Pyrococcus furiosus enzyme engineering by directed evolution of selected TrpB mutant PfTrpB4D11 results in an modified improved enzyme, L-threonine may substitute for L-serine in the beta-substitution reaction of the engineered subunit of tryptophan synthase yielding (2S,3S)-beta-methyltryptophan (beta-MeTrp) in a single step. The trace activity of the wild-type beta-subunit on this substrate is enhanced more than 1000fold by directed evolution. Structural and spectroscopic data indicate that this increase is correlated with stabilization of the electrophilic aminoacrylate intermediate. The engineered biocatalyst also reacts with a variety of indole analogues and thiophenol for diastereoselective C-C, C-N, and C-S bond forming reactions. The new activity circumvents the 3-enzyme pathway that produces beta-MeTrp in nature and offers a simple and expandable route to preparing derivatives of this valuable building block | Pyrococcus furiosus |
4.2.1.122 | T292S | site-directed mutagenesis, the mutant exhibits a 6fold higher activity with L-Thr compared to wild-type | Pyrococcus furiosus |
4.2.1.122 | T292S/E17G/I68V/F274S/T321A | site-directed mutagenesis, the mutant exhibits a 660old higher activity with L-Thr compared to wild-type | Pyrococcus furiosus |
EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
4.2.1.122 | L-serine + indole | Pyrococcus furiosus | - |
L-tryptophan + H2O | - |
? | |
4.2.1.122 | L-threonine + indole | Pyrococcus furiosus | - |
(2S,3S)-beta-methyltryptophan + H2O | - |
? |
EC Number | Organism | UniProt | Comment | Textmining |
---|---|---|---|---|
4.2.1.20 | Pyrococcus furiosus | Q8U093 | beta chain 1 | - |
4.2.1.122 | Pyrococcus furiosus | - |
- |
- |
EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
4.2.1.20 | L-threonine + 2-methylindole | - |
Pyrococcus furiosus | 2-methyl-(2S,3S)-beta-methyltryptophan + H2O | - |
? | |
4.2.1.20 | L-threonine + 4-fluoroindole | reaction of mutant I16V/E17G/I68V/F95L/F274S/T292S/T321A/V384A | Pyrococcus furiosus | 4-fluoro-(2S,3S)-beta-methyltryptophan + H2O | - |
? | |
4.2.1.20 | L-threonine + 5-fluoroindole | reaction of mutant I16V/E17G/I68V/F95L/F274S/T292S/T321A/V384A | Pyrococcus furiosus | 5-fluoro-(2S,3S)-beta-methyltryptophan + H2O | - |
? | |
4.2.1.20 | L-threonine + 6-methylindole | reaction of mutant I16V/E17G/I68V/F95L/F274S/T292S/T321A/V384A | Pyrococcus furiosus | 6-methyl-(2S,3S)-beta-methyltryptophan + H2O | - |
? | |
4.2.1.20 | L-threonine + 7-azaindole | reaction of mutant I16V/E17G/I68V/F95L/F274S/T292S/T321A/V384A | Pyrococcus furiosus | 7-aza-(2S,3S)-beta-methyltryptophan + H2O | - |
? | |
4.2.1.20 | L-threonine + indole | - |
Pyrococcus furiosus | (2S,3S)-beta-methyltryptophan + H2O | - |
? | |
4.2.1.122 | L-serine + indole | - |
Pyrococcus furiosus | L-tryptophan + H2O | - |
? | |
4.2.1.122 | L-serine + indole | L-serine is the preferred substrate | Pyrococcus furiosus | L-tryptophan + H2O | - |
? | |
4.2.1.122 | L-threonine + 7-azaindole | engineered mutant enzyme PfTrpB2B9 | Pyrococcus furiosus | (2S,3S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)butanoic acid + H2O | - |
? | |
4.2.1.122 | L-threonine + indole | - |
Pyrococcus furiosus | (2S,3S)-beta-methyltryptophan + H2O | - |
? | |
4.2.1.122 | L-threonine + indole | veryl low activity of the wild-type enzyme, higher activity with engineered mutant enzymes | Pyrococcus furiosus | (2S,3S)-beta-methyltryptophan + H2O | - |
? | |
4.2.1.122 | additional information | no activity of engineered mutant enzyme with 5-chloro-, 5-bromo, and 6-hydroxyindoles combined with L-threonine | Pyrococcus furiosus | ? | - |
? |
EC Number | Synonyms | Comment | Organism |
---|---|---|---|
4.2.1.122 | PfTrpB | - |
Pyrococcus furiosus |