Application | Comment | Organism |
---|---|---|
synthesis | synthesis of analogues of L-phenylalanine, that are incorporated as pharmacophores in several peptidomimetic drug molecules and are therefore of particular interest to the fine chemical industry. Engineering of phenylalanine ammonia lyase from Rhodotorula graminis and identification of variants with very high levels of activity towards a panel of substituted cinnamic acids including; 4-bromo-, 3-bromo-, 4-fluoro-, 3-fluoro- and 3-nitro-cinnamic acid. Optimisation studies for use of one of these variants in the preparative synthesis of related variants of L-phenylalanine. Identification of variants used in a preparative scale biotransformation resulting in a 94% conversion to L-4-Br-phenylalanine (more than 99% enantiomeric excess) | Rhodotorula graminis |
Cloned (Comment) | Organism |
---|---|
expression in Escherichia coli | Rhodotorula graminis |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Rhodotorula graminis | A0A194SF92 | - |
- |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
(2E)-3-bromo-cinnamic acid + NH3 | 112% compared to activity with trans-cinnamate | Rhodotorula graminis | 3-bromo-L-phenylalanine | - |
? | |
(2E)-3-fluoro-cinnamic acid + NH3 | 128% compared to activity with trans-cinnamate | Rhodotorula graminis | 3-fluoro-L-phenylalanine | - |
? | |
(2E)-3-nitro-cinnamic acid + NH3 | 54% compared to activity with trans-cinnamate | Rhodotorula graminis | 4-nitro-L-phenylalanine | - |
? | |
(2E)-4-bromo-cinnamic acid + NH3 | 17% compared to activity with trans-cinnamate | Rhodotorula graminis | 4-bromo-L-phenylalanine | - |
? | |
(2E)-4-fluoro-cinnamic acid + NH3 | 109% compared to activity with trans-cinnamate | Rhodotorula graminis | 4-fluoro-L-phenylalanine | - |
? | |
L-phenylalanine | - |
Rhodotorula graminis | trans-cinnamate + NH3 | - |
r | |
trans-cinnamate + NH3 | - |
Rhodotorula graminis | L-phenylalanine | - |
r |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.33 | - |
(2E)-4-bromo-cinnamic acid | pH 9.0, 30°C, wild-type enzyme | Rhodotorula graminis | |
0.78 | - |
(2E)-3-nitro-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143N/Q144S | Rhodotorula graminis | |
1.06 | - |
(2E)-3-nitro-cinnamic acid | pH 9.0, 30°C, wild-type enzyme | Rhodotorula graminis | |
1.31 | - |
(2E)-3-nitro-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143S/Q144N | Rhodotorula graminis | |
1.97 | - |
trans-cinnamate | pH 9.0, 30°C, wild-type enzyme | Rhodotorula graminis | |
2.14 | - |
(2E)-4-fluoro-cinnamic acid | pH 9.0, 30°C, wild-type enzyme | Rhodotorula graminis | |
2.22 | - |
(2E)-3-bromo-cinnamic acid | pH 9.0, 30°C, wild-type enzyme | Rhodotorula graminis | |
2.52 | - |
(2E)-3-fluoro-cinnamic acid | pH 9.0, 30°C, wild-type enzyme | Rhodotorula graminis | |
2.53 | - |
(2E)-4-bromo-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143N/Q144S | Rhodotorula graminis | |
2.64 | - |
trans-cinnamate | pH 9.0, 30°C, mutant enzyme H143C/Q144N | Rhodotorula graminis | |
2.71 | - |
(2E)-3-nitro-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143N/Q144C | Rhodotorula graminis | |
2.76 | - |
(2E)-3-nitro-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143C/Q144N | Rhodotorula graminis | |
2.97 | - |
(2E)-3-bromo-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143C/Q144N | Rhodotorula graminis | |
3.12 | - |
(2E)-3-bromo-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143S/Q144N | Rhodotorula graminis | |
3.46 | - |
trans-cinnamate | pH 9.0, 30°C, mutant enzyme H143N/Q144C | Rhodotorula graminis | |
4.66 | - |
(2E)-3-fluoro-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143S/Q144N | Rhodotorula graminis | |
4.83 | - |
(2E)-4-bromo-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143S/Q144N | Rhodotorula graminis | |
5.43 | - |
(2E)-3-fluoro-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143N/Q144C | Rhodotorula graminis | |
5.99 | - |
(2E)-4-bromo-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143N/Q144C | Rhodotorula graminis | |
6.7 | - |
(2E)-3-bromo-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143N/Q144C | Rhodotorula graminis | |
6.7 | - |
(2E)-3-fluoro-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143N/Q144S | Rhodotorula graminis | |
8.63 | - |
(2E)-3-fluoro-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143C/Q144N | Rhodotorula graminis | |
9.26 | - |
(2E)-4-bromo-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143C/Q144N | Rhodotorula graminis |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.22 | - |
(2E)-4-bromo-cinnamic acid | pH 9.0, 30°C, wild-type enzyme | Rhodotorula graminis | |
0.53 | - |
(2E)-3-nitro-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143N/Q144S | Rhodotorula graminis | |
0.55 | - |
(2E)-3-nitro-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143S/Q144N | Rhodotorula graminis | |
0.89 | - |
(2E)-3-bromo-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143S/Q144N | Rhodotorula graminis | |
1.24 | - |
(2E)-3-nitro-cinnamic acid | pH 9.0, 30°C, wild-type enzyme | Rhodotorula graminis | |
1.26 | - |
(2E)-4-fluoro-cinnamic acid | pH 9.0, 30°C, wild-type enzyme | Rhodotorula graminis | |
1.27 | - |
trans-cinnamate | pH 9.0, 30°C, mutant enzyme H143N/Q144C | Rhodotorula graminis | |
1.55 | - |
trans-cinnamate | pH 9.0, 30°C, mutant enzyme H143C/Q144N | Rhodotorula graminis | |
1.79 | - |
(2E)-3-fluoro-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143N/Q144S | Rhodotorula graminis | |
2.17 | - |
(2E)-3-nitro-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143N/Q144C | Rhodotorula graminis | |
2.72 | - |
(2E)-3-bromo-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143C/Q144N | Rhodotorula graminis | |
2.75 | - |
trans-cinnamate | pH 9.0, 30°C, wild-type enzyme | Rhodotorula graminis | |
2.81 | - |
(2E)-3-fluoro-cinnamic acid | pH 9.0, 30°C, wild-type enzyme | Rhodotorula graminis | |
3.38 | - |
(2E)-3-fluoro-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143S/Q144N | Rhodotorula graminis | |
3.45 | - |
(2E)-3-bromo-cinnamic acid | pH 9.0, 30°C, wild-type enzyme | Rhodotorula graminis | |
4.19 | - |
(2E)-3-nitro-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143C/Q144N | Rhodotorula graminis | |
4.29 | - |
(2E)-3-fluoro-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143N/Q144C | Rhodotorula graminis | |
5.04 | - |
(2E)-3-bromo-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143N/Q144C | Rhodotorula graminis | |
5.76 | - |
(2E)-4-bromo-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143N/Q144S | Rhodotorula graminis | |
7.2 | - |
(2E)-4-bromo-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143S/Q144N | Rhodotorula graminis | |
8.68 | - |
(2E)-3-fluoro-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143C/Q144N | Rhodotorula graminis | |
14.97 | - |
(2E)-4-bromo-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143C/Q144N | Rhodotorula graminis | |
29.38 | - |
(2E)-4-bromo-cinnamic acid | pH 9.0, 30°C, mutant enzyme H143N/Q144C | Rhodotorula graminis |