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Literature summary for 4.3.1.24 extracted from

  • Rowles, I.; Groenendaal, B.; Binay, B.; Malone, K.; Willies, S.; Turner, N.
    Engineering of phenylalanine ammonia lyase from Rhodotorula graminis for the enhanced synthesis of unnatural L-amino acids (2016), Tetrahedron, 72, 7343-7347 .
No PubMed abstract available
Search for more literature for 4.3.1.24

Application

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(Commentary)

Cloned (Comment) Organism
expression in Escherichia coli Rhodotorula graminis

Organism

Organism UniProt Comment Textmining
Rhodotorula graminis A0A194SF92
-
-

Substrates and Products (Substrate)

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 [1/s]

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 [mM/s]

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