L-amino acid. In contrast to imine reductases, amino acid dehydrogenases catalyse
both imine formation and C¼N bond reduction. Since this mechanism is highly
tuned for α-keto/α-amino acids, it is clear that only ammonia is accepted as amine
donor and a neutral imine (Schiff base) lacking the carboxylate moiety on the imine
carbon atom cannot be accepted as substrate. Due to the importance of α-amino
acids, both D- and L-amino acid dehydrogenases are important enzymes in industrial
processes.
Among the various amino acid dehydrogenases, Leu-DH has captured an important role for the synthesis of nonproteinogenic L-α-amino acids via asymmetric
reductive amination of the corresponding α-ketoacids [1073, 1074]. A range
of protease inhibitors used for the treatment of tumors and viral infections
contain sterically hindered amino acids as key element for their biological action.
The latter cannot be synthetized via the conventional (protease-dependent) methods
(Sect. 2.1.2), but they are produced on industrial-scale making use of the relaxed
substrate specificity of LeuDH in combination with NADH recycling using the
formate dehydrogenase/formate system [1075]. In particular, L-t-leucine is a key
intermediate for the synthesis of the HIV protease inhibitor Atazanavir.
In analogy to α-amino acid dehydrogenases, nicotinamide-depending enzymes
for the reductive amination of ketones lacking the carboxylate moiety would be
termed ‘amine dehydrogenases’. Since naturally occurring enzymes of this type are
NH 2
CO 2 H
R
O
CO 2 H
R
D or L
NAD(P)
+
NAD(P)H
Amino acid dehydrogenase
H 2 O
NH 3
O
L-Leu DH (mutant)
H 2 O
NH 3
NH 2
R
93% conv.,
99.8 % e.e.
HO
NH 2
CO 2 H
R
O
CO 2 H
R
Lys
R
CO 2
NH 3
NH 3
H
NH 2
Lys
Lys NH 3
NH 2
Lys
NH 2
CO 2
R
Lys
R
CO 2
NH 3
NH 2
HO
NH 3
Lys
Lys NH 3
NH 3
Lys
O
CO 2
R
( )n
n = 0, 1, 2
Sterically hindered non-natural substrates:
L
+H +
+NAD(P)H
+NH 3
[H
- ]
H 2 O
NH 3
L-Leu DH
NADH
NAD +
NADH (recycling)
H 2 O
NH 3
Scheme 2.131 Reductive amination of α-ketocarboxylic acids using D- and L-amino acid dehydrogenases (top); reductive amination of a methyl ketone using a L-leucine dehydrogenase mutant
(center); mechanism of L-leucine dehydrogenase
160
2 Biocatalytic Applications
both imine formation and C¼N bond reduction. Since this mechanism is highly
tuned for α-keto/α-amino acids, it is clear that only ammonia is accepted as amine
donor and a neutral imine (Schiff base) lacking the carboxylate moiety on the imine
carbon atom cannot be accepted as substrate. Due to the importance of α-amino
acids, both D- and L-amino acid dehydrogenases are important enzymes in industrial
processes.
Among the various amino acid dehydrogenases, Leu-DH has captured an important role for the synthesis of nonproteinogenic L-α-amino acids via asymmetric
reductive amination of the corresponding α-ketoacids [1073, 1074]. A range
of protease inhibitors used for the treatment of tumors and viral infections
contain sterically hindered amino acids as key element for their biological action.
The latter cannot be synthetized via the conventional (protease-dependent) methods
(Sect. 2.1.2), but they are produced on industrial-scale making use of the relaxed
substrate specificity of LeuDH in combination with NADH recycling using the
formate dehydrogenase/formate system [1075]. In particular, L-t-leucine is a key
intermediate for the synthesis of the HIV protease inhibitor Atazanavir.
In analogy to α-amino acid dehydrogenases, nicotinamide-depending enzymes
for the reductive amination of ketones lacking the carboxylate moiety would be
termed ‘amine dehydrogenases’. Since naturally occurring enzymes of this type are
NH 2
CO 2 H
R
O
CO 2 H
R
D or L
NAD(P)
+
NAD(P)H
Amino acid dehydrogenase
H 2 O
NH 3
O
L-Leu DH (mutant)
H 2 O
NH 3
NH 2
R
93% conv.,
99.8 % e.e.
HO
NH 2
CO 2 H
R
O
CO 2 H
R
Lys
R
CO 2
NH 3
NH 3
H
NH 2
Lys
Lys NH 3
NH 2
Lys
NH 2
CO 2
R
Lys
R
CO 2
NH 3
NH 2
HO
NH 3
Lys
Lys NH 3
NH 3
Lys
O
CO 2
R
( )n
n = 0, 1, 2
Sterically hindered non-natural substrates:
L
+H +
+NAD(P)H
+NH 3
[H
- ]
H 2 O
NH 3
L-Leu DH
NADH
NAD +
NADH (recycling)
H 2 O
NH 3
Scheme 2.131 Reductive amination of α-ketocarboxylic acids using D- and L-amino acid dehydrogenases (top); reductive amination of a methyl ketone using a L-leucine dehydrogenase mutant
(center); mechanism of L-leucine dehydrogenase
160
2 Biocatalytic Applications