unknown to date,
30 artificial amine dehydrogenases have been created by semirational protein design using existing α-amino acid dehydrogenase scaffolds. In a
pioneering study, L-LeuDH from Bacillus stearothermophilus was chosen as
starting point for directed evolution [1077]. A total of 19 amino acid residues was
selected for mutagenesis including combinatorial active-site saturation (CAST).
After several rounds of directed evolution, a quadruple variant (K68S, E114V,
N261L, V291C) was obtained, which showed a reasonable specific activity of
0.69 U/mg in the reductive amination of 4-methyl-2-pentanone forming the
corresponding (R)-amine in 99.8% e.e. Not surprisingly, two of the mutations
introduced (K68S, N261L) involved those in binding of the natural substrate’s
carboxylate group. Their replacement by more unpolar amino acid residues
completely abolished the restriction to α-amino/α-ketocarboxylic acids. This strategy was later successfully extended to L-phenylalanine dehydrogenase [1078].
2.2.5 Reduction of C¼C-Bonds
The asymmetric (bio)catalytic reduction of C¼C-bonds goes in hand with the
creation of (up to) two chiral centers and is thus one of the most widely employed
strategies for the production of chiral compounds. Whereas cis-hydrogenation
using transition-metal based homogeneous catalysts has been developed to an
impressive standard [1079], stereocomplementary asymmetric trans-hydrogenation
is less sophisticated [1080].
The biocatalytic counterpart for the stereoselective reduction of alkenes is
catalyzed by flavin-dependent ene-reductases [EC 1.3.1.31], which are members
of the ‘old yellow enzyme’ family (OYE, Scheme 2.132) [1081, 1082], first
described in the 1930s by O. Warburg, who first demonstrated the requirement of
a low molecular weight ‘cofactor’ for enzymatic catalysis [1083]. These enzymes
are widely distributed in microorganisms and in plants. Some of them occur in welldefined pathways, e.g., in the biosynthesis of secondary metabolites, such as
morphine [1084] and jasmonic acid [1085]. Others are involved in the detoxification of xenobiotics [1086], such as nitro esters [1087] and nitro-aromatics [1088]
like trinitrotoluene (TNT) [1089]. Ene-reductases should not be confused with
‘enoate reductases’,
31 which contain an Fe 4 S 4 -cluster in addition to flavin. These
enzymes are found in strict anaerobic organisms and are very sensitive towards
molecular oxygen, which makes them of limited use for preparative biotransformations [1090, 1091].
The catalytic mechanism of the asymmetric reduction of alkenes catalyzed by
ene-reductases from the old yellow enzyme family has been studied in great detail
30 For a rare exception see [1076].
31 More precisely 2-enoate reductase. Since these enzymes belong to the same EC class 1.3.1.X,
ene-reductases and enoate reductases are often confused.
2.2 Reduction Reactions
161
30 artificial amine dehydrogenases have been created by semirational protein design using existing α-amino acid dehydrogenase scaffolds. In a
pioneering study, L-LeuDH from Bacillus stearothermophilus was chosen as
starting point for directed evolution [1077]. A total of 19 amino acid residues was
selected for mutagenesis including combinatorial active-site saturation (CAST).
After several rounds of directed evolution, a quadruple variant (K68S, E114V,
N261L, V291C) was obtained, which showed a reasonable specific activity of
0.69 U/mg in the reductive amination of 4-methyl-2-pentanone forming the
corresponding (R)-amine in 99.8% e.e. Not surprisingly, two of the mutations
introduced (K68S, N261L) involved those in binding of the natural substrate’s
carboxylate group. Their replacement by more unpolar amino acid residues
completely abolished the restriction to α-amino/α-ketocarboxylic acids. This strategy was later successfully extended to L-phenylalanine dehydrogenase [1078].
2.2.5 Reduction of C¼C-Bonds
The asymmetric (bio)catalytic reduction of C¼C-bonds goes in hand with the
creation of (up to) two chiral centers and is thus one of the most widely employed
strategies for the production of chiral compounds. Whereas cis-hydrogenation
using transition-metal based homogeneous catalysts has been developed to an
impressive standard [1079], stereocomplementary asymmetric trans-hydrogenation
is less sophisticated [1080].
The biocatalytic counterpart for the stereoselective reduction of alkenes is
catalyzed by flavin-dependent ene-reductases [EC 1.3.1.31], which are members
of the ‘old yellow enzyme’ family (OYE, Scheme 2.132) [1081, 1082], first
described in the 1930s by O. Warburg, who first demonstrated the requirement of
a low molecular weight ‘cofactor’ for enzymatic catalysis [1083]. These enzymes
are widely distributed in microorganisms and in plants. Some of them occur in welldefined pathways, e.g., in the biosynthesis of secondary metabolites, such as
morphine [1084] and jasmonic acid [1085]. Others are involved in the detoxification of xenobiotics [1086], such as nitro esters [1087] and nitro-aromatics [1088]
like trinitrotoluene (TNT) [1089]. Ene-reductases should not be confused with
‘enoate reductases’,
31 which contain an Fe 4 S 4 -cluster in addition to flavin. These
enzymes are found in strict anaerobic organisms and are very sensitive towards
molecular oxygen, which makes them of limited use for preparative biotransformations [1090, 1091].
The catalytic mechanism of the asymmetric reduction of alkenes catalyzed by
ene-reductases from the old yellow enzyme family has been studied in great detail
30 For a rare exception see [1076].
31 More precisely 2-enoate reductase. Since these enzymes belong to the same EC class 1.3.1.X,
ene-reductases and enoate reductases are often confused.
2.2 Reduction Reactions
161