Amino acid dehydrogenases reaction scheme

A variation on the transamination approach that also starts with an a-keto acid substrate is to perform a reductive amination catalyzed by amino acid dehydrogenases (dHs) (Scheme 9.31) in combination with the formate dH cofactor recycling system, although other reducing systems can be used. " The generation of carbon dioxide from formate drives the coupled reactions to completion. 

In an original application, Yasuda et al have used both l-AAO and d-AAO, and L-lysine oxidase to oxidize o ,Ci -diamino acids. The reactions produce the expected a-keto w-amino acid products, but these then spontaneously cyclize to form cyclic a-imino acids. These compounds are then substrates for the authors recently discovered A methyl amino acid dehydrogenase (NMAADH) from Pseudomonas putida, producing the pure L-cyclic amino acid (Scheme 5). 

The only preparative-scale reactions of synthetic value in this category are those catalyzed by the amino acid dehydrogenases. These enzymes catalyze the reductions of a-imino acids to a-amino acids. This can be done on a very large scale, as demonstrated by the LeuDH-catalyzed reduction of (120) to f-butyl leucine (121) shown in Scheme 59. Another enzyme of this group with preparative promise is... 

The (reversible) transformation of an a-ketocarboxyhc acid in presence of ammonia and one equivalent of NAD(P)H furnishes the corresponding a-amino acid and is catalyzed by amino acid dehydrogenases [EC 1.4.1.X] [962]. Despite major differences in its mechanism, this reaction bears a strong resemblance to carbonyl group reduction and it formally respresents a reductive amination (Scheme 2.133). As deduced for L-Leu-dehydrogenase [963], the a-ketoacid substrate is positioned in the active site between two Lys-residues. Nucleophihc attack by NH3 leads to a hemiaminal intermediate, which eliminates H2O to form an iminium species. The latter is reduced by a hydride from nicotinamide forming the L-amino acid. Since this mechanism is highly tuned for a-keto/a-amino acids, it is clear that a neutral Schiff base cannot be accepted as substrate. 

More recently, the focus has been put on formal nucleophilic substitution of —OH or —NH2 groups. To perform this biocatalytic variant of the Mitsunobu reaction, an oxidation-nucleophilic addition-reduction sequence is necessary, for which linked NAD-dependent oxidoreductases are ideally suited. The early contributions from the Forschungszentrum Jiilich [79] have been recently rediscovered by Kroutil and coworkers [80]. By combining a mandelate racemase (MR) with a mandelate dehydrogenase and an L-amino acid dehydrogenase, the authors could completely transform racemic mandelic acid into enantiopure (S)-phenyl-glycine (Scheme 8.16). 

Scheme 8.16 Formal Mitsunobu reaction by combining an alcohol dehydrogenase (here mandelate dehydrogenase, D-MDH) with an amino acid dehydrogenase (here l-AADH).

Scheme 11.12 Synthesis of enantiopure amino acids by cascade reactions, including amino acid dehydrogenase-catalyzed reactions.

Scheme 11.13 Deracemization of racemic amino acids by cascade reactions, including DAAOs-catalyzed reactions and reductive amination reactions catalyzed either by amino acid dehydrogenases (a) or transaminases (b).

In a similar exercise with D-methionine, Findrik and Vasic-Racki used the D-AAO of Arthrobacter, and for the second-step conversion of oxoacid into L-amino acid, used L-phenylalanine dehydrogenase (L-PheDH), which has a sufficiently broad specificity to accept L-methionine and its corresponding oxoacid as substrates. Efficient quantitative conversion in this latter reaction requires recycling of the cofactor NAD into NADH, and for this the commercially available formate dehydrogenase (FDH) was used (Scheme 2). 

Lactate dehydrogenase is a pyridine nucleotide oxidoreductase, a tetramer of 140 kD molecular weight, which has been extensively investigated (Bloxham et al., 1975 Eventoff et al., 1977). It catalyses the reversible oxidation of L-lactate to pyruvate using NAD+ as a coenzyme. The reaction scheme with a view of the active site with bound substrate and essential amino-acid side chains are depicted in Equation (3) and in Figure 17. The probable reaction mechanism, involving proton and hydride transfers,... 

The reductive half-reaction of methylamine dehydrogenase is shown in Scheme 10. The methylamine substrate initiates a nucleophilic attack on the quinone carbon at the C6 position of the TTQ cofactor displacing the oxygen to form a substrate-TTQ Schiff base adduct (29). The reactivity of the C6 position was demonstrated by covalent adduct formation at this position by hydrazines which are inactivators of methylamine dehydrogenase. Deprotonation of the substrate-derived carbon of 29 by an active-site amino acid residue results in reduction of the cofactor and yields an intermediate in which the Schiff base is now between the nitrogen and substrate-derived carbon (30). Hydrolysis of 30 releases the formaldehyde product and yields the aminoquinol form of the cofactor with the substrate-derived amino group still covalently bound (31). 

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