Unnatural amino acids (UAA) play an important role in the production of pharmaceuticals, with an increasingly large pool used as modular building blocks in drug development, broadening their future applications [1]. However, the latter strictly depends on UAA being synthesized at a high purity-enantiopure level prior to the drug formulation process proceedings [2], [3]. Enzymatic methods are widely preferred for cost-effective asymmetric synthesis of UAA. Asymmetric synthesis of L-alanine (L-Ala) derivatives (as the smallest member of the unnatural amino acid family) is invaluable since the derivatives are the basic units for structural modification and novel molecules for medicinal chemistry. In particular, enzymatic reductive amination reaction of NAD+- dependent alanine dehydrogenase (AlaDH) has shown considerable potential for asymmetric synthesis of L-Ala derivatives [4], [5], [6], [7]. In microorganisms, AlaDH assists in redox balancing as its deamination/amination reaction is linked to the reduction/oxidation of NAD+/NADH. Moreover, AlaDH [L-alanine: nicotinamide adenine dinucleotide (NAD)+-oxidoreductase (deaminating) EC 1.4.1.1] is a catalyst for the oxidizing deamination of L-Ala and the reductive amination of oxyacid (Fig. 1).

Numerous L-AlaDH enzymes, mainly from mesophilic organisms, have been identified via purification, and partial kinetic and biochemical characterizations have been performed, although most studies were limited to the analysis of oxidative deamination activity [6], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19]. To date, AlaDH reductive amination activity studies have been limited to the use of pyruvate [19], [20], [21], [22], while a biochemical and computational study that proposes an AlaDH reductive amination activity mechanism model for α-keto acid derivatives of pyruvate has not yet been reported.

The present study investigates, for the first time, a comparative biochemical, computational study of the potential of Thermomicrobium roseum alanine dehydrogenase (TrAlaDH) for reductive amination activity with α-ketobutyrate, α-ketovalerate and α-ketocaproate as pyruvate derivatives. This can be more efficient to synthesize their matching L-alanine derivatives as unnatural amino acids. A computationally developed paradigm for the TrAlaDH catalytic reductive amination mechanism model was also studied in respect to the observed biochemical-kinetic oxidative deamination activity against L-alanine and selected derivatives L-α-aminobutyrate, L-norvaline, and L-norleucine.