Comparison of L-Threonine Aldolase Variants in the Aldol and Retro-Aldol Reactions

Abstract

Most of biochemical and mutagenesis studies performed with L-threonine aldolases were done with respect to natural activity, the cleavage of L-threonine and sometimes L-β-phenylserine. However, the properties of variants and the impact of mutations on the product synthesis are more interesting from an applications point of view. Here we performed site-directed mutagenesis of active site residues of L-threonine aldolase from Aeromonas jandaei to analyze their impact on the retro-aldol activity and on the aldol synthesis of L-β-phenylserine and L-α-alkyl-β-phenylserines. Consequently, reduced retro-aldol activity upon mutation of catalytically important residues led to increased conversions and diastereoselectivities in the synthetic direction. Thus, L-β-phenylserine can be produced with conversions up to 60% and d.e .‘s up to 80% ( syn ) under kinetic control. Furthermorem, the donor specificity of L-threonine aldolase was increased upon mutation of active site residues, which enlarged the pocket size for an efficient binding and stabilization of donor molecules in the active site. This study broadens the knowledge about L-threonine aldolase catalyzed reactions and improves the synthetic protocols for the biocatalytic asymmetric synthesis of unnatural amino acids.

Scalable and Selective β‐Hydroxy‐α‐Amino Acid Synthesis Catalyzed by Promiscuous l‐Threonine Transaldolase ObiH

“…[21] By running these transformations in reverse with very high concentrations of glycine, TAs and SHMTs have been leveraged for the synthesis of β-hydroxy-α-amino acids. [22][23][24][25][26][27] However, most native TAs catalyze aldol reactions with low stereoselectivity at Cβ, leading to poor diastereoselectivity profiles. [25,28,29] These challenges can be overcome in exceptional cases, either through intensive directed evolution or through the use of diastereoselective crystallization, but these routes are limited to one or a handful of substrates.…”

Improving the Cβ Stereoselectivity of l‐Threonine Aldolase for the Synthesis of l‐threo‐4‐Methylsulfonylphenylserine by Modulating the Substrate‐Binding Pocket To Control the Orientation of the Substrate Entrance

“…There are 25 amino residues (Phe12, Ala13, Ser14, Asp15, Asn16, Tyr17, Ala38, Tyr39, Thr91, His93, Val96, Asp97, His136, Glu150, Leu151, Arg177, Arg180, Lys210, Lys235, Ser242, Phe296, Tyr314, Trp316, Arg324, Met326) located within 6 Å surrounded (2 S ,3 R )‐ 1 b (Figure 1A). Among these residues, Lys210 covalently bound with the PLP cofactor in the resting state (referred to as the internal aldimine), and the mutation of Lys210 would result in the inactive of the enzyme [14] . Residues His93 and His136 play a vital role in substrate specificity and retro‐aldol reaction promotion, and their close residues Asp97 and Glu150, were reported for substrate recognition [3b,5] .…”

Directed Evolution of l-Threonine Aldolase for the Diastereoselective Synthesis of β-Hydroxy-α-amino Acids

“…l -Threonine aldolase (LTA) is a 5′-phosphate (PLP)-dependent aldolase that catalyzes the formation of β-hydroxy-α-amino acids, important chiral building blocks for pharmaceuticals such as antibiotics and proteasome inhibitors, from glycine (Gly) and aromatic or aliphatic aldehydes (Scheme ). − The process is attractive for producing chiral products with two chiral centers (C α and C β ) in one step. − However, although LTA has a strict selectivity for C α , the selectivity for C β is moderate, hampering its wide applications in stereoselective synthesis of carbon–carbon bonds. ,− LTA has been engineered for improved C β stereoselectivity in several studies but with limited success, mainly due to an unclear mechanism determining diastereoselectivity and the lack of an appropriate directed evolution strategy. ,− Salvo et al crystallized LTA from Escherichia coli and took rational design to improve diastereoselectivity . Based on the result of docking threonine to the LTA, several amino acid residues located in the active center were mutated to other polar or nonpolar amino acid residues.…”