Expanding the threonine aldolase toolbox for the asymmetric synthesis of tertiary α-amino acids

Abstract

The direct biochemical synthesis of tertiary α-amino acids with a wide range of diversity was recently reported using natural threonine aldolases LTA from Aeromonas jandei and DTA from Pseudomonas sp. Here, we describe the identification of five novel threonine aldolases which accept alanine and serine as amino acid donors. The enzymes were found by sequence database analysis using known aldolases as template. All enzymes were overexpressed in Escherichia coli and purified, and their biochemical properties were characterized. The new enantiocomplementary L- and D-threonine aldolases catalyze the asymmetric synthesis of β-hydroxy α-methyl- and α-hydroxymethyl-α-amino acids with good conversion and perfect enantioselectivity at α-carbon of the products (e.e. >99 %). The structural basis for the broad donor specificity of these threonine aldolases is analyzed based on crystal structure alignments and amino acid sequences comparison.

Threonine aldolases: perspectives in engineering and screening the enzymes with enhanced substrate and stereo specificities

“…To a similar conclusion came the authors, trying to identify the residues responsible for the broader donor specificity of ajTA compared to those which only accept glycine as the donor (Fesko et al 2015 ). The sequence and crystal structure alignment of available l TAs reveal that the catalytically important residues were conserved among l TAs from different organisms, whereas their orientation in the active site was different in some cases.…”

“…were found to accept d -alanine, d -serine and d -cysteine as donors, whereas formerly TAs had been known to be highly specific towards glycine donor (Fesko et al 2010 ). Subsequent search for homologues using these enzymes as templates could reveal a set of other l TAs and d TAs with broad donor specificities (Fesko et al 2015 ). The respective genes were selected based on 50–80 % sequence similarities to the templates.…”

Asymmetric assembly of high-value α-functionalized organic acids using a biocatalytic chiral-group-resetting process

“…The biocatalytic process in this study is elegant and nicely exploits the power of cascades: (i) it provides a universal, efficient, and renewable catalyst to replace precious transition metals such as Pd 10 , 11 in the modern chemical C–H functionalization-mediated asymmetric assembly of α-functionalized organic acids and (ii) when compared to other enzymatic methods 3 – 5 , 9 , 26 , 28 , 36 , 39 , the universality and practicality of this biocatalytic cascade is more attractive owing to the more diverse product range that it supports, including liphatic, aromatic, heteroaromatic, and heterocyclic products from three classes (α-keto/hydroxy/amino acids). Furthermore, since enantiocomplementary ʟ- and D -specific TAs were identified to accept nucleophilic substrates other than glycine (such as alanine, serine, and cysteine) 42 , 43 , the expansion of this biocatalytic process could enable the synthesis of a vast array of α-functionalized products simply by combinatorically integrating different components such as other catalyst modules and substrate units.…”

Nucleophile Promiscuity of Natural and Engineered Aldolases

“…During syntheses, both TAs were able to accept 5 a , d ‐Ser ( 5 b ), and d ‐Cys ( 5 c ) as nucleophiles, with similar electrophile tolerance to that reported for glycine as a nucleophile (Scheme ). By means of homology searching with the amino acid sequence of both TAs, five new enantiocomplementary l ‐ and d ‐specific TAs were identified to accept nucleophilic substrates other than glycine …”

“…By means of homology searching with the amino acid sequence of both TAs, five new enantiocomplementary l-a nd d-specific TAsw ere identified to accept nucleophilic substrates other than glycine. [24] PLP-dependent SHMT Sth (EC 2.1.2.1) was found to catalyze the aldol addition of glycinet oa ldehydes with remarkable diastereoselectivity under kineticc ontrol. To broaden the nucleophile scope, the analogy of SHMT with a-methylserine hydroxymethyltransferase from Paracoccus sp.…”