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

Kateryna Fesko

doi:10.3389/fbioe.2019.00119

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.

Highlights

L-threonine aldolases (LTAs) catalyse the reversible cleavage of L-threonine and non-natural L-βhydroxy-α-amino acids to produce glycine and the corresponding aldehydes
The active site residues, which are located within 5Å from PLP-Glycine complex in L-threonine aldolase from Aeromonas jandaei (LTAaj), were selected for the site-directed mutagenesis to test their influence on the outcome in aldol reactions (Figure 3)
LTAaj is a unique enzyme with broad donor specificity, accepting other small-sized D-amino acids as donors (Fesko et al, 2010; Blesl et al, 2018)

Summary

Introduction

L-threonine aldolases (LTAs) catalyse the reversible cleavage of L-threonine and non-natural L-βhydroxy-α-amino acids to produce glycine and the corresponding aldehydes. The aldol addition reactions catalyzed by LTAs have great biotechnological potential for asymmetric carbon-carbon bond formation toward amino acids (Figure 1) (Fesko and Gruber-Khadjawi, 2013). The products of such reaction, β-hydroxy-α-amino acids, are important building blocks for many complex natural products and pharmaceuticals (Breuer et al, 2004; Baltz et al, 2005; Panke and Wubbolts, 2005; Goldstein, 2006). LTAs show high stereospecificity at the α-carbon, strictly only Lproducts are formed This makes LTA a promising candidate for the stereoselective asymmetric synthesis of unnatural L-amino acids (Dückers et al, 2010; Franz and Stewart, 2014). These studies allowed scientists to elucidate key functional residues in LTAs and draw general conclusions on the mechanism of catalysis involved (Kielkopf and Burley, 2002; di Salvo et al, 2014; Qin et al, 2014)

Methods

Results

Conclusion