Discovery and Engineering of the l-Threonine Aldolase from Neptunomonas marine for the Efficient Synthesis of β-Hydroxy-α-amino Acids via C–C Formation

Abstract

l-Threonine aldolases (LTAs) are attractive biocatalysts for synthesizing β-hydroxy-α-amino acids (HAAs) via C–C bond formation in pharmaceuticals, although their industrial applications suffer from low activity and diastereoselectivity. Herein, we describe the discovery of an LTA from Neptunomonas marine (NmLTA) that displays both ideal enzymatic activity (64.8 U mg–1) and diastereoselectivity (89.5% diastereomeric excess, de) for the desired product l-threo-4-methylsulfonylphenylserine (l-threo-MTPS). Using X-ray crystallography, site-directed mutagenesis, and computational modeling, we propose a “dual-conformation” mechanism for the diastereoselectivity control of NmLTA, whereby the incoming 4-methylsulfonylbenzaldehyde (4-MTB) could potentially bind at the NmLTA active site in two distinct orientations, potentially forming two diastereoisomers (threo- or erythro-form products). Importantly, two key NmLTA residues H140 and Y319 play critical roles in fine-tuning the binding mode of 4-MTB, supported by our site-mutagenesis assays. Uncovering of the catalytic mechanism in NmLTA guides us to further improve the diastereoselectivity of this enzyme. A triple variant of NmLTA (N18S/Q39R/Y319L, SRL) exhibited both improved diastereoselectivity (de value > 99%) and enzymatic activity (95.7 U mg–1) for the synthesis of l-threo-MTPS compared with that of the wild type. The preparative gram-scale synthesis for l-threo-MTPS with the SRL variant produced a space-time yield of up to 9.0 g L–1 h–1, suggesting a potential role as a robust C–C bond synthetic tool for the industrial synthesis of HAAs at a preparative scale. Finally, the SRL variant accepted a wider range of aromatic aldehyde derivatives as substrates and exhibited improved diastereoselectivity toward para-site substituents. This work provides deep structural insights into the molecular mechanism underlying the catalysis in NmLTA and pinpoints the key structural motifs responsible for regulating the diastereoselectivity control, thereby guiding future attempts for protein engineering of various LTAs from different sources.