Discovery and Engineering of the l-Threonine Aldolase fromNeptunomonas Marinefor Efficient Synthesis of β-Hydroxy-α-Amino Acids via C–C Formation

Abstract

ABSTRACTl-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 a new LTA fromNeptunomonas marine(NmLTA) that displays both ideal enzymatic activity (64.8 U/mg) and diastereoselectivity (89.5% diastereomeric excess; de) for the desired productl-threo-4-methylsulfonylphenylserine (l-threo-MPTS). Using X-ray crystallography, site-directed mutagenesis, and computational modeling, we propose a “dual-conformation” mechanism for the diastereoselectivity control ofNmLTA, whereby the incoming 4-methylsulfonylbenzaldehyde (4-MTB) could potentially bind at theNmLTA active site in two distinct orientations, potentially forming two diastereoisomers (threo- orerythro-form products). Importantly, two keyNmLTA 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 inNmLTA guides us to further improve the diastereoselectivity of this enzyme. A triple variant ofNmLTA (N18S/Q39R/Y319L; SRL) exhibited both improved diastereoselectivity (de value > 99%) and enzymatic activity (95.7 U/mg) for the synthesis ofl-threo-MPTS compared with that of wild type. The preparative gram-scale synthesis forl-threo-MPTS with the SRL variant produced a space-time yield of up to 9.0 g L−1h−1, suggesting a potential role as a robust C–C bond synthetic tool for 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 towardpara-site substituents. This work provides deep structural insights into the molecular mechanism underlying the catalysis inNmLTA 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.