Multi-Enzymatic Synthesis of Optically Pure beta-Hydroxy alpha-Amino Acids

A novel enzymatic production system of optically pure beta-hydroxy alpha-amino acids was developed. Two enzymes were used for the system: an N-succinyl l-amino acid beta-hydroxylase (SadA) belonging to the iron(II)/alpha-ketoglutarate-dependent dioxygenase superfamily and an N-succinyl l-amino acid desuccinylase (LasA). The genes encoding the two enzymes are part of a gene set responsible for the biosynthesis of peptidyl compounds found in the Burkholderia ambifaria AMMD genome. SadA stereoselectively hydroxylated several N-succinyl aliphatic l-amino acids and produced N-succinyl beta-hydroxy l-amino acids, such as N-succinyl-l-beta-hydroxyvaline, N-succinyl-l-threonine, (2S, 3R)-N-succinyl-l beta-hydroxyisoleucine, and N-succinyl-l-threo-beta-hydroxyleucine. LasA catalyzed the desuccinylation of various N-succinyl-l-amino acids. Surprisingly, LasA is the first amide bond-forming enzyme belonging to the amidohydrolase superfamily, and has succinylation activity towards the amino group of l-leucine. By combining SadA and LasA in a preparative scale production using N-succinyl-l-leucine as substrate, 2.3 mmol of l-threo-beta-hydroxyleucine were successfully produced with 93% conversion and over 99% of diastereomeric excess. Consequently, the new production system described in this study has advantages in optical purity and reaction efficiency for application in the mass production of several beta-hydroxy alpha-amino acids.