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A novel enzymatic production system of optically pure β-hydroxy α-amino acids was developed. Two enzymes were used for the system: an N-succinyl L-amino acid β-hydroxylase (SadA) belonging to the iron(II)/α-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 β-hydroxy L-amino acids, such as N-succinyl- L-β-hydroxyvaline, N-succinyl- L-threonine, (2 S,3 R)- N-succinyl- L-β-hydroxyisoleucine, and N-succinyl- L- threo-β-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-β-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 β-hydroxy α-amino acids. [ABSTRACT FROM AUTHOR]