Multi‐Enzymatic Synthesis of Optically Pure β‐Hydroxy α‐Amino Acids

Abstract

A novel enzymatic production system of optically pure b-hydroxy a-amino acids was developed. Two enzymes were used for the system: an Nsuccinyl l-amino acid b-hydroxylase (SadA) belonging to the iron(II)/a-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 b-hydroxy l-amino acids, such as N-succinyl-l-b-hydroxyvaline, N-succinyl-l-threonine, (2S,3R)-N-succinyl-lb-hydroxyisoleucine, and N-succinyl-l-threo-b-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-b-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 b-hydroxy a-amino acids.Keywords: enzymatic synthesis; b-hydroxy a-amino acids; iron(II)/a-ketoglutarate-dependent dioxygenase; N-succinyl l-amino acid desuccinylase

Recent examples of α-ketoglutarate-dependent mononuclear non-haem iron enzymes in natural product biosyntheses

“…9F). 179 The crystal structures of SadA•Zn(II) and SadA•Zn(II)•αKG complexes were reported, 112 and showed a bidentate coordination of the αKG molecule to the Zn(II) metal ion. In addition, N -succinyl-L-leucine and N -succinyl-L-phenylalanine were modelled into the active site of SadA and revealed that the binding pocket of the N -succinyl group is located in an electropositive-rich cavity formed by the side chains of Arg83, Arg163 and Arg203.…”

An unusual metal-bound 4-fluorothreonine transaldolase from Streptomyces sp. MA37 catalyses promiscuous transaldol reactions

“…We found that FTaseMA displays considerable substrate promiscuity to generate 4-fluorothreonine as well as β-hydroxy-α-amino acids using various aldehydes and Lthreonine as substrates. β-Hydroxy-α-amino acids are key building blocks and important biomolecules (Delle Monache et al 1997;Fanning et al 2005;Hibi et al 2015;Pirrung et al 1993;Steinreiber et al 2007), suggesting that FTaseMA could be used as a potential versatile biocatalyst. Strikingly, the enzyme can also utilise L-allo-threonine as substrates albeit less efficient than the natural substrate L-threonine, a clear difference compared to other L-threonine transaldolases that catalyse the similar transaldol reaction to FTases.…”

Cofactor Generation Cascade for α-Ketoglutarate and Fe(II)-Dependent Dioxygenases

“…Therefore, either a desuccinylase or chemical desuccinylation can be used as reported by Hibi and co-workers. 23 , 27 The enzyme LasA can be used in a subsequent module to perform the final reaction step.…”