Novel Enzyme Family Found in Filamentous Fungi Catalyzing trans -4-Hydroxylation of l -Pipecolic Acid

Abstract

ABSTRACT Hydroxypipecolic acids are bioactive compounds widely distributed in nature and are valuable building blocks for the organic synthesis of pharmaceuticals. We have found a novel hydroxylating enzyme with activity toward l -pipecolic acid ( l -Pip) in a filamentous fungus, Fusarium oxysporum c8D. The enzyme l -Pip trans -4-hydroxylase (Pip4H) of F. oxysporum ( Fo Pip4H) belongs to the Fe(II)/α-ketoglutarate-dependent dioxygenase superfamily, catalyzes the regio- and stereoselective hydroxylation of l -Pip, and produces optically pure trans -4-hydroxy- l -pipecolic acid ( trans -4- l -HyPip). Amino acid sequence analysis revealed several fungal enzymes homologous with Fo Pip4H, and five of these also had l -Pip trans -4-hydroxylation activity. In particular, the homologous Pip4H enzyme derived from Aspergillus nidulans FGSC A4 ( An Pip4H) had a broader substrate specificity spectrum than other homologues and reacted with the l and d forms of various cyclic and aliphatic amino acids. Using Fo Pip4H as a biocatalyst, a system for the preparative-scale production of chiral trans -4- l -HyPip was successfully developed. Thus, we report a fungal family of l -Pip hydroxylases and the enzymatic preparation of trans -4- l -HyPip, a bioactive compound and a constituent of secondary metabolites with useful physiological activities.