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

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 (FoPip4H) 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 FoPip4H, and five of these also had L-Pip trans-4-hydroxylation activity. In particular, the homologous Pip4H enzyme derived from Aspergillus nidulans FGSC A4 (AnPip4H) 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 FoPip4H 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.