Cryptic production of trans-3-hydroxyproline in echinocandin B biosynthesis

Abstract

Echinocandins are antifungal nonribosomal hexapeptides produced by fungi. Two of the amino acids are hydroxy-L-prolines: trans-4-hydroxy-L-proline and, in most echinocandin structures, (trans-2,3)-3-hydroxy-(trans-2,4)-4-methyl-Lproline. In the case of echinocandin biosynthesis by Glarea lozoyensis, both amino acids are found in pneumocandin A0, while in pneumocandin B0 the latter residue is replaced by trans-3-hydroxy-L-proline (3-Hyp). We have recently reported that all three amino acids are generated by the 2-oxoglutarate-dependent proline hydroxylase GloF. In echinocandin B biosynthesis by Aspergillus species, 3-Hyp derivatives have not been reported. Here we describe the heterologous production and kinetic characterization of HtyE, the 2-oxoglutarate-dependent proline hydroxylase from the echinocandin B biosynthetic cluster in Aspergillus pachycristatus. Surprisingly, L-proline hydroxylation with HtyE resulted in an even higher proportion (~30%) of 3-Hyp than that with GloF. This suggests that the selectivity for methylated 3-Hyp in echinocandin B biosynthesis is due solely to a substrate-specific adenylation domain of the nonribosomal peptide synthetase. Moreover, we observed that one product of HtyE catalysis, 3-hydroxy-4-methyl-L-proline, is slowly further oxidized at the methyl group, giving 3-hydroxy-4-hydroxymethyl-Lproline, upon prolonged incubation with HtyE. This dihydroxylated amino acid has been reported as a building block of cryptocandin, an echinocandin produced by Cryptosporiopsis.