Pneumocandin Biosynthesis: Involvement of a trans‐Selective Proline Hydroxylase

Abstract

Echinocandins are cyclic nonribosomal hexapeptides based mostly on nonproteinogenic amino acids and displaying strong antifungal activity. Despite previous studies on their biosynthesis by fungi, the origin of three amino acids, trans-4- and trans-3-hydroxyproline, as well as trans-3-hydroxy-4-methylproline, is still unknown. Here we describe the identification, overexpression, and characterization of GloF, the first eukaryotic α-ketoglutarate/Fe(II) -dependent proline hydroxylase from the pneumocandin biosynthesis cluster of the fungus Glarea lozoyensis ATCC 74030. In in vitro transformations with L-proline, GloF generates trans-4- and trans-3-hydroxyproline simultaneously in a ratio of 8:1; the latter reaction was previously unknown for proline hydroxylase catalysis. trans-4-Methyl-L-proline is converted into the corresponding trans-3-hydroxyproline. All three hydroxyprolines required for the biosynthesis of the echinocandins pneumocandins A0 and B0 in G. lozoyensis are thus provided by GloF. Sequence analyses revealed that GloF is not related to bacterial proline hydroxylases, and none of the putative proteins with high sequence similarity in the databases has been characterized so far.

Structural diversity in echinocandin biosynthesis: the impact of oxidation steps and approaches toward an evolutionary explanation

“…For one of them, thought to be a glutamine hydroxylase (GH), no homologue has been found in Ecd/Hty. Another dioxygenase was characterized as an αKG-dependent PH [18]. More recently, this and three other oxygenases were deleted in G. lozoyensis and the production of pneumocandins was analysed [19,20].…”

“…About a decade later, our group characterized the corresponding PH (GloF) [18] in the genome of G. lozoyensis mutant strain ATCC 74030 [21] (GloF is identical with GLOXY2 in the genome of wild-type G. lozoyensis ATCC 20868 [17,20]). It is distantly related to a group of fungal pipecolic acid hydroxylases discovered only recently [22].…”

“…In vitro experiments with the heterologously expressed and purified enzyme showed that proline is converted into trans-4-hydroxyproline and a minor amount of the trans-3-hydroxy isomer. Additionally, GloF hydroxylates trans-4-methylproline to provide (S,S)-4-methyl-3-hydroxyproline, so that all three hydroxyproline building blocks for the biosynthesis of pneumocandins A and B are provided by this enzyme [18]. The ratio of trans-4/trans-3 hydroxyproline was about 8:1.…”

“…An additional gene for an αKG/Fe(II)-dependent dioxygenase was found in pneumocandin biosynthesis, which has no homologue in echinocandin B biosynthesis. As residue 6 in the pneumocandins, but not in echinocandin B, is hydroxyglutamine it can be concluded that the enzyme is a GH [18]. Currently, two genome sequences of the pneumocandin producer G. lozoyensis are available: one of the wild-type strain ATCC 20868 [17], which is a producer of pneumocandins A and B, and the other from mutant strain ATCC 74030 which produces pneumocandin B 0 predominantly [21].…”

“…a Protein nomenclature for wildtype [20] and in brackets: mutant strain ATCC 74030 [18]. b The highest sequence identity to a protein of the A. pachychristatus cluster was 33% (EcdG).…”