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: -4-hydroxy-l-proline and, in most echinocandin structures, (-2,3)-3-hydroxy-(-2,4)-4-methyl-l-proline. In the case of echinocandin biosynthesis by , both amino acids are found in pneumocandin A, while in pneumocandin B the latter residue is replaced by -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 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 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-l-proline, upon prolonged incubation with HtyE. This dihydroxylated amino acid has been reported as a building block of cryptocandin, an echinocandin produced by Secondary metabolites from bacteria and fungi are often produced by sets of biosynthetic enzymes encoded in distinct gene clusters. Usually, each enzyme catalyzes one biosynthetic step, but multiple reactions are also possible. Pneumocandins A and B are produced by the fungus They belong to the echinocandin family, a group of nonribosomal cyclic lipopeptides that exhibit a strong antifungal activity. Chemical derivatives are important drugs for the treatment of systemic fungal infections. We have recently shown that in the biosynthesis of pneumocandins A and B, three hydroxyproline building blocks are provided by one proline hydroxylase. Here we demonstrate that the proline hydroxylase from echinocandin B biosynthesis in produces the same hydroxyprolines, with an increased proportion of-3-hydroxyproline. However, echinocandin B biosynthesis does not require -3-hydroxyproline; its formation remains cryptic. While one can only speculate on the evolutionary background of this unexpected finding, proline hydroxylation in and provides an unusual insight into peptide antibiotic biosynthesis-namely, the complex interplay between the selectivity of a hydroxylase and the substrate specificity of a nonribosomal peptide synthetase.

Biochemical and genetic characterization of fungal proline hydroxylase in echinocandin biosynthesis

“…Due to the lack of their respective standards, the identity of these products was not confirmed. Our observation was, however, consistent with recently reported data (Mattay et al 2018), wherein they confirmed that the major product catalyzed by Ap-HtyE was 4-Me-3-Hyp while the minor product was 3-hydroxy-4-hydroxymethyl-L-proline (4-HyMe-3-Hyp).…”

“…Moreover, both Ap-HtyE and Aa-HtyE were shown to effectively hydroxylate C-3 of 4-Me-Pro in vitro, confirming their involvement in the generation of both trans-4-Hyp and 4-Me-3-Hyp. During the preparation of this manuscript, Mattay et al independently published the characterization of Ap-HtyE and reported some of the results discussed above (Mattay et al 2018). We further demonstrated that Ap-HtyE and Aa-HtyE are also capable of hydroxylating D-proline though with relatively lower efficiency.…”

“…The reason for the dual Fig. 5 Anti-Candida activity test of the culture extract from the wild-type A. pachycristatus strain and two independent htyE deletion mutants (△htyE-1 and △htyE-2) regioselectivity of fungal proline hydroxylase remains unknown, probably resulting from an evolutionary relief for HtyE which is otherwise taken on by NRPS in echinocandin biosynthesis (Mattay et al 2018).…”

Echinocandins: structural diversity, biosynthesis, and development of antimycotics

“…All these findings suggest that in pneumocandin biosynthesis, Hyp incorporation at position 6 is largely driven by the availability of Hyp substrates rather than a distinct substrate selectivity of the NRPS adenylation domain (A-domain). This is different in echinocandin B biosynthesis by A. pachychristatus : here, the proline hydroxylase HtyE produces even more 3Hyp (3Hyp/4Hyp ≈ 1:2.5–3.0; (Mattay et al 2018 ; Zhang et al 2018 )). However, 3Hyp is not found in echinocandin products.…”

Integrated strategy of temperature shift and mannitol feeding for enhanced production of echinocandin B by Aspergillus nidulans CCTCC M2012300

“…Echinocandins, a family of cyclic hexlipopeptides, are a new class of antifungal agents with broad-spectrum activity against Pneumocystis carinii, Aspergillus species, and Candida (Emri et al 2013). These compounds can disturb the biosynthesis of β-1,3-glucan in the fungal cell walls by inhibiting β-1,3-glucan synthase activity (Bauer and Bronstrup 2014;Mattay et al 2018). Thus, echinocandins have very few toxic side effects in humans because glucan polymers are not present in mammalian cells.…”