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Cryptic Sulfur Incorporation in Thioangucycline Biosynthesis.

ABSTRACT: Sulfur incorporation into natural products is a critical area of biosynthetic studies. Recently, a subset of sulfur-containing angucyclines has been discovered, and yet, the sulfur incorporation step is poorly understood. In this work, a series of thioether-bridged angucyclines were discovered, and a cryptic epoxide Michael acceptor intermediate was revealed en route to thioangucyclines (TACs) A and B. However, systematic gene deletion of the biosynthetic gene cluster (BGC) by CRISPR/Cas9 could not identify any gene responsible for the conversion of the epoxide intermediate to TACs. Instead, a series of in vitro and in vivo experiments conclusively showed that the conversion is the result of two non-enzymatic steps, possibly mediated by endogenous hydrogen sulfide. Therefore, the TACs are proposed to derive from a detoxification process. These results are expected to contribute to the study of both angucyclines and the utilization of inorganic sulfur in natural product biosynthesis.

SUBMITTER: Cao M

PROVIDER: S-EPMC7969429 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Cryptic Sulfur Incorporation in Thioangucycline Biosynthesis.

Cao Mingming M Zheng Chengjian C Yang Dong D Kalkreuter Edward E Adhikari Ajeeth A Liu Yu-Chen YC Rateb Mostafa E ME Shen Ben B

Angewandte Chemie (International ed. in English) 20210217 13

Sulfur incorporation into natural products is a critical area of biosynthetic studies. Recently, a subset of sulfur-containing angucyclines has been discovered, and yet, the sulfur incorporation step is poorly understood. In this work, a series of thioether-bridged angucyclines were discovered, and a cryptic epoxide Michael acceptor intermediate was revealed en route to thioangucyclines (TACs) A and B. However, systematic gene deletion of the biosynthetic gene cluster (BGC) by CRISPR/Cas9 could ...[more]

PMID: 33465268

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Project description: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-l-proline. 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-l-proline, upon prolonged incubation with HtyE. This dihydroxylated amino acid has been reported as a building block of cryptocandin, an echinocandin produced by CryptosporiopsisIMPORTANCE 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 A0 and B0 are produced by the fungus Glarea lozoyensis 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 A0 and B0, three hydroxyproline building blocks are provided by one proline hydroxylase. Here we demonstrate that the proline hydroxylase from echinocandin B biosynthesis in Aspergillus pachycristatus produces the same hydroxyprolines, with an increased proportion of trans-3-hydroxyproline. However, echinocandin B biosynthesis does not require trans-3-hydroxyproline; its formation remains cryptic. While one can only speculate on the evolutionary background of this unexpected finding, proline hydroxylation in G. lozoyensis and A. pachycristatus 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.

Dataset Information

Cryptic Sulfur Incorporation in Thioangucycline Biosynthesis.

Publications

Cryptic Sulfur Incorporation in Thioangucycline Biosynthesis.

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