Abstract
Non-heme iron and α-ketoglutarate-dependent (Fe/αKG) oxygenases catalyze various oxidative biotransformations. Due to their catalytic flexibility and high efficiency, Fe/αKG oxygenases have attracted keen attention for their application as biocatalysts. Here, we report the biochemical and structural characterizations of the unusually promiscuous and catalytically versatile Fe/αKG oxygenase SptF, involved in the biosynthesis of fungal meroterpenoid emervaridones. The in vitro analysis revealed that SptF catalyzes several continuous oxidation reactions, including hydroxylation, desaturation, epoxidation, and skeletal rearrangement. SptF exhibits extremely broad substrate specificity toward various meroterpenoids, and efficiently produced unique cyclopropane-ring-fused 5/3/5/5/6/6 and 5/3/6/6/6 scaffolds from terretonins. Moreover, SptF also hydroxylates steroids, including androsterone, testosterone, and progesterone, with different regiospecificities. Crystallographic and structure-based mutagenesis studies of SptF revealed the molecular basis of the enzyme reactions, and suggested that the malleability of the loop region contributes to the remarkable substrate promiscuity. SptF exhibits great potential as a promising biocatalyst for oxidation reactions.
Highlights
Non-heme iron and α-ketoglutarate-dependent (Fe/αKG) oxygenases catalyze various oxidative biotransformations
In addition to the typical hydroxylation reactions, Fe/αKG oxygenases catalyze a wide range of chemical transformations in the biosynthesis of natural products2–27, including complex skeletal rearrangement11,12, ringexpansion15, and C–C bond formation21–23
SptF is an Fe/αKG oxygenase first discovered in the biosynthesis of the fungal meroterpenoid emervaridones40
Summary
To determine the reaction sequence, putative intermediates, including 2, 3, and emeridone F were used as substrates of the SptF enzyme reactions As a result, both 2 and 3 were efficiently converted into 4 and 5, which clearly indicated that SptF converts 1 into 3 via 2, and 4 and 5 are generated from 3 (Supplementary Fig. 11c, d). Double labeling of H218O and 18O2 revealed that the enzyme generated +2 of 3 (m/z 428) as a major product together with +0 (m/z 426) and +4 (m/z 430) of 3 as minor products (m/z 426 (+0): 428 (+2): 430 (+4) = 1.3%: 92.0%: 6.8%) These results indicated that an epoxide group introduced by SptF is mostly from O2, while ~5% of water-derived oxygen atom are incorporated into 3.
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