Structure function and engineering of multifunctional non-heme iron dependent oxygenases in fungal meroterpenoid biosynthesis

Abstract

Non-heme iron and α-ketoglutarate (αKG) oxygenases catalyze remarkably diverse reactions using a single ferrous ion cofactor. A major challenge in studying this versatile family of enzymes is to understand their structure–function relationship. AusE from Aspergillus nidulans and PrhA from Penicillium brasilianum are two highly homologous Fe(II)/αKG oxygenases in fungal meroterpenoid biosynthetic pathways that use preaustinoid A1 as a common substrate to catalyze divergent rearrangement reactions to form the spiro-lactone in austinol and cycloheptadiene moiety in paraherquonin, respectively. Herein, we report the comparative structural study of AusE and PrhA, which led to the identification of three key active site residues that control their reactivity. Structure-guided mutagenesis of these residues results in successful interconversion of AusE and PrhA functions as well as generation of the PrhA double and triple mutants with expanded catalytic repertoire. Manipulation of the multifunctional Fe(II)/αKG oxygenases thus provides an excellent platform for the future development of biocatalysts.

A pharmaceutical model for the molecular evolution of microbial natural products

“…This catalytic promiscuity promotes the ability to catalyze fortuitous chemical transformations and may form the seed for the emergence of new enzymatic activities . Furthermore, natural product biosynthetic pathways often encode multifunctional enzymes that catalyze either several consecutive reactions with different regiospecificity or entirely different chemical transformations .…”

“…This is most apparent in comparisons of related secondary metabolite biosynthetic gene clusters, where highly conserved enzymes can nonetheless catalyze distinct chemistry. Perhaps the most illustrative examples are found from evolution‐inspired protein engineering experiments, where the activities of functionally distinct enzymes can be interchanged with single point mutations .…”

“…Studies on fungal meroterpenoids (Fig. B) have illustrated how highly divergent products may be obtained through dynamic skeletal rearrangement . AusE and PrhA both utilize preaustinoid A1 as a substrate, but whereas the former catalyzes desaturation at C1–C2 and carbocyclic rearrangement to preaustinoid A3, the latter first desaturates at C5–C6, followed by rearrangement of the A/B‐ring to form berkeleydione.…”

“…AusE and PrhA both utilize preaustinoid A1 as a substrate, but whereas the former catalyzes desaturation at C1–C2 and carbocyclic rearrangement to preaustinoid A3, the latter first desaturates at C5–C6, followed by rearrangement of the A/B‐ring to form berkeleydione. The functions of the two proteins could be interchanged by manipulating two amino acids in the vicinity of the A‐ring . The importance of enzyme promiscuity was evident as a single point mutation AusE‐S232A was sufficient for gaining a PrhA‐type ‘B‐ring expansion’ activity, while still retaining its natural ‘spiro‐lactone forming’ activity.…”

“…The importance of enzyme promiscuity was evident as a single point mutation AusE‐S232A was sufficient for gaining a PrhA‐type ‘B‐ring expansion’ activity, while still retaining its natural ‘spiro‐lactone forming’ activity. In case of a PhrA‐V150L‐A232S double mutant, the enzyme gained unnatural hydroxylation functionality as a third activity, while the addition of a third M241V mutation to this scaffold led to the formation of even more novel reaction products .…”