Published in
Angewandte Chemie Int Ed, Wiley-VCH
Content
Angewandte Chemie International Edition, EarlyView.
In a study of the biosynthesis of the polyether ionophore lysocellin, the P450 enzyme LyoI was found to catalyze a rare oxidation of a hydro‐2,2′‐bifuran moiety to its hemiketal form. The oxidation was identified as the key for unlocking the bioactivity of lysocellin. The sequence of LyoI lacks a key acidic residue, essential for its oxidative capabilities, which was utilized to identify a range of non‐canonical P450 enzymes.
Abstract
Polyether ionophores are potent antimicrobials, albeit also cytotoxic against mammalian cells. We have identified several polyether ionophores containing a common hydro‐2,2′‐bifuran‐2‐ol (hemiketal) moiety, which cannot be derived from the canonical biosynthetic steps observed for the compound class, suggesting an unusual oxidative transformation. To identify the responsible enzyme, we applied CRISPR–BEST to knock out genes in the lysocellin‐producing strain S. longwoodensis. This allowed us to propose the first annotation of the lysocellin biosynthetic gene cluster and identify the responsible P450 enzyme, LyoI, through reconstitution of the function in vivo. LyoI knockout provided access to the non‐oxidized precursor (pre‐lysocellin) which allowed both in vitro validation of the unusual direct hydro‐2,2′‐bifuran to hydro‐2,2′‐bifuran‐2‐ol oxidation and investigation of the impact on biological activity. Interestingly, absence of the LyoI‐mediated oxidation greatly reduced the biological potency of the compound. Closer investigation of the sequence revealed that LyoI lacks a key conserved acidic residue, which proved essential for the unusual oxidative function of the enzyme. Through a sequence similarity network of LyoI, we were able to identify a wide range of non‐canonical P450 enzymes, highlighting the possibilities of a biosynthesis‐focused approach to discovering novel enzymes.
In a study of the biosynthesis of the polyether ionophore lysocellin, the P450 enzyme LyoI was found to catalyze a rare oxidation of a hydro-2,2′-bifuran moiety to its hemiketal form. The oxidation was identified as the key for unlocking the bioactivity of lysocellin. The sequence of LyoI lacks a key acidic residue, essential for its oxidative capabilities, which was utilized to identify a range of non-canonical P450 enzymes.
Abstract
Polyether ionophores are potent antimicrobials, albeit also cytotoxic against mammalian cells. We have identified several polyether ionophores containing a common hydro-2,2′-bifuran-2-ol (hemiketal) moiety, which cannot be derived from the canonical biosynthetic steps observed for the compound class, suggesting an unusual oxidative transformation. To identify the responsible enzyme, we applied CRISPR–BEST to knock out genes in the lysocellin-producing strain S. longwoodensis. This allowed us to propose the first annotation of the lysocellin biosynthetic gene cluster and identify the responsible P450 enzyme, LyoI, through reconstitution of the function in vivo. LyoI knockout provided access to the non-oxidized precursor (pre-lysocellin) which allowed both in vitro validation of the unusual direct hydro-2,2′-bifuran to hydro-2,2′-bifuran-2-ol oxidation and investigation of the impact on biological activity. Interestingly, absence of the LyoI-mediated oxidation greatly reduced the biological potency of the compound. Closer investigation of the sequence revealed that LyoI lacks a key conserved acidic residue, which proved essential for the unusual oxidative function of the enzyme. Through a sequence similarity network of LyoI, we were able to identify a wide range of non-canonical P450 enzymes, highlighting the possibilities of a biosynthesis-focused approach to discovering novel enzymes.
Michelle H. Rasmussen, Søren L. B. Møller, Esben B. Svenningsen, Thomas Tørring, Thomas B. Poulsen
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