Regiodivergent Oxyfuctionalization of Non-natural Tricyclic Terpenoids Using Sequence Divergent Unspecific Peroxygenases (UPOs)

Abstract

Unspecific peroxygenases (UPOs) catalyze the H2O2-dependent monooxygenation of organic substrates with regioselectivity and chemoselectivity; however, their application to sterically demanding, non-natural terpene skeletons has remained largely unexplored to date. Here, we present a computationally-guided, multiparametric screening pipeline that was used to prioritize 17 structurally distinct UPOs and UPO-like enzymes for the late-stage oxyfunctionalization of chemoenzymatically synthesized tricyclic terpenoids not reported from natural sources. These are accessible via BcBOT2-catalyzed cyclization of non-natural farnesyl diphosphate analogs. The computer-aided pre-screening included an evaluation of expression levels, an analysis of the tunnel architecture for substrate access, and hierarchical docking using geometric criteria close to the attack conformation to rank candidate enzymes. Experimental screening of the prioritized enzymes identified productive UPO-substrate combinations showed correspondence between the docking-based regioselectivity predictions and the product profiles confirmed by GC–MS. MthUPO FuncLib_18 catalyzed an “orchestrated” linear cascade of three consecutive oxidations of a furan sesquiterpenoid, converting it into an α-hydroxylactone—a transformation that mimics the natural oxidative diversification that biosynthetically follows terpene cyclization. In addition, a single inversion of the stereocenter in the furano sesquiterpenoids diastereomer pair redirected the regioselectivity of MspUPO-mg133 and ChiUPO-II to opposite sides of the furan ring. This finding thus represents a proof-of-concept for regioselectivity reversal through stereochemical changes in the substrate during UPO-catalyzed terpenoid oxidation. Additionally, we were able to access two structures with tertiary alcohols, difficult to achieve by chemical means. In silico comparison of AlphaFold 3 models, supported by multiple-sequence alignment, suggested that the Monosporascus UPOs characterized here form a distinct UPO/UPO-like subgroup characterized by an EHR catalytic motif, a shortened α-helix, an elongated loop, and an active-site lacking aromatic residues and exhibit comparatively the lowest predicted active-site hydrophobicity among all characterized UPOs. Molecular dynamics simulations, combined with an analysis of the near-attack-conformations of selected enzyme-substrate complexes, confirmed that only the experimentally observed oxidation positions achieve sustained proximity to the ferryl oxygen of compound I with catalytically suitable approach angles over timescales in the microsecond timescale. This work thus expands both the known sequence-structure space of catalytically competent UPOs and the accessible chemical space of terpenoids as substrates.