Hunting for the Intermolecular Diels-Alderase

Area of Science:

• Biocatalysis and Natural Product Biosynthesis
• Enzyme Engineering and Evolution
• Organic Chemistry and Synthetic Biology

Background:

• The Diels-Alder reaction is a fundamental [4+2] cycloaddition crucial for synthesizing cyclic compounds, including natural products and pharmaceuticals.
• While microbial Diels-Alderases have been identified, enzymes catalyzing intermolecular Diels-Alder reactions with concerted mechanisms are rare.
• Plants utilize Diels-Alder reactions, but identifying specific plant-derived enzymes has been challenging.

Purpose of the Study:

• To discover naturally occurring intermolecular Diels-Alderases from plants.
• To elucidate the catalytic mechanisms and evolutionary pathways of these enzymes.
• To engineer novel Diels-Alderases for applications in chemoenzymatic synthesis.

Main Methods:

• Biomimetic synthesis of Diels-Alder-type terpenoids and flavonoids to guide enzyme discovery.
• Biosynthetic intermediate probe-based target identification strategy to isolate enzymes from Morus plants.
• Functional characterization, mechanistic studies (including stereoselectivity), evolutionary analysis, and enzyme engineering.

Main Results:

• Identification of MaDA, an endo-selective intermolecular Diels-Alderase from mulberry, functioning via a concerted but asynchronous mechanism.
• Discovery of exo-selective intermolecular Diels-Alderases in Morus plants with broad substrate scope but distinct stereochemical control.
• Phylogenetic analysis revealed these enzymes as a unique subgroup of FAD-dependent enzymes in moraceous plants, evolved from oxidocyclases via specific amino acid mutations.

Conclusions:

• Plants possess unique endo- and exo-selective intermolecular Diels-Alderases, explaining the prevalence of specific cycloadducts in moraceous plants.
• The evolutionary trajectory from FAD-dependent oxidocyclases to Diels-Alderases provides insights into enzyme adaptation.
• Engineered Diels-Alderases offer new avenues for diversity-oriented chemoenzymatic synthesis, expanding biocatalytic toolkits for organic synthesis.