Tandem intermolecular [4 + 2] cycloadditions are catalysed by glycosylated enzymes for natural product biosynthesis

Tandem Diels–Alder reactions are frequently used in the construction of polycyclic ring systems in complex organic compounds. Unlike the many Diels−Alderases (DAases) that catalyse a single cycloaddition, enzymes for multiple Diels–Alder reactions are rare. Here we demonstrate that two calcium-ion-dependent glycosylated enzymes, EupfF and PycR1, independently catalyse sequential, intermolecular Diels–Alder reactions in the biosynthesis of bistropolone-sesquiterpenes. We elucidate the origins of catalysis and stereoselectivity within these DAases through analysis of enzyme co-crystal structures, together with computational and mutational studies. These enzymes are secreted as glycoproteins with diverse N-glycans. The N-glycan at N211 in PycR1 significantly increases the affinity to the calcium ion, which in turn regulates the active cavity, making it specifically interact with substrates to accelerate the tandem [4 + 2] cycloaddition. The synergistic effect of the calcium ion and N-glycan on the catalytic centre of enzymes involved in secondary metabolism, especially for complex tandem reactions, can extend our understanding of protein evolution and improve the artificial design of biocatalysts.