Engineering the Entrance of a Flavonoid Glycosyltransferase Promotes the Glycosylation of Etoposide Aglycone

Enzyme entrances, which function as the first molecular filters, influencesubstrate selectivity and enzymatic activity. Because of low binding affinities, engineeringenzyme entrances that recognize non-natural substrates is a major challenge for artificialbiocatalyst design. Here, the entrance offlavonoid glycosyltransferase UGT78D2 wasengineered to promote the recognition of the aglycone of etoposide, a chemotherapeuticagent. We found that Q258, S446, R444, and R450, the key residues surrounding thesubstrate entrance, specifically guide theflux of etoposide aglycone, which has a highsteric hindrance, into the active site; this activity was inferred to be determined by theentrance size and hydrophobic and electrostatic interactions. Engineering thecoordination of Q258 and S446 to increase the entrance size and hydrophobicinteraction between UGT78D2 and etoposide aglycone increased the affinity by 10.10-fold and the conversion by 10%. The entrance-engineering strategy applied in this study can improve the design of artificial biocatalysts

Automated summary

The study engineered the entrance of an enzyme to improve substrate recognition and enzymatic activity. By manipulating the positions of key residues and the size of the entrance, as well as hydrophobic and electrostatic interactions, the study successfully increased the affinity and conversion rate between the enzyme and substrate.