Characterizing Family 1 Glycosyltransferases (GT1, UGT) by Reverse Glycosylation: Fast Determination of Acceptor Specificity, Donor Specificity, Hydrolysis, and Enzyme Stability

Manufacturing glycoside conjugates through conventional chemistry is a labor-intensive process that involves multiple reaction steps, generates substantial toxic waste, and often results in low yields with poor control over regioselectivity. In contrast, glycosyltransferase family 1 enzymes offer an efficient alternative, enabling the transfer of glycosyl groups in a single step with excellent regioselective control and high yields. However, the factors governing acceptor specificity and the scope of individual enzymes remain unclear, limiting their broader application. To date, there is no widely implemented, low-cost approach to the high-throughput screening of these enzymes. Here, we use reverse glycosylation with 2-chloro-4-nitrophenyl-β-d-glycosides as a generic high-throughput screening method for UGT characterization. We investigate reverse glycosylation kinetics of 34 UGTs and their donor specificity toward the glucose and galactose moieties. Then, the method is utilized in an acceptor screening with 44 acceptors against 32 enzymes, and we determined the <i>K</i> _M parameters for 11 enzyme/substrate pairs. We showcase how this method can be employed for assaying other UGT phenomena, specifically hydrolysis of the glycosyl donor and inactivation upon enzyme dilution. The reverse glycosylation reaction with 2-chloro-4-nitrophenyl-β-d-glycosides can be catalyzed by all 33 assayed UGTs, and therefore the methodology qualifies as a cheap generic tool for high-throughput assays of these enzymes. Moreover, we provide a script for automatic determination of rates and kinetics and present an acceptor data set consisting of 2280 UGT/acceptor pairs.