Variants of glycosyl hydrolase family 2 β-glucuronidases have increased activity on recalcitrant substrates

Glucuronidated drug metabolites can be quantified from urine samples by first hydrolyzing conjugates with ?-glucuronidase (?-GUS) and then separating free drug molecules by liquid chromatography and mass spectrometry detection (LC?MS). To improve the activity and specificity of various ?-GUS, we designed enzyme chimeras and generated site-saturation variants based on structural analyses, then screened them for improved activity on drug metabolites important to clinical and forensic drug-testing laboratories. Often, an increase of activity on one substrate of interest was countered by loss of activity against another, and there was no strong correlation of activity on standard ?-glucuronidase substrates to activity on recalcitrant drug glucuronides. However, we discovered a chimera of two enzymes from different species of Aspergillus that displays a 27 % increase in activity on morphine-3-glucuronide than the parent proteins. Furthermore, mutations in the M-loop, which is a loop near the active site, resulted in numerous variants with dramatically increased rates of hydrolysis on drug glucuronides. Specifically, the M-loop variant Q451D/A452E of a ?-GUS from Brachyspira pilosicoli has a 50-fold and 25-fold increase in activity on the recalcitrant substrates codeine-6-glucuronide and dihydrocodeine6-glucuronide, respectively, compared to the parent enzyme.

Automated summary

Researchers improved the activity of β-GUS on drug metabolites by designing enzyme chimeras and site-saturation variants, and discovered a chimera from enzymes of different species that exhibited a significant increase in activity on morphine-3-glucuronide.