The Nicotiana tabacum UGT89A2 enzyme catalyzes the glycosylation of di- and trihydroxylated benzoic acid derivatives.

ABSTRACT

Glycosyltransferases catalyze the transfer of a glycoside group to a wide range of acceptor compounds to produce glycoconjugates with diverse biological and pharmacological activities. The present work reports the identification and biochemical characterization of Nicotiana tabacum UGT89A2 glycosyltransferase (NtUGT89A2). The enzyme is a monomer in solution that catalyzes the O-ß-glucosylation of di- and tri-hydroxylated and chlorinated derivatives of benzoic acid. NtUGT89A2 has a preference for 2,5-dihydroxybenzoic acid (2,5-DHBA) over 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,4-dihydroxybenzoic acid (2,4-DHBA). Other substrates that can be used by NtUGT89A2 include 3,4,5-trihydroxybenzoic acid and chlorinated derivatives such as 2-chloro-5-hydroxybenzoic acid (2-Cl-5-HBA). The substrates of NtUGT89A2 were identified by thermal stability experiments, where we observed a maximum increase of the thermal denaturation midpoint (Tm) of 10 °C in the presence of 2,5-DHBA and UDP-glucose. On the other hand, the highest specific activity was obtained with 2,5-DHBA (225 ± 1.7 nkat/mg). Further characterization revealed that the enzyme has a micromolar affinity for its substrates. Notably, the enzyme retains full activity after incubation at 70 °C for 1 h. These results provide a basis for future functional and structural studies of NtUGT89A2.