Crystal structures of rhamnosyltransferase UGT89C1 from Arabidopsis thaliana reveal the molecular basis of sugar donor specificity for UDP‐β‐l‐rhamnose and rhamnosylation mechanism

Abstract

Glycosylation is a key modification for most molecules including plant natural products, for example, flavonoids and isoflavonoids, and can enhance the bioactivity and bioavailability of the natural products. The crystal structure of plant rhamnosyltransferase UGT89C1 from Arabidopsis thaliana was determined, and the structures of UGT89C1 in complexes with UDP-b-L-rhamnose and acceptor quercetin revealed the detailed interactions between the enzyme and its substrates. Structural and mutational analysis indicated that Asp356, His357, Pro147 and Ile148 are key residues for sugar donor recognition and specificity for UDPb-L-rhamnose. The mutant H357Q exhibited activity with both UDP-b-L-rhamnose and UDP-glucose. Structural comparison and mutagenesis confirmed that His21 is a key residue as the catalytic base and the only catalytic residue involved in catalysis independently as UGT89C1 lacks the other catalytic Asp that is highly conserved in other reported UGTs and forms a hydrogen bond with the catalytic base His. Ser124 is located in the corresponding position of the catalytic Asp in other UGTs and is not able to form a hydrogen bond with His21. Mutagenesis further showed that Ser124 may not be important in its catalysis, suggesting that His21 and acceptor may form an acceptor-His dyad and UGT89C1 utilizes a catalytic dyad in catalysis instead of catalytic triad. The information of structure and mutagenesis provides structural insights into rhamnosyltransferase substrate specificity and rhamnosylation mechanism.

Complete Biosynthesis of the Anti-Diabetic Plant Metabolite Montbretin A

“…F3GGT1 (flavonoid 3-O-glucoside GT 1) catalyze 1,2-xylosylation of anthocyanidin 3-O-glycosides (Montefiori et al, 2011;Yonekura-Sakakibara et al, 2012;Itkin et al, 2013). A few studies investigated the molecular basis of sugar-donor specificity of UGTs, but much more remains to be learned (Osmani et al, 2009;Chen and Li, 2017;Zong et al, 2019). For example, a single amino acid was identified to confer a change in sugar donor specificity in two UGT89A2 isoforms in Arabidopsis (Chen and Li, 2017).…”

“…CcUGT4, which is specific for UDP-Xyl, contains a serine in this position. Using the first crystal structure of a rhamnosyltransferase, UGT89C1, Zong et al (2019) identified the histidine in position 357 in the PSPG-motif as a key amino acid for UDP-Rha specificity (Zong et al, 2019). Exchange with glutamine resulted in an enzyme that accepted both UDP-Rha and UDP-Glc.…”

Qualitative and Quantitative NAD+ Metabolomics Lead to Discovery of Multiple Functional Nicotinate N-Glycosyltransferase in Arabidopsis

“…PSPG motif (total 44 amino acids in almost all identified family 1 GTs) is highly conserved in all glycosyltransferases involved in a plant specialized metabolism, as this motif is thought to correspond to a UDP-binding site (Caputi et al, 2012). Variations in PSPG motif had been demonstrated to play an important role in the UDP-sugar donor specificity of plant family 1 GTs (Noguchi et al, 2009; Ono et al, 2010; Zong et al, 2019). There are three amino acids difference in the PSPG motif of UGT76C4 and UGT76C5: position 6 (D in UGT76C5 vs .…”

Structure-based enzyme engineering improves donor-substrate recognition of Arabidopsis thaliana glycosyltransferases

“…A thorough look at plant UGTs capable of recognizing diverse donor sugars of UDP, GDP, and dTDP base such as UGT89C1 [ 58 ] could help into understanding the difference in the substrate flexibility. This UGT's crystal structure in complex with UDP-rhamnose and acceptor quercetin has been recently solved [ 16 ], which improves our understanding of the mechanism of glycosylation and UGT engineering to aid the production of bioactive glycosides. In plant UGTs, the C-terminal domains are highly similar leading to their recognition of same or similar donors.…”