A kinetic analysis of regiospecific glucosylation by two glycosyltransferases of Arabidopsis thaliana: domain swapping to introduce new activities

Abstract

Plant Family 1 glycosyltransferases (GTs) recognize a wide range of natural and non-natural scaffolds and have considerable potential as biocatalysts for the synthesis of small molecule glycosides. Regiospecificity of glycosylation is an important property, given that many acceptors have multiple potential glycosylation sites. This study has used a domain-swapping approach to explore the determinants of regiospecific glycosylation of two GTs of Arabidopsis thaliana, UGT74F1 and UGT74F2. The flavonoid quercetin was used as a model acceptor, providing five potential sites for O-glycosylation by the two GTs. As is commonly found for many plant GTs, both of these enzymes produce distinct multiple glycosides of quercetin. A high performance liquid chromatography method has been established to perform detailed steady-state kinetic analyses of these concurrent reactions. These data show the influence of each parameter in determining a GT product formation profile toward quercetin. Interestingly, construction and kinetic analyses of a series of UGT74F1/F2 chimeras have revealed that mutating a single amino acid distal to the active site, Asn-142, can lead to the development of a new GT with a more constrained regiospecificity. This ability to form the 4 '-O-glucoside of quercetin is transferable to other flavonoid scaffolds and provides a basis for preparative scale production of flavonoid 4 '-O-glucosides through the use of whole-cell biocatalysis.

FIGURE 1.

Amino acid sequence alignment and secondary structure prediction of UGT74F1 and UGT74F2. Sequence identity (76%) is shaded light gray, similarity (90%) dark gray. The diagnostic PSPG motif of plant Family 1 GTs is underlined. The predicted secondary structure (analyzed using the JPred server) (45) of UGT74F1 and UGT74F2 is identical and is illustrated by an arrow for a β-sheet and a cylinder for an α-helix. Each of the four amino acid positions (67, 112, 153, 239) corresponding to a domain-swapping shuffle point is indicated by an asterisk.

FIGURE 2.

An HPLC-based assay to determine kinetic parameters of UGT74F1 and UGT74F2. A, the flavonoid quercetin (Q). B, recombinant UGT74F1 and UGT74F2 (2μg of each) analyzed by 10% (w/v) SDS-PAGE. C and D, HPLC analysis of UGT74F1 and UGT74F2 activity toward 100 μm quercetin. E and F, Hanes plot of UGT74F1 and UGT74F2 activity toward the OH groups of quercetin. The error bars in E and F represent the S.D. of independent triplicate data sets. The kinetic parameters k_cat and K_m for UGT74F1 and UGT74F2 activity are shown in Table 1.

FIGURE 3.

UGT74F1 and UGT74F2 chimeras. A, schematic representation of parent UGT74F1*, UGT74F2*, and the generated chimeras. The introduced shuffle points are given their equivalent amino acid position, partitioning the primary sequence into five segments. Chimeras are named according to their composition of UGT74F1 or UGT74F2 sequence. B, 10% (w/v) SDS-PAGE analysis of UGT74F1/F2 chimeric proteins (2 μg of each). C, thermal denaturation curves of UGT74F1(•), UGT74F2(+), F22221(▴), and F22211(⋄) recombinant proteins with no GST fusion tag. The percentage change in CD signal at 220 nm, relative to 10 °C, was measured over the temperature range 10 to 80 °C.

FIGURE 4.

The kinetic parameters k_cat, K_m, and the specificity constant (k_cat/K_m) of UGT74F1*, UGT74F2*, and chimeras toward quercetin. Enzyme (0.2–2 μg), quercetin concentration (4–200 μm), and reaction time (0.5–10 min) were varied to analyze product using the pseudo-single substrate assumptions described under “Experimental Procedures.” UDPG concentration was maintained in excess (7 mm). Error bars represent S.D. of independent triplicate data sets.

FIGURE 5.

Regiospecificity of UGT74F1 mutants toward quercetin. A, amino acid alignment of UGT74F1 and UGT74F2 from position 112 to 153. Amino acid identity is shown in light gray, similarity in dark gray. B, the site-directed mutagenesis of UGT74F1 focused on Ser-135 and Asn-142. Error bars represent the S.D. of independent triplicate data sets.

FIGURE 6.

Regiospecificity of UGT74F1 and UGT74F1 N142Y toward a range of flavonoid substrates. Error bars represent the S.D. of triplicate data sets.

FIGURE 7.

Whole-cell biotransformation of quercetin by E. coli expressing UGT74F1 N142Y and purification of quercetin 4 ′-O-glucoside. A, time course of quercetin 4′-O-glucoside (Q4′-O-Glc) formation by 1 liter of shake flask cultures of E. coli BL21 (DE3) harboring pGEX-2T/UGT74F1 N142Y. Time point zero equates to quercetin (Q) addition. B, analysis of the amount of Q 4′-O-Glc during the initial concentration and purification of the fermentation medium on an Amberlite XAD-2 column. The panel shows the total amount of Q4′-O-Glc in the filtrate (F) before application to the column, the flow-through (FT), H₂O wash (W), and the eluates of increasing acetonitrile concentration. Error bars represent the S.D. of triplicate data sets. C, preparative HPLC spectra. Analysis of individual fractions identified quercetin 7,4′-di-O-gluco-side (Q 7,4′-di-O-Glc),Q4′-O-Glc, Q 3′-O-glucoside (Q3′-O-Glc), and Q by comparison to known standards. D, HPLC analysis of purified Q 4′-O-Glc.