Isolation of Legume Glycosyltransferases and Active Site Mapping of the <i>Phaseolus lunatus</i> Zeatin O-glucosyltransferase ZOG1

O-Glycosides of the cytokinin zeatin are found in many plant tissues. They provide protection against degradative enzymes and may serve as cytokinin reserves. Two zeatin glycosyltransferase (GT) genes, an O-glucosyltransferase ( ZOG1 ) from Phaseolus lunatus and an O-xylosyltransferase ( ZOX1 ) from P. vulgaris , were previously isolated. Five novel bean and soybean GT genes with high sequence identity to ZOG1 were isolated, sequenced, and expressed, along with two such genes from rice and one from tomato. None of the recombinant proteins showed GT activity with zeatin. By comparing the ZOG1 sequence to the 3D model of Medicago truncatula UGT71G1, four regions possibly important to zeatin binding were identified, and mutation studies identified one amino acid within each region (R59, D87, L127, and F149) whose mutation strongly impaired enzyme activity. The new bean and soybean GTs differ from ZOG1 in one ( Pl GT2 and Gm GT2) to three ( Gm GT1) of these residues. Mutation of one such GT ( Pl GT2) to render it identical to ZOG1 at the four implicated residues conferred low enzyme activity, providing further support for the importance of these amino acids in recognizing zeatin as substrate.