UWA School of Agriculture and Environment, The University of Western Australia, 35-Stirling Highway, Perth, WA 6009, Australia.
Int J Mol Sci. 2023 Aug 2;24(15):12356. doi: 10.3390/ijms241512356.
Uridine diphosphate glycosyltransferases (UGTs) are known for promiscuity towards sugar acceptors, a valuable characteristic for host plants but not desirable for heterologous biosynthesis. UGTs characterized for the O-glycosylation of isoflavonoids have shown a variable efficiency, substrate preference, and OH site specificity. Thus, 22 UGTs with reported isoflavonoid O-glycosylation activity were analyzed and ranked for OH site specificity and catalysis efficiency. Multiple-sequence alignment (MSA) showed a 33.2% pairwise identity and 4.5% identical sites among selected UGTs. MSA and phylogenetic analysis highlighted a comparatively higher amino acid substitution rate in the N-terminal domain that likely led to a higher specificity for isoflavonoids. Based on the docking score, OH site specificity, and physical and chemical features of active sites, selected UGTs were divided into three groups. A significantly high pairwise identity (67.4%) and identical sites (31.7%) were seen for group 1 UGTs. The structural and chemical composition of active sites highlighted key amino acids that likely define substrate preference, OH site specificity, and glycosylation efficiency towards selected (iso)flavonoids. In conclusion, physical and chemical parameters of active sites likely control the position-specific glycosylation of isoflavonoids. The present study will help the heterologous biosynthesis of glycosylated isoflavonoids and protein engineering efforts to improve the substrate and site specificity of UGTs.
尿苷二磷酸糖基转移酶（UGTs）以对糖受体的混杂性而闻名，这对宿主植物来说是一个有价值的特性，但对异源生物合成来说则不是。已经对黄酮类化合物的 O-糖基化进行了表征的 UGTs 表现出了不同的效率、底物偏好和 OH 位点特异性。因此，对具有报道的黄酮类 O-糖基化活性的 22 个 UGT 进行了分析和排序，以确定其 OH 位点特异性和催化效率。多重序列比对（MSA）显示，选定的 UGT 之间具有 33.2%的成对同一性和 4.5%的相同位点。MSA 和系统发育分析突出了 N 端结构域中氨基酸替代率相对较高，这可能导致对黄酮类化合物具有更高的特异性。根据对接评分、OH 位点特异性以及活性位点的物理和化学特性，选择了 UGT 分为三组。第 1 组 UGT 的成对同一性（67.4%）和相同位点（31.7%）非常高。活性位点的结构和化学组成突出了关键的氨基酸，这些氨基酸可能决定了对所选（异）黄酮类化合物的底物偏好、OH 位点特异性和糖基化效率。总之，活性位点的物理和化学参数可能控制黄酮类化合物的位置特异性糖基化。本研究将有助于异源生物合成糖基化黄酮类化合物和蛋白质工程努力，以提高 UGT 的底物和位点特异性。
