The UDP-glycosyltransferase UGT88F subfamily has been described first in Malus x domestica with the characterization of UGT88F1. Up to now UGT88F1 was one of the most active UGT glycosylating dihydrochalcones in vitro. The involvement of UGT88F1 in phloridzin (phloretin 2'-O-glucoside) synthesis, the main apple tree dihydrochalcone, was further confirmed in planta. Since the characterization of UGT88F1, this new UGT subfamily has been poorly studied probably because it seemed restricted to Maloideae. In the present study, we investigate the apple tree genome to identify and biochemically characterize the whole UGT88F subfamily. The apple tree genome contains five full-length UGT88F genes out of which three newly identified members (UGT88F6, UGT88F7 and UGT88F8) and a pseudogene. These genes are organized into two genomic clusters resulting from the recent global genomic duplication event in the apple tree. We show that recombinant UGT88F8 protein specifically glycosylates phloretin in the 2'OH position to synthetize phloridzin in vitro and was therefore named UDP-glucose: phloretin 2'-O-glycosyltransferase. The Km values of UGT88F8 are 7.72 μM and 10.84 μM for phloretin and UDP-glucose respectively and are in the same range as UGT88F1 catalytic parameters thus constituting two isoforms. Co-expression patterns of both UGT88F1 and UGT88F8 argue for a redundant function in phloridzin biosynthesis in planta. Contrastingly, recombinant UGT88F6 protein is able to glycosylate in vitro a wide range of flavonoids including flavonols, flavones, flavanones, chalcones and dihydrochalcones, although flavonols are the preferred substrates, e.g. Km value for kaempferol is 2.1 μM. Depending on the flavonoid, glycosylation occurs at least on the 3-OH and 7-OH positions. Therefore UGT88F6 corresponds to an UDP-glucose: flavonoid 3/7-O-glycosyltransferase. Finally, a molecular modeling study highlights a very high substitution rate of residues in the acceptor binding pocket between UGT88F8 and UGT88F6 which is responsible for the enzymes divergence in substrate and regiospecificity, despite an overall high protein homology.

中文翻译：

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Malus xdomestica Borkh UGT88F 亚科的全基因组鉴定和生化表征

UDP-糖基转移酶UGT88F 亚家族首先在Malus xdomestica 中被描述，并具有UGT88F1 的特征。到目前为止，UGT88F1 是体外最活跃的 UGT 糖基化二氢查耳酮之一。UGT88F1 参与根皮苷（根皮素 2'-O-葡萄糖苷）合成，主要的苹果树二氢查耳酮，在植物中得到进一步证实。自从对 UGT88F1 进行表征以来，这个新的 UGT 亚科一直没有得到很好的研究，可能是因为它似乎仅限于 Maloideae。在本研究中，我们调查了苹果树基因组以鉴定和生化表征整个 UGT88F 亚科。苹果树基因组包含五个全长 UGT88F 基因，其中三个新鉴定的成员（UGT88F6、UGT88F7 和 UGT88F8）和一个假基因。这些基因被组织成两个基因组簇，这是由最近苹果树中的全球基因组复制事件产生的。我们显示重组 UGT88F8 蛋白在 2'OH 位置特异性糖基化根皮素以在体外合成根皮苷，因此被命名为 UDP-葡萄糖：根皮素 2'-O-糖基转移酶。UGT88F8 的 Km 值对于根皮素和 UDP-葡萄糖分别为 7.72 μM 和 10.84 μM，与 UGT88F1 催化参数在同一范围内，因此构成了两个同种型。UGT88F1 和 UGT88F8 的共表达模式证明了植物根皮苷生物合成的冗余功能。相比之下，重组 UGT88F6 蛋白能够在体外糖基化多种黄酮类化合物，包括黄酮醇、黄酮、黄烷酮、查耳酮和二氢查耳酮，尽管黄酮醇是优选的底物，例如山奈酚的 Km 值为 2.1 μM。根据类黄酮，糖基化至少发生在 3-OH 和 7-OH 位置。因此UGT88F6对应于UDP-葡萄糖：类黄酮3/7-O-糖基转移酶。最后，分子建模研究强调了 UGT88F8 和 UGT88F6 之间受体结合口袋中残基的非常高的取代率，尽管总体蛋白质同源性很高，但这是导致酶在底物和区域特异性方面出现差异的原因。