Weissensteiner Julia, Molitor Christian, Marinovic Silvija, Führer Lisa, Waqas Hassan Syed, Hutabarat Olly Sanny, Spornberger Andreas, Stich Karl, Hausjell Johanna, Spadiut Oliver, Haselmair-Gosch Christian, Halbwirth Heidi
Institute of Chemical, Environmental and Bioscience Engineering, Technische Universität Wien, Getreidemarkt 9, 1060 Vienna, Austria.
Department of Bio-Sciences, University of Wah, Quaid Avenue, Wah 47040, Pakistan.
Plants (Basel). 2021 Sep 19;10(9):1956. doi: 10.3390/plants10091956.
× (apple) accumulates particularly high amounts of dihydrochalcones in various tissues, with phloridzin (phloretin 2'--glucoside) being prevalent, although small amounts of 3-hydroxyphloretin and 3-hydroxyphloridzin are also constitutively present. The latter was shown to correlate with increased disease resistance of transgenic × plants. Two types of enzymes could be involved in 3-hydroxylation of dihydrochalcones: polyphenol oxidases or the flavonoid 3'-hydroxylase (F3'H), which catalyzes B-ring hydroxylation of flavonoids. We isolated two F3'H cDNA clones from apple leaves and tested recombinant F3'Hs for their substrate specificity. From the two isolated cDNA clones, only encoded a functionally active enzyme. In the F3'HI sequence, we identified two putatively relevant amino acids that were exchanged in comparison to that of a previously published F3'HI. Site directed mutagenesis, which exchanged an isoleucine into methionine in position 211 restored the functional activity, which is probably because it is located in an area involved in interaction with the substrate. In contrast to high activity with various flavonoid substrates, the recombinant enzymes did not accept phloretin under assay conditions, making an involvement in the dihydrochalcone biosynthesis unlikely.
×（苹果）在各种组织中积累了特别大量的二氢查耳酮，其中根皮苷（根皮素2'-葡萄糖苷）最为普遍，不过也有少量的3-羟基根皮素和3-羟基根皮苷组成性存在。后者已被证明与转基因×植物抗病性的提高相关。二氢查耳酮的3-羟基化可能涉及两种类型的酶：多酚氧化酶或类黄酮3'-羟化酶（F3'H），后者催化类黄酮B环的羟基化。我们从苹果叶片中分离出两个F3'H cDNA克隆，并测试了重组F3'H的底物特异性。从分离出的两个cDNA克隆中，只有一个编码具有功能活性的酶。在F3'HI序列中，我们鉴定出两个与先前发表的F3'HI相比发生了交换的假定相关氨基酸。将第211位的异亮氨酸替换为甲硫氨酸的定点诱变恢复了功能活性，这可能是因为它位于与底物相互作用的区域。与对各种类黄酮底物具有高活性相反，重组酶在测定条件下不接受根皮素，因此不太可能参与二氢查耳酮的生物合成。
