Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
Institute of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan.
Biosci Biotechnol Biochem. 2023 Dec 19;88(1):63-69. doi: 10.1093/bbb/zbad142.
MdDOX-Co, the ectopic expression of which is considered to cause the apple columnar tree shape, belongs to the 2-oxoglutarate-dependent dioxygenase (2ODD) family. It adds a hydroxyl group to position 12 of gibberellins (GAs). However, the 2ODD enzymes related to GA biosynthesis and catabolism are phylogenetically distinct from MdDOX-Co. Thus, it is possible that substrates other than GAs exist in MdDOX-Co. To identify the previously unidentified substrate(s) of MdDOX-Co, we searched for MdDOX-Co-specific inhibitors. Chemical screening using gas chromatography-mass spectrometry was performed to investigate the effects of 2400 compounds that inhibited the catalytic reaction of MdDOX-Co, but not the catabolic reaction of GA 2-oxidase, an enzyme involved in GA catabolism. By applying two positive compounds in Arabidopsis, a chemical 3-((2-chloro-6-fluorobenzyl)thio)-5,7-dimethyl-5H-pyrazolo[3,4-e][1,4,2]dithiazine-1,1-dioxide designated as TPDD that did not inhibit GA biosynthesis was selected. The structure-activity relationships among the TPDD analogs were also obtained.
MdDOX-Co 的异位表达被认为会导致苹果树柱状树形，属于 2- 酮戊二酸依赖性双加氧酶（2ODD）家族。它在赤霉素（GAs）的 12 位添加一个羟基。然而，与 GA 生物合成和分解代谢相关的 2ODD 酶在系统发育上与 MdDOX-Co 明显不同。因此，除了 GAs 之外，MdDOX-Co 可能存在其他底物。为了鉴定 MdDOX-Co 以前未识别的底物，我们寻找了 MdDOX-Co 特异性抑制剂。使用气相色谱-质谱联用进行化学筛选，以研究抑制 MdDOX-Co 催化反应但不抑制 GA2-氧化酶（参与 GA 分解代谢的酶）分解反应的 2400 种化合物的影响。通过在拟南芥中应用两种阳性化合物，选择了一种化学物质 3-((2-氯-6-氟苄基)硫基)-5,7-二甲基-5H-吡唑并[3,4-e][1,4,2]二噻嗪-1,1-二氧化物，指定为 TPDD，它不抑制 GA 生物合成。还获得了 TPDD 类似物的构效关系。
