銅耐性腐朽菌に特有なシュウ酸合成複合酵素系の機能解析とその制御法の開発

耐铜木材腐烂真菌（大部分属于褐腐担子菌）已被认为可以通过草酸盐的生物合成将铜沉淀为草酸铜，从而对含铜木材防腐剂进行解毒。因此，阐明这些耐铜真菌草酸生物合成的生化机制非常重要。本研究的新发现总结如下： 1．我们发现，大多数腐木担子菌虽然是在富含葡萄糖的培养基上生长的，但它们却具有乙醛酸循环酶，这与传统的认为在葡萄糖上生长的微生物中乙醛酸循环酶受到抑制的观点形成鲜明对比。 2．我们首次从耐铜真菌Tyromyces (Fomitopsis) palustris中成功纯化并表征了乙醛酸循环的关键酶，如异柠檬酸裂解酶(ICL)和苹果酸合酶(MS)。 3．重要的是，我们可以使用 ICL 酶抑制剂成功控制耐铜真菌 Tyromyces palustris，该抑制剂还可以有效抑制草酸合酶。我们发现 TCA 和乙醛酸循环在草酸生物合成中发挥着重要作用，为真菌生长产生能量4。褐腐真菌子实体形成过程中，草酸生物合成停止，乙醛酸循环酶和草酸产生酶消失，而异柠檬酸脱氢酶(ICDH)被激活，并在ICL上占主导地位。结果进一步表明，在真菌生长后期，ICDH比ICL发挥了更重要的作用，支持GABA途径进行TCA循环的运行。 5．重要的是，在这种真菌的整个生命周期中，我们发现它缺乏α-酮戊二酸脱羧酶，而α-酮戊二酸脱羧酶是植物和动物中正常TCA循环功能的关键酶。因此，乙醛酸循环和 GABA 途径交替发挥重要作用，以补偿这些耐铜真菌中正常 TCA 循环的功能障碍。较少的

Copper-tolerant wood rotting fungi most of which belong to a group of brown-rot basidiomycestes have been recognized to detoxicate copper-contining wood preservertives by precipitating copper into copper oxalate by biosynthesis of oxlate. Thus, it is very important to elucidate biochemical mechanisms for oxalate biosyntheis in these copper-tolerant fungi.New findings obtained by this ivestigation are summized as follows.1. We have discovered that most of the wood-rotting basidiomyceses have the glyoxylate cycle enzymes although thy were grown on gluose-rich medium, which is sharply contrasted with the clasical view that the glyolxylate cycle enzymes are repressed in microorganisms grown on glucose.2. We sucessfuly purified and characterized the key enzymes of the glyxolylate cycle such as isocitrate lyase (ICL) and malate synthase (MS) for the first time from a basidiomycetous fungus Tyromyces (Fomitopsis) palustris, which is a copper-tolerant fungus.3. Importanlty, we could successful … More ly control the copper-tolerant fungus Tyromyces palustris with ICL enzyme inhibior, which also found potently inhibit the oxalate bioxynthesis. We have found that both TCA and glyoxylate cycles play an important role in biosynthesis of oxalic acid to produce energy for the fungal growth4. During the fruitbody formation of the brown-rot fungus, the oxalate biosynthesis was ceased and the glyoxylate cycle enzymes and oxalate producing enzynes disappeared, whereas isocitate dehydrogenase (ICDH) was activated and predominate over the ICL.The results further indicate that at the later stage of thefungal growth ICDH played more important role than ICL to support the GABA route for operation of the TCA cycle.5. Importantly, throughout the life cycle of thisfungus, it was found to lack a-ketoglutarate decarbxylase which is a key enzyme of normal TCA cycle functioning in both plants and animals. Thus, the glyoxylate cycle and GABA route play alternately importantl role to compensate the desfunction of the normal TCA cycle in these copper-tolerant fungi. Less