Kuorelahti Satu, Kalkkinen Nisse, Penttilä Merja, Londesborough John, Richard Peter
VTT Biotechnology, P.O. Box 1500, 02044 VTT, Espoo, Finland.
Biochemistry. 2005 Aug 23;44(33):11234-40. doi: 10.1021/bi050792f.
A d-galacturonic acid reductase and the corresponding gene were identified from the mold Hypocrea jecorina (Trichoderma reesei). We hypothesize that the enzyme is part of a fungal d-galacturonic acid catabolic pathway which has not been described previously and which is distinctly different from the bacterial pathway. H. jecorina grown on d-galacturonic acid exhibits an NADPH-dependent d-galacturonic acid reductase activity. This activity is absent when the mold is grown on other carbon sources. The d-galacturonic acid reductase was purified, and tryptic digests of the purified protein were sequenced. The open reading frame of the corresponding gene was then cloned from a cDNA library. The open reading frame was functionally expressed in the yeast Saccharomyces cerevisiae. A histidine-tagged protein was purified, and the enzyme kinetics were characterized. The enzyme converts in a reversible reaction from d-galacturonic acid and NADPH to l-galactonic acid and NADP. The enzyme also exhibits activity with d-glucuronic acid and dl-glyceraldehyde.
从霉菌里氏木霉（Hypocrea jecorina，即Trichoderma reesei）中鉴定出了一种D-半乳糖醛酸还原酶及其相应基因。我们推测该酶是真菌D-半乳糖醛酸分解代谢途径的一部分，此途径此前尚未被描述，且与细菌途径明显不同。在D-半乳糖醛酸上生长的里氏木霉表现出一种依赖NADPH的D-半乳糖醛酸还原酶活性。当该霉菌在其他碳源上生长时，这种活性不存在。对D-半乳糖醛酸还原酶进行了纯化，并对纯化后的蛋白质的胰蛋白酶消化产物进行了测序。然后从cDNA文库中克隆了相应基因的开放阅读框。该开放阅读框在酿酒酵母（Saccharomyces cerevisiae）中进行了功能表达。纯化了一种带有组氨酸标签的蛋白质，并对其酶动力学进行了表征。该酶在一个可逆反应中能将D-半乳糖醛酸和NADPH转化为L-半乳糖酸和NADP。该酶对D-葡萄糖醛酸和D,L-甘油醛也表现出活性。
