Bian Mingjie, Li Shan, Wei Huanhuan, Huang Shiping, Zhou Feng, Zhu Youming, Zhu Guoping
Institute of Molecular Biology and Biotechnology and the Research Center of Life Omics and Health, Anhui Normal University, No.1 Beijing East Road, Wuhu 241000, Anhui, China.
Department of Oral and Maxillofacial Surgery, School of Stomatology, Stomatological Hospital, Anhui Medical University, No.81 Mei Shan Road, Hefei 230032, Anhui, China.
Protein Expr Purif. 2018 Aug;148:1-8. doi: 10.1016/j.pep.2018.03.007. Epub 2018 Mar 23.
Yarrowia lipolytica, a nonpathogenic, nonconventional, aerobic and dimorphic yeast, is considered an oleaginous microorganism due to its excellent ability to accumulate large amounts of lipids. Glucose-6-phosphate dehydrogenase (G6PD) is one of two key enzymes involved in the lipid accumulation in this fungi, which catalyzes the oxidative dehydrogenation of glucose-6-phosphate to 6-phosphoglucono-δ-lactone with the reduction of NADP to NADPH. In this study, the full-length gene of G6PD from Y. lipolytica (YlG6PD) was cloned without intron and heterogeneously expressed in E. coli. Then, YlG6PD was purified and biochemically characterized in details. Kinetic analysis showed that YlG6PD was completely dependent on NADP and its apparent K for NADP was 33.3 μM. The optimal pH was 8.5 and the maximum activity was around 47.5 °C. Heat-inactivation profiles revealed that it remained 50% of maximal activity after incubation at 48 °C for 20 min YlG6PD activity was competitively inhibited by NADPH with a K value of 56.04 μM. Most of the metal ions have no effect on activity, but Zn was a strong inhibitor. Furthermore, the determinants in the coenzyme specificity of YlG6PD were investigated. Kinetic analysis showed that the single mutant R52D completely lost the ability to utilize NADP as its coenzyme, suggesting that Arg-52 plays a decisive role in NADP binding in YlG6PD. The identification of Y. lipolytica G6PD may provide useful scientific information for metabolic engineering of this yeast as a model for bio-oil production.
解脂耶氏酵母是一种非致病性、非常规、需氧的二态性酵母，因其具有出色的大量积累脂质的能力而被视为产油微生物。葡萄糖-6-磷酸脱氢酶（G6PD）是参与这种真菌脂质积累的两种关键酶之一，它催化葡萄糖-6-磷酸氧化脱氢生成6-磷酸葡萄糖酸-δ-内酯，同时将NADP还原为NADPH。在本研究中，克隆了解脂耶氏酵母G6PD的全长基因（YlG6PD），该基因无内含子，并在大肠杆菌中进行了异源表达。然后，对YlG6PD进行了纯化并详细进行了生化特性分析。动力学分析表明，YlG6PD完全依赖于NADP，其对NADP的表观K值为33.3μM。最适pH为8.5，最大活性温度约为47.5°C。热失活曲线表明，在48°C孵育20分钟后，其仍保留50%的最大活性。YlG6PD活性受到NADPH的竞争性抑制，K值为56.04μM。大多数金属离子对活性没有影响，但锌是一种强抑制剂。此外，还研究了YlG6PD辅酶特异性的决定因素。动力学分析表明，单突变体R52D完全丧失了利用NADP作为辅酶的能力，这表明精氨酸-52在YlG6PD与NADP的结合中起决定性作用。解脂耶氏酵母G6PD的鉴定可能为将这种酵母作为生物油生产模型进行代谢工程改造提供有用的科学信息。
