Institute of Biological Chemistry, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei, 11529, Taiwan.
National Center for High-performance Computing, National Applied Research Laboratories, No. 7, R&D 6th Road, Hsinchu Science Park, Hsinchu, 30076, Taiwan.
Enzyme Microb Technol. 2020 Sep;139:109585. doi: 10.1016/j.enzmictec.2020.109585. Epub 2020 May 5.
The undecaprenyl pyrophosphate phosphatase (UppP) is an integral membrane pyrophosphatase. In bacteria, UppP catalyzes the dephosphorylation of undecaprenyl pyrophosphate (C-pp) to undecaprenyl phosphate (C-P) in the periplasmic space, which is an essential step for the isoprenyl lipid carrier to reenter the peptidoglycan synthesis cycle. Besides bacteria, the UppP homologs are widely distributed in archaea genome. However, all archaea lack peptidoglycan structure in their cell wall components, and the major archaeal lipid carriers are dolichol phosphate (Dol-p) and dolichol pyrophosphate (Dol-pp), so the functions of the UppP homolog in archaea remain unclear. Here, we purified a recombinant polyisoprenyl pyrophosphatase of a thermoacidophilic archaeon, Saccharolobus solfataricus (SsUppP), and characterized its enzymatic properties. Two isoprenyl pyrophosphate, farnesyl pyrophosphate (Fpp) and geranylgeranyl pyrophosphate (Ggpp), were used as the surrogate substrates, simulating the bacterial and archaeal lipid carriers. SsUppP dephosphorylated Fpp and Ggpp at 37 °C, but retained the phosphatase activity at high temperatures. The optimal condition for the enzymatic activity was found to be at pH 7 and 70 °C. The thermostability of SsUppP was also supported by molecular dynamics simulation studies. Our results indicated that the archaeal SsUppP can dephosphorylate isoprenyl pyrophosphates at the natural environment of high temperature, and the possibility to catalyze the dephosphorylation of archaeal lipid carriers.
十一异戊烯焦磷酸磷酸酶（UppP）是一种整合膜焦磷酸酶。在细菌中，UppP 在周质空间中将十一异戊烯焦磷酸（C-pp）催化去磷酸化为十一异戊烯磷酸（C-P），这是异戊烯脂质载体重新进入肽聚糖合成循环的必要步骤。除了细菌，UppP 同源物广泛分布于古菌基因组中。然而，所有古菌的细胞壁成分中都缺乏肽聚糖结构，而主要的古菌脂质载体是焦磷酸多萜醇（Dol-p）和焦磷酸双萜醇（Dol-pp），因此 UppP 同源物在古菌中的功能尚不清楚。在这里，我们纯化了一种嗜热嗜酸古菌 Saccharolobus solfataricus 的重组多异戊烯焦磷酸酶（SsUppP），并对其酶学性质进行了表征。两种异戊烯焦磷酸，法呢基焦磷酸（Fpp）和香叶基焦磷酸（Ggpp）被用作替代底物，模拟细菌和古菌的脂质载体。SsUppP 在 37°C 下可使 Fpp 和 Ggpp 去磷酸化，但在高温下仍保留磷酸酶活性。发现酶活性的最佳条件为 pH7 和 70°C。分子动力学模拟研究也支持了 SsUppP 的热稳定性。我们的结果表明，古菌 SsUppP 可以在高温的自然环境中使异戊烯焦磷酸去磷酸化，并且有可能催化古菌脂质载体的去磷酸化。
