Jeon Jeong Ho, Lim Jae Kyu, Kim Min-Sik, Yang Tae-Jun, Lee Seong-Hyuk, Bae Seung Seob, Kim Yun Jae, Lee Sang Hee, Lee Jung-Hyun, Kang Sung Gyun, Lee Hyun Sook
Marine Biotechnology Research Division, Korea Institute of Ocean Science and Technology, Ansan, South Korea.
Extremophiles. 2015 Jan;19(1):109-18. doi: 10.1007/s00792-014-0689-y. Epub 2014 Aug 21.
The F420-reducing hydrogenase has been known as a key enzyme in methanogenesis. Its homologs have been identified in non-methanogenic hyperthermophilic archaea, including Thermococcus onnurineus NA1, but neither physiological function nor biochemical properties have been reported to date. The enzyme of T. onnurineus NA1 was distinguished from those of other methanogens and the members of the family Desulfurobacteriaceae with respect to the phylogenetic distribution of the α and β subunits, organization of frhAGB genes and conservation of F420-coordinating residues. RT-qPCR and Western blot analyses revealed frhA gene is not silent but is expressed in T. onnurineus NA1 grown in the presence of sulfur, carbon monoxide, or formate. The trimeric enzyme complex was purified to homogeneity via affinity chromatography from T. onnurineus NA1 and exhibited catalytic activity toward the electron acceptors such as viologens and flavins but not the deazaflavin coenzyme F420. This is the first biochemical study on the function of the frhAGB-encoding enzyme from a non-methanogenic archaea.
F420还原氢化酶一直被认为是产甲烷过程中的关键酶。其同源物已在非产甲烷嗜热古菌中被鉴定出来，包括嗜热栖热袍菌NA1，但迄今为止，其生理功能和生化特性均未见报道。嗜热栖热袍菌NA1的这种酶在α和β亚基的系统发育分布、frhAGB基因的组织以及F420配位残基的保守性方面，与其他产甲烷菌和脱硫杆菌科的成员有所不同。逆转录定量聚合酶链反应（RT-qPCR）和蛋白质免疫印迹分析表明，frhA基因并非沉默基因，而是在以硫、一氧化碳或甲酸盐为生长底物的嗜热栖热袍菌NA1中表达。通过亲和层析从嗜热栖热袍菌NA1中纯化得到了三聚体酶复合物，该复合物对诸如紫精和黄素等电子受体具有催化活性，但对脱氮黄素辅酶F420没有催化活性。这是首次对非产甲烷古菌中编码frhAGB的酶的功能进行生化研究。
