A Highly Active Formate Dehydrogenase Fdh3 toward NADP+ and Its Diaphorase Subunit from Hyperthermophilic Archaeon Thermococcus onnurineus NA1

Title
A Highly Active Formate Dehydrogenase Fdh3 toward NADP+ and Its Diaphorase Subunit from Hyperthermophilic Archaeon Thermococcus onnurineus NA1
Author(s)
Yang, Ji-in; Choi, Bo Gyoung; Lee, Hyun Sook; Kang, Sung Gyun
KIOST Author(s)
Yang, Ji In(양지인)Choi, Bo Gyoung(최보경)Lee, Hyun Sook(이현숙)Kang, Sung Gyun(강성균)
Alternative Author(s)
양지인; 최보경; 이현숙; 강성균
Publication Year
2023-06-21
Abstract
It has been identified that the genome of T. onnurineus NA1 possesses three genes, fdh1, fdh2, and fdh3, which were annotated as formate dehydrogenases (FDHs). Fdh2 forms a putative formate-dependent respiratory complex with Mfh2 hydrogenase and plays an essential role in formate-dependent energy metabolism. On the other hand, until recently, the functions and properties of Fdh1 and Fdh3 had not been revealed. In a recent study, we reported that the novel cytoplasmic NADP+-dependent Fdh3 consist of a tungsten-containing catalytic subunit (Fdh3A), a flavin-containing subunit (Fdh3B), and two Fe-S proteins (Fdh3G1 and Fdh3G2). In contrast to the most NAD+-dependent FDHs, Fdh3 displayed a prominent preference for NADP+ as a cofactor. The catalytic efficiency (kcat/Km) of Fdh3 for NADP+ was measured to be 5,281 mM-1s-1, which is suitable for NADPH regeneration. In this study, we separately expressed and purified a putative NAD(P)+-reducing subunit Fdh3B from Escherichia coli Rosetta and T. onnurineus NA1, respectively. According to the structural modeling, Arg204 and Arg205 residues of Fdh3B may contribute to the stabilization of the 2-phosphate group of NADP(H). Fdh3B alone exhibited NADPH-diaphorase activity with a specific activity of 404 U/mg using benzyl viologen, which was determined at the optimal conditions of 70 ℃ and pH 9.5. These results suggest that the high NADP+ conversion rate of Fdh3 is derived from the properties of Fdh3B.
URI
https://sciwatch.kiost.ac.kr/handle/2020.kiost/44376
Bibliographic Citation
2023년 학회 창립 50주년 기념 국제학술대회 및 정기학술대회, pp.40, 2023
Publisher
한국미생물생명공학회
Type
Conference
Language
English
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