Electron Donor Systems to Facilitate Development of Assays for Two Flavoproteins Involved in Tetrahydromethanopterin Biosynthesis

• 作者: Madeline E. Rasche
• 来源期刊: 微生物学(英文)
• 学科: 生物学
• 关键词: Methanopterin Coenzyme Biosynthesis Dihydromethanopterin Reductase FLAVOPROTEIN ARCHAEA METHANOGENESIS
• 年，卷（期）: 2020,（6）
• 页码范围: 275-294
• 页数: 20页
• 分类号: Q78

摘要

<span style="font-family:;" "=""><span style="font-family:Verdana;">Methane production by archaea depends on tetrahydromethanopterin (H</span>_{<span style="font-family:Verdana;">4</span>}<span style="font-family:Verdana;">MPT), a pterin-containing cofactor that carries one-carbon units. Two redox reactions within the nine steps of H</span>_{<span style="font-family:Verdana;">4</span>}<span style="font-family:Verdana;">MPT side chain biosynthesis have been hypothesized. Biochemical assays have demonstrated that the archaeal iron-sulfur flavoprotein dihydromethanopterin reductase X (DmrX or MM1854) catalyzes the final reaction of the pathway, the reduction of dihydromethanopterin to H</span>_{<span style="font-family:Verdana;">4</span>}<span style="font-family:Verdana;">MPT</span></span><span style="font-family:Verdana;">,</span><span style="font-family:;" "=""><span style="font-family:Verdana;"> using dithiothreitol (DTT) as an artificial electron donor. The crystal structure of DmrB, a bacterial DmrX homolog that lacks iron-sulfur clusters, has led to a proposed ping-pong mechanism of electron transfer between FMNH</span>_{<span style="font-family:Verdana;">2</span>}<span style="font-family:Verdana;"> and the FMN prosthetic group of DmrB. However, an enzymatic assay to test the hypothetical DmrB mechanism is lacking because a suitable electron donor has not previously been identified. Furthermore, a second uncharacterized archaeal flavoprotein (MM1853) has been hypothesized to function in H</span>_{<span style="font-family:Verdana;">4</span>}<span style="font-family:Verdana;">MPT side chain biosynthesis. In this </span><span style="font-family:Verdana;">work, to facilitate the development of assays to elucidate the functions of DmrB </span><span style="font-family:Verdana;">and MM1853, we tested a variety of electron donors, including dithiothreitol, ferredoxin, and a system consisting of NADH and an NADH-dependent fla</span><span style="font-family:Verdana;">vin-reducing