Purification, kinetic characterization, and site-directed mutagenesis of Methanothermobacter thermautotrophicus RFAP Synthase Produced in Escherichia coli
Purification, kinetic characterization, and site-directed mutagenesis of Methanothermobacter thermautotrophicus RFAP Synthase Produced in Escherichia coli
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DOI：
10.3934/microbiol.2019.3.186
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发表时间：
2019-01-01
影响因子：
4.8
通讯作者：
Rasche, Madeline E.
中科院分区：
文献类型：
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作者：
Bechard, Matthew E.;Farahani, Payam;Rasche, Madeline E.
产生甲烷的古细菌是一组精选的微生物，它们利用四氢甲烷蝶呤 (H4MPT) 作为单碳载体而不是四氢叶酸。在 H4MPT 生物合成中，β-呋喃核糖基氨基苯 5'-磷酸 (RFAP) 合酶催化从对氨基苯甲酸 (pABA) 和磷酸核糖基焦磷酸 (PRPP) 生成 RFAP、CO2 和焦磷酸盐。在这项工作中，为了深入了解底物结合所需的氨基酸残基，在大肠杆菌中产生了来自嗜热甲烷嗜热杆菌的 RFAP 合酶，并使用定点诱变来改变精氨酸 26 (R26) 和天冬氨酸 19 (D19)，它们位于类似于二氢叶酸合酶的 pABA 结合位点的保守氨基酸序列中。用赖氨酸替换 R26 使 pABA 的 K-M 相对于野生型酶增加了一个数量级，而没有显着改变 PRPP 的 K-M。虽然用丙氨酸取代 D19 会产生无活性的酶，但天冬酰胺取代可保留一些活性，并且 pABA 的 K-M 相对于野生型酶增加约三倍。通过将 RFAP 合酶连接到高丝氨酸激酶的晶体结构上而开发的分子模型将 R26 置于拟议的活性位点。在静态模型中，D19 位于靠近活性位点的位置，但看起来距离太远而无法直接影响配体结合。这可能表明先前在 Bi-Ter 动力学机制中预测的蛋白质构象变化和/或两个亚基界面处活性位点的形成。由于 RFAP 合酶在 H4MPT 生物合成中的重要作用，深入了解底物结合模式和机制可能有利于开发旨在减少温室气体甲烷产生的 RFAP 合酶抑制剂。
Methane-producing archaea are among a select group of microorganisms that utilize tetrahydromethanopterin (H4MPT) as a one-carbon carrier instead of tetrahydrofolate. In H4MPT biosynthesis, beta-ribofuranosylaminobenzene 5'-phosphate (RFAP) synthase catalyzes the production of RFAP, CO2, and pyrophosphate from p-aminobenzoic acid (pABA) and phosphoribosyl-pyrophosphate (PRPP). In this work, to gain insight into amino acid residues required for substrate binding, RFAP synthase from Methanothermobacter thermautotrophicus was produced in Escherichia coli, and site-directed mutagenesis was used to alter arginine 26 (R26) and aspartic acid 19 (D19), located in a conserved sequence of amino acids resembling the pABA binding site of dihydropteroate synthase. Replacement of R26 with lysine increased the K-M for pABA by an order of magnitude relative to wild-type enzyme without substantially altering the K-M for PRPP. Although replacement of D19 with alanine produced inactive enzyme, asparagine substitution allowed retention of some activity, and the K-M for pABA increased about threefold relative to wild-type enzyme. A molecular model developed by threading RFAP synthase onto the crystal structure of homoserine kinase places R26 in the proposed active site. In the static model, D19 is located close to the active site, yet appears too far away to influence ligand binding directly. This may be indicative of the protein conformational change predicted previously in the Bi-Ter kinetic mechanism and/or formation of the active site at the interface of two subunits. Due to the vital role of RFAP synthase in H4MPT biosynthesis, insights into the mode of substrate binding and mechanism could be beneficial for developing RFAP synthase inhibitors designed to reduce the production of methane as a greenhouse gas.