Bechard Matthew E, Farahani Payam, Greene Dina, Pham Anna, Orry Andrew, Rasche Madeline E
Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN 37232.
Chemistry and Biochemistry Department, California State University at Fullerton, 800 North State College Blvd., Fullerton, CA 92834.
AIMS Microbiol. 2019 Jul 23;5(3):186-204. doi: 10.3934/microbiol.2019.3.186. eCollection 2019.
Methane-producing archaea are among a select group of microorganisms that utilize tetrahydromethanopterin (HMPT) as a one-carbon carrier instead of tetrahydrofolate. In HMPT biosynthesis, β-ribofuranosylaminobenzene 5'-phosphate (RFAP) synthase catalyzes the production of RFAP, CO, and pyrophosphate from -aminobenzoic acid (ABA) and phosphoribosyl-pyrophosphate (PRPP). In this work, to gain insight into amino acid residues required for substrate binding, RFAP synthase from was produced in , 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 ABA binding site of dihydropteroate synthase. Replacement of R26 with lysine increased the for ABA by an order of magnitude relative to wild-type enzyme without substantially altering the for PRPP. Although replacement of D19 with alanine produced inactive enzyme, asparagine substitution allowed retention of some activity, and the for ABA 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 HMPT 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.
产甲烷古菌是一类特殊的微生物，它们利用四氢甲烷蝶呤（HMPT）作为一碳载体，而非四氢叶酸。在HMPT生物合成过程中，β-核糖呋喃氨基苯5'-磷酸（RFAP）合酶催化由对氨基苯甲酸（ABA）和磷酸核糖焦磷酸（PRPP）生成RFAP、CO和焦磷酸。在本研究中，为深入了解底物结合所需的氨基酸残基，在大肠杆菌中表达了来自某菌的RFAP合酶，并利用定点诱变改变位于一段保守氨基酸序列中的精氨酸26（R26）和天冬氨酸19（D19），该保守序列类似于二氢蝶酸合酶的ABA结合位点。用赖氨酸取代R26使ABA的Km相对于野生型酶增加了一个数量级，而对PRPP的Km没有显著改变。虽然用丙氨酸取代D19产生了无活性的酶，但用天冬酰胺取代则保留了一些活性，且ABA的Km相对于野生型酶增加了约三倍。通过将RFAP合酶嵌入高丝氨酸激酶的晶体结构构建的分子模型显示，R26位于推测的活性位点。在静态模型中，D19位于活性位点附近，但似乎距离太远而无法直接影响配体结合。这可能预示着先前在双底物动力学机制中预测的蛋白质构象变化和/或在两个亚基界面处形成活性位点。由于RFAP合酶在HMPT生物合成中起着至关重要的作用，深入了解底物结合模式和机制可能有助于开发旨在减少作为温室气体的甲烷产生的RFAP合酶抑制剂。
