Purification, kinetic characterization, and site-directed mutagenesis of RFAP Synthase Produced in .

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.