| Most methanogenic archaea, including Methanothermobacter thermautrophicus, derive the energy for growth from the H2-dependent reduction of CO2 to methane in the process of methanogenesis. In these microorganisms, the coenzyme tetrahydromethanopterin serves as a one-carbon carrier instead of folate during catabolism. The biosynthesis of tetrahydromethanopterin consists of eighteen steps. The first step is catalyzed by RFAP synthase, which condenses p-aminobenzoic acid (pABA) and phosphoribosylpyrophosphate (PRPP) to form ribofuranosylaminobenzene 5'-phosphate (RFAP). Bioinformatic analysis of homologous methanogen RFAP synthase sequences revealed highly conserved regions of amino acids. One of these conserved sequences resembles a region of dihydropteroate synthase proposed to be involved in pABA binding and two other conserved motifs were proposed to be involved in phosphate binding. In this work, the residues histidine 98 (H98), glutamic acid 153 (D153) and arginine 134 (R134) that are proposed to be in the phosphate binding region were altered by site-directed mutagenesis. The resulting altered RFAP synthase genes were overexpressed in Escherichia coli. The altered RFAP synthases were purified and biochemically characterized using an enzymatic assay and isothermal calorimetry (ITC). The kinetic data indicated that the apparent dissociation constant (KD) for pABA for H98A, R134K and D153A increased by 1.5, 5 and 16-fold, respectively; while the KD for PRPP increased by 8, 3 and 13-fold, respectively. Molecular modeling revealed that these conserved residues reside in the RFAP synthase binding pocket and could play a role in substrate binding. ITC data and molecular modeling also support the ordered Bi-Ter mechanism in which PRPP binds first followed by pABA. |
