The first committed step in methanopterin biosynthesis is catalyzed by 4-(beta-D-ribofuranosyl)aminobenzene 5'-phosphate (RFA-P) synthase. Unlike all known phosphoribosyltransferases, beta-RFA-P synthase catalyzes the unique formation of a C-riboside instead of an N-riboside in the condensation of p-aminobenzoic acid (pABA) and 5-phospho-alpha-D-ribosyl-1-pyrophosphate (PRPP) to produce 4-(beta-D-ribofuranosyl)aminobenzene 5'-phosphate (beta-RFA-P), CO(2), and inorganic pyrophosphate (PP(i)). Here we report the successful cloning, active overexpression in Escherichia coli, and purification of this homodimeric enzyme containing two 36.2-kDa subunits from the methanogen Methanococcus jannaschii. Steady-state initial velocity and product inhibition kinetic studies indicate an ordered Bi-Ter mechanism involving binding of PRPP, then pABA, followed by release of the products CO(2), then beta-RFA-P, and finally PP. The Michaelis parameters are as follows: K(m)pABA, 0.15 mm; K(m)PRPP, 1.50 mm; V(max), 375 nmol/min/mg; k(cat), 0.23 s(-1). CO(2) showed uncompetitive inhibition, K(i) = 0.990 mm, under varied PRPP and saturated pABA, and a mixed type of inhibition, K(1) = 1.40 mm and K = 3.800 mm, under varied pABA and saturated PRPP. RFA-P showed uncompetitive inhibition, K(i) = 0.210 mm, under varied PRPP and saturated pABA, and again uncompetitive, K(i) = 0.300 mm, under saturated PRPP and varied pABA. PP(i) exhibits competitive inhibition, K(i) = 0.320 mm, under varied PRPP and saturated pABA, and a mixed type of inhibition, K(1) = 0.60 mm and K(2) = 1.900 mm, under saturated PRPP and varied pABA. Synthase lacks any chromogenic cofactor, and the presence of pyridoxal phosphate and the mechanistically related pyruvoyl cofactors has been strictly excluded.