Mechanism of 4-(beta-D-ribofuranosyl)aminobenzene 5'-phosphate synthase, a key enzyme in the methanopterin biosynthetic pathway.
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.
Dumitru
Razvan V
RV
Department of Biochemistry, Beadle Center, University of Nebraska, Lincoln, Nebraska 68588-0664, USA.
Ragsdale
Stephen W
SW
D002384:Catalysis; D003001:Cloning, Molecular; D004789:Enzyme Activation; D007700:Kinetics; D017017:Methanococcus / Q000201:enzymology* / Q000235:genetics; D010430:Pentosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011622:Pterins / Q000378:metabolism*; D011732:Pyridoxal Phosphate / Q000737:chemistry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D011622:Pterins; D011732:Pyridoxal Phosphate; C036686:methanopterin; C114050:4-(beta-D-ribofuranosyl)aminobenzene 5'-phosphate synthase; D010430:Pentosyltransferases