Inhibition and mechanism of 4-(beta-D-ribofuranosyl)aminobenzene 5′-phosphate synthase, a key enzyme in the methanopterin biosynthetic pathway

Abstract

The first committed step in methanopterin biosynthesis is catalyzed by 4-(beta-D-ribofuranosyl)aminobenzene 5 ′-phosphate (RFA-P) synthase. Unlike all known phosphor-ribosyltransferases, 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-α-D-ribosyl-1-pyrophosphate (PRPP) to produce 4-(β-D-ribofuranosyl)aminobenzene 5 ′-phosphate (β-RFA-P), CO2, and inorganic pyrophosphate (PPi). Herein, we report the successful cloning, active overexpression in Escherichia coli, and purification of this enzyme 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 CO2, then β-RFA-P, and finally PPi. β-RFA-P synthase lacks any chromogenic cofactor, and the presence of pyridoxal phosphate and a mechanistically related pyruvoyl cofactor have been strictly excluded. Collaborative work in Dr. Takacs\u27s laboratory has involved the design and synthesis of inhibitors of β-RFA-P synthase. These include N-substituted pABA analogs and 4-(N-alkylamino)-phenyl derivatives bearing in the 1-position either a boronic acid, sulfonic acid, phosphonic acid or monoester and diester derivatives. Based on comparisons of the inhibition constants among the N-substituted derivatives, we propose that the pABA binding site in β-RFA-P synthase has a relatively large hydrophobic pocket near the amino group. These enzyme-targeted inhibitors arrest the methanogenesis and growth of pure cultures of methanogens. The inhibitors do not adversely affect the growth of pure cultures of acetogenic bacteria that play a beneficial role in the rumen. Collaborative work with Dr. Miner\u27s group demonstrated that inhibitors added to dense ruminal fluid cultures (the artificial rumen) halt methanogenesis; however, they do not inhibit volatile fatty acid (VFA) production and, in some cases, VFA levels are slightly elevated in the methanogenesis-inhibited cultures. The design of tight-binding inhibitors of the synthase is an important objective since it provides information about the enzyme\u27s active site and could lead to development of practical antimicrobial agents that eradicate methanogenesis in domesticated ruminant animals