Mechanism for the Enzymatic Formation of 4-(β-d-Ribofuranosyl)aminobenzene 5‘-Phosphate during the Biosynthesis of Methanopterin

Abstract

A central step in the biosynthesis of the modified folate methanopterin is the condensation of p-aminobenzoic acid (pAB) and 5-phospho-α-d-ribosyl-1-pyrophosphate (PRPP) which produce 4-(β-d-ribofuranosyl)aminobenzene 5‘-phosphate (β-RFA-P) [White, R. H. (1996) Biochemistry 35, 3447−3456]. This reaction, catalyzed by the enzyme β-RFA-P synthase, is unique among known phosphoribosyltransferases in that a decarboxylation of one of the substrates (pAB) occurs during the reaction and a C-riboside rather than an N-riboside is the product. In this work, the reaction catalyzed by the enzyme from Methanosarcina thermophila is shown to be analogous to other phosphoribosyltransferase reactions in that pyrophosphate is released as a product of the reaction, which is dependent upon magnesium ions. The molecular weight of the enzyme was estimated to be 65 000 using gel filtration chromatography, and the pH optimum was 4.8. Kinetic analysis indicated that the reaction involved a sequential pattern of substrate binding. Benzoic acid and several para-substituted benzoic acids inhibited β-RFA-P synthase activity, while aniline, 4-aminobenzamide, and the methyl ester of pAB did not, indicating that an ionized carboxylic group plays a role in the binding of pAB. The observation that the enzyme was not inhibited by carbonyl reagents and that 4-hydroxybenzoic acid served as an alternate substrate, producing 4-(β-d-ribofuranosyl)hydroxybenzene 5‘-phosphate as the product, indicated that pyridoxal phosphate was not directly involved in the reaction mechanism. Incubation of the enzyme with PRPP and either pAB or 4-aminothiobenzoic acid in the presence of sodium cyanoborohydride led to the decreased production of β-RFA-P and the accumulation of a reduced form of the proposed cyclohexadienimine reaction intermediates. These compounds were characterized by their acid-catalyzed decomposition which produces β-d-ribofuranosylbenzene 5‘-phosphate. On the basis of these results, a concerted mechanism is proposed for β-RFA-P synthase in which an S_N1-like reaction produces oxonium ion character at C-1 of PRPP which undergoes an ipso electrophilic aromatic substitution reaction at the carboxylic acid-bound carbon of pAB. Decarboxylation of the resulting cyclohexadienimine intermediate leads to the formation of β-RFA-P.