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-alpha-D-ribosyl-1-pyrophosphate (PRPP) which produce 4-(beta-D-ribofuranosyl)aminobenzene 5'-phosphate (beta-RFA-P) [White, R. H. (1996) Biochemistry 35, 3447-3456]. This reaction, catalyzed by the enzyme beta-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 beta-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-(beta-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 beta-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 beta-D-ribofuranosylbenzene 5'-phosphate. On the basis of these results, a concerted mechanism is proposed for beta-RFA-P synthase in which an SN1-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 beta-RFA-P.

Mechanism of 4-(β-D-Ribofuranosyl)aminobenzene 5′-Phosphate Synthase, a Key Enzyme in the Methanopterin Biosynthetic Pathway

“…What would stabilize this negative charge on a carbon that already contains a negatively charged carboxyl group? It was suggested that ␤-RFA-P synthase contains PLP, which is known to stabilize similar intermediates during decarboxylation or deamination of amino acids, and the partially purified enzyme appeared to contain a chromophore with properties similar to those of PLP (3). However, we show here that ␤-RFA-P synthase lacks PLP and apparently any other cofactor that would be capable of stabilizing negative charge developing during the reaction.…”

“…Furthermore, ␤-RFAP synthase is inhibited by pyridoxol phosphate, an analog of PLP (3), and nonenzymatic decarboxylation of pABA by PLP has been observed (16). However, ␤-RFAP synthase is not inhibited by cysteine or by several carbonyl reactive reagents known to inactivate PLP-dependent enzymes (3). In addition, the partially purified ␤-RFAP synthase from A. fulgidus showed no UV-visible absorbance at wavelengths characteristic of pyridoxal phosphate and a consensus pyridoxal phosphate-binding motif was not found in the amino acid sequence of ␤-RFAP synthase.…”

“…For example, in an early step in tetrahydrofolate biosynthesis, dihydropteroate synthase catalyzes a condensation between the amino group of pABA and dihydropterin pyrophosphate to generate dihydropteroate, eliminating PP i . Thus, ␤-RFA-P synthase and dihydropteroate synthase both use pABA as a substrate and produce PP i as product; however, the amino group is the nucleophile in dihydropteroate synthase, whereas the aromatic ring carbon 4 (C-4) is the nucleophile in ␤-RFA-P synthase (2,3).…”

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

“…12A, compound m). The enzyme catalyzing this reaction has affinity for both 4-hydroxy-and 4-aminobenzoate (304,305). In vitro and in vivo analyses used 4-aminobenzoate to elucidate the metabolic pathway.…”

“…Some kinetic constants were determined; the apparent K m 4-aminobenzoate was 58 M and the apparent K m PRPP was 3.6 mM. Pyridoxal phosphate is suggested to be involved in the reaction, but data are inconclusive in this respect, and it has been suggested that pyridoxal phosphate may be involved in the reaction in a way that does not require the carbonyl of pyridoxal phosphate (301,305).…”