Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli

Abstract

Tetrahydromethanopterin (H(4)MPT) is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H(4)MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H(4)MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase). Given the importance of RFAP synthase in H(4)MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H(4)MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

Fig. 1

The structures of folate and methanopterin. The arrow indicates the position of the carbonyl residue of folate. The absence of the carbonyl residue in methanopterin (*) is the defining structural feature of methanopterin and its derivatives.

Fig. 2

The reaction of RFAP synthase. Abbreviations: pAB, p-aminobenzoic acid; PRPP, phosphoribosylpyrophosphate; PPi, inorganic pyrophosphate.

Fig. 3

SDS-PAGE of steps in the purification of RFAP synthase from A. fulgidus. E. coli BL21(DE3) with pJWS1 was induced for the production of RFAP synthase encoded by the AF2089 gene. Samples were boiled for 10 min with reducing SDS-PAGE sample buffer prior to loading on a 12% polyacrylamide gel, and the gel was stained with Coomassie blue. Lane 1: uninduced cells (20 µg protein); lane 2: cells induced at 30˚C with 1 mM IPTG (20 µg protein); lane 3: cell-free extract (26 µg); lane 4: heated cell-free extract (27 µg); lane 5: hydroxyapatite fraction (17 µg); lane 6: MonoQ fraction (2 µg). The molecular mass markers (97.4, 66.2, 45, 31, 21.5, and 14 kilodaltons) are shown in lane 7. The protein encoded by AF2089 has a predicted molecular mass of 34 kilodaltons and is indicated by the arrow.

Fig. 4

Time course for the reaction of purified RFAP synthase (AF2089) from A. fulgidus. The reaction mixtures (125 µL) contained 100 mM PIPES, pH 7.0, 8 mM MgCl2, 2 mM dithiothreitol, 12 mM pAB, 8.3 mM PRPP, and 1.8 µg of protein. The assays were incubated at 70˚C for the indicated times and terminated by placing the reaction vials on ice and adjusting the pH to 3.6. Samples were processed as described in the detailed protocol.

Fig. 5

SDS-PAGE of soluble versus insoluble fractions of KB1 cells induced for the production of RFAP synthase from M. thermautotrophicus. KB1 cells were grown at 37˚C and then induced with IPTG at the indicated temperatures. Cells were broken with a French press and centrifuged, producing a soluble fraction (supernatant) and insoluble fraction (pellet). Induction at 37˚C for 2 h, soluble (lane 1) and insoluble (lane 2); induction at 30˚C for 6 h, soluble (lane 3) and insoluble (lane 4); induction at 20˚C for 16 h, soluble (lane 5) and insoluble (lane 6). Induction of chaperone, followed by induction of MTH0830 and incubation at 20˚C for 16 h, soluble (lane 7) and insoluble (lane 8). Each lane contained between 15 and 30 µg of protein. The arrow indicates the position of RFAP synthase from M. thermautotrophicus.

Fig. 6

Diagram of the C18 column used to separate RFAP from the substrate pAB.