Purification, overproduction, and partial characterization of beta-RFAP synthase, a key enzyme in the methanopterin biosynthesis pathway

Abstract

Methanopterin is a folate analog involved in the C1 metabolism of methanogenic archaea, sulfate-reducing archaea, and methylotrophic bacteria. Although a pathway for methanopterin biosynthesis has been described in methanogens, little is known about the enzymes and genes involved in the biosynthetic pathway. The enzyme beta-ribofuranosylaminobenzene 5'-phosphate synthase (beta-RFAP synthase) catalyzes the first unique step to be identified in the pathway of methanopterin biosynthesis, namely, the condensation of p-aminobenzoic acid with phosphoribosylpyrophosphate to form beta-RFAP, CO2, and inorganic pyrophosphate. The enzyme catalyzing this reaction has not been purified to homogeneity, and the gene encoding beta-RFAP synthase has not yet been identified. In the present work, we report on the purification to homogeneity of beta-RFAP synthase. The enzyme was purified from the methane-producing archaeon Methanosarcina thermophila, and the N-terminal sequence of the protein was used to identify corresponding genes from several archaea, including the methanogen Methanococcus jannaschii and the sulfate-reducing archaeon Archaeoglobus fulgidus. The putative beta-RFAP synthase gene from A. fulgidus was expressed in Escherichia coli, and the enzymatic activity of the recombinant gene product was verified. A BLAST search using the deduced amino acid sequence of the beta-RFAP synthase gene identified homologs in additional archaea and in a gene cluster required for C1 metabolism by the bacterium Methylobacterium extorquens. The identification of a gene encoding a potential beta-RFAP synthase in M. extorquens is the first report of a putative methanopterin biosynthetic gene found in the Bacteria and provides evidence that the pathways of methanopterin biosynthesis in Bacteria and Archaea are similar.

FIG. 1.

The reaction catalyzed by β-RFAP synthase.

FIG. 2.

Purification of β-RFAP synthase from M. thermophila. Protein samples were boiled in the presence of 7.5% β-mercaptoethanol in SDS-PAGE sample buffer and loaded onto a 12% polyacrylamide gel. The gel was stained by using the Bio-Rad Silver Stain Plus kit. Lane 1, cell extract; lane 2, Q-Sepharose; lane 3, hydroxyapatite; lane 4, Phenyl Sepharose; lane 5, Mono Q; lane 6, Superdex 75 gel filtration. Arrows indicate the positions of the following molecular mass markers from top to bottom: phosphorylase b (97.4 kDa), BSA (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), and soybean trypsin inhibitor (21.5 kDa).

FIG. 3.

Phylogenetic relationships of putative β-RFAP synthases. The amino acid sequences of putative β-RFAP synthases were aligned with ClustalX (32), and the unrooted tree was generated by the program TREEVIEW (21). The numbers in the figure indicate the bootstrap values (as a percentage of 1,000) calculated by ClustalX. The GenBank accession numbers were as follows: M. jannaschii MJ1427 (Mja), gi:3183371; A. fulgidus AF2089 (Afu), gi:11499671; M. thermautotrophicum ΔH MTH0830 (Mth), gi:3183231; M. kandleri (Mka), gi:20093996; M. mazei Orf 322 (Mma), gi:2909376; M. acetivorans (Mac), gi:20089237; S. solfataricus (Sso), gi:13813516; S. tokodaii (Sto), gi:15622465; P. abyssi PAB0141 (Pab-1), gi:7445271, and PAB1694 (Pab-2), gi:14521221; P. horikoshii PH0227 (Pho-1), gi:7429728, and PH1228 (Pho-2), gi:14591047; P. furiosus (Pfu), gi:18892945; A. pernix APE2425 (Ape-1), gi:14602055, and APE1512 (Ape-2), gi:7445269; and M. extorquens AM-1 Orf 4 (Mex), gi:3237329.