Application of a colorimetric assay to identify putative ribofuranosylaminobenzene 5-phosphate synthase genes expressed with activity inEscherichia coli

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 H4MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H4MPT 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 H4MPT 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.