Abstract
There is variability as to how archaea catalyze the final step of de novo purine biosynthesis to form inosine 5’-monophosphate (IMP) from 5-formamidoimidazole-4-carboxamide ribonucleotide (FAICAR). Although non-archaea almost uniformly use the bifunctional PurH protein, which has an N-terminal IMP cyclohydrolase (PurH2) fused to a C-terminal folate-dependent aminoimidazole-4-carboxamide ribonucleotide (AICAR) formyltransferase (PurH1) domain, a survey of the genomes of archaea reveals use of PurH2 (with or without fusion to PurH1), the “euryarchaeal signature protein” PurO, or an unidentified crenarchaeal IMP cyclohydrolase. In this report, we present the cloning and functional characterization of two representatives of the known IMP cyclohydrolase families. The locus TK0430 in Thermococcus kodakarensis encodes a PurO-type IMP cyclohydrolase with demonstrated activity despite its position in a cluster of apparently redundant biosynthetic genes, the first functional characterization of a PurO from a non-methanogen. Kinetic characterization reveals a Km for FAICAR of 1.56 ± 0.39 μM and a kcat of 0.48 ± 0.04 s-1. The locus AF1811 from Archaeoglobus fulgidus encodes a PurH2-type IMP cyclohydrolase. This Archaeoglobus fulgidus PurH2 has a Km of 7.8 ± 1.8 μM and kcat of 1.32 ± 0.14 s-1, representing the first characterization of an archaeal PurH2 and the first characterization of PurH2 that naturally occurs unfused to an AICAR formyltransferase domain. Each of these two characterized IMP cyclohydrolases converts FAICAR to IMP in vitro, and each cloned gene allows the growth on purine-deficient media of an E. coli purine auxotroph lacking the purH2 gene.
Highlights
A de novo purine biosynthesis pathway is found in complete form in the genomes of all but a few free-living organisms
Synthetically-produced formamidoimidazole-4-carboxamide ribonucleotide (FAICAR) incubated at 60 ̊C with either TK0430 or AF1811 enzymes was analyzed by HPLC (Fig 2)
An E. coli strain with the purH gene replaced with a kanamycin resistance cassette, ΔpurH, exhibited the expected growth requirement for purines, which could be relieved by rich media or by minimal media supplemented with inosine-5’-monosphosphate
Summary
A de novo purine biosynthesis pathway is found in complete form in the genomes of all but a few free-living organisms. In most Bacteria and Eukarya, the final two steps from 5’-phosphoribosyl-5-amino-4-imidazolecarboxamide (AICAR, CID 65110) to inosine 5’-monophosphate (IMP, CID 135398640) in the de novo pathway are catalyzed by the bifunctional PurH protein, containing a C-terminal 10-formyltetrahydrofolate-dependent AICAR formyltransferase
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