A Novel Function for the N-Terminal Nucleophile Hydrolase Fold Demonstrated by the Structure of an Archaeal Inosine Monophosphate Cyclohydrolase^,

Abstract

Inosine 5′-monophosphate (IMP) cyclohydrolase catalyzes the cyclization of 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) to IMP in the final step of de novo purine biosynthesis. Two major types of this enzyme have been discovered to date: PurH in Bacteria and Eukarya, and PurO in Archaea. The structure of the MTH1020 gene product from Methanothermobacter thermoautotrophicus was previously solved without functional annotation but shows high amino acid sequence similarity to other PurOs. We determined the crystal structure of the MTH1020 gene product in complex with either IMP or 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) at 2.0 Å and 2.6 Å resolution, respectively. Based on the sequence analysis, ligand-bound structures, and biochemical data, MTH1020 is confirmed as an archaeal IMP cyclohydrolase, thus designated as MthPurO. MthPurO has a four-layered αββα core structure, showing an N-terminal nucleophile (NTN) hydrolase fold. The active site is located at the deep pocket between two central β-sheets and contains residues strictly conserved within PurOs. Comparisons of the two types of IMP cyclohydrolase, PurO and PurH, revealed that there are no similarities in sequence, structure, or the active site architecture, suggesting that they are evolutionarily not related to each other. The MjR31K mutant of PurO from Methanocaldococcus jannaschii showed 76% decreased activity and MjE102Q mutation completely abolished enzymatic activity, suggesting that these highly conserved residues play critical roles in catalysis. Interestingly, green fluorescent protein (GFP), which has no structural homology to either PurO or PurH but catalyzes a similar intramolecular cyclohydrolase reaction required for chromophore maturation, utilizes Arg96 and Glu222 in a mechanism analogous to that of PurO.The final two steps of de novo purine biosynthesis are the conversion of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) and the subsequent conversion of FAICAR to inosine 5′-monophosphate (IMP). These two reactions occur in different ways depending on the domains of life (Figure 1). In Bacteria and Eukarya, one bifunctional enzyme, AICAR transformylase/ IMP cyclohydrolase (PurH), encoded by the purH gene, catalyzes both steps (1). The AICAR † This work was supported by a National Institutes of Health grant GM073220 to SEE and National Science Foundation grant MCB-0231319 to RHW. Beamline 8-BM is supported by NIH grant RR-015301. SEE is indebted to the Lucille P. Markey Charitable Trust and W.M. Keck Foundation. ‡ The Brookhaven Protein Data Bank code for MthPurO is 2NTM, for the MthPurO/IMP complex is 2NTK and for the MthPurO/AICAR complex is 2NTL. * To whom correspondence should be addressed at the Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853. Telephone: (607) (7). Therefore, in all Archaea PurP appears to catalyze the penultimate step of purine biosynthesis, while the final step is catalyzed by P...

Structural Analyses of a Purine Biosynthetic Enzyme from Mycobacterium tuberculosis Reveal a Novel Bound Nucleotide

“…The 13 C resonances for C2 and the formamide C7 of 134.1 (major), 164.8 (major), and 168.2 (minor) ppm, respectively, are particularly close to the 134.9, 164.5, and 167.6 ppm for FAICAR. Notably, there is no equivalent 13 C resonance evident in the nucleotide spectra at the 165.4 ppm reported for the amide C6 of FAICAR, with the remaining unassigned peaks in this region occurring at 162.8 and 168.7 ppm.…”

“…A range of one-and two-dimensional 1 H and 13 C NMR experiments was carried out on the ligand in D 2 O (supplemental Figs. S2 and S3).…”

“…H2 of the imidazole ring was observed to undergo slow exchange with deuterium from the solvent. Through a combination of 1 H-13 C HSQC and 1 H- 13 C HMBC experiments, a nearly complete assignment was also obtained for 13 C NMR resonances of the major isomer. However, there was not sufficient signal to unequivocally assign the 13 C peak for the significant C6 carbonyl.…”

“…Through a combination of 1 H-13 C HSQC and 1 H- 13 C HMBC experiments, a nearly complete assignment was also obtained for 13 C NMR resonances of the major isomer. However, there was not sufficient signal to unequivocally assign the 13 C peak for the significant C6 carbonyl. Overall, the 1 H and 13 C chemical shifts for the nucleotide (Tables 3 and 4) are similar to those previously reported for FAICAR (37).…”

“…1A). In archaea, however, these two steps are carried out by quite distinct enzymes, unrelated to PurH, and without the use of tetrahydrofolate (THF) (13).…”