Publications

• Crystal structure of Methanobacterium thermoautotrophicum phosphoribosyl-AMP cyclohydrolase HisI.

  Sivaraman J., Myers R.S., Boju L., Sulea T., Cygler M., Jo Davisson V., Schrag J.D.

  The metabolic pathway for histidine biosynthesis is interesting from an evolutionary perspective because of the diversity of gene organizations and protein structures involved. Hydrolysis of phosphoribosyl-AMP, the third step in the histidine biosynthetic pathway, is carried out by PR-AMP cyclohyd ... >> More

  The metabolic pathway for histidine biosynthesis is interesting from an evolutionary perspective because of the diversity of gene organizations and protein structures involved. Hydrolysis of phosphoribosyl-AMP, the third step in the histidine biosynthetic pathway, is carried out by PR-AMP cyclohydrolase, the product of the hisI gene. The three-dimensional structure of PR-AMP cyclohydrolase from Methanobacterium thermoautotrophicum was solved and refined to 1.7 A resolution. The enzyme is a homodimer. The position of the Zn(2+)-binding site that is essential for catalysis was inferred from the positions of bound Cd(2+) ions, which were part of the crystallization medium. These metal binding sites include three cysteine ligands, two from one monomer and the third from the second monomer. The enzyme remains active when Cd(2+) is substituted for Zn(2+). The likely binding site for Mg(2+), also necessary for activity in a homologous cyclohydrolase, was also inferred from Cd(2+) positions and is comprised of aspartic acid side chains. The putative substrate-binding cleft is formed at the interface between the two monomers of the dimer. This fact, combined with the localization of the Zn(2+)-binding site, indicates that the enzyme is an obligate dimer. << Less

  Biochemistry 44:10071-10080(2005) [PubMed] [EuropePMC]

• N1-(5'-phosphoribosyl)adenosine-5'-monophosphate cyclohydrolase: purification and characterization of a unique metalloenzyme.

  D'Ordine R.L., Klem T.J., Davisson V.J.

  N1-(5'-Phosphoribosyl)adenosine-5'-monophosphate cyclohydrolase (HisI, PR-AMP cyclohydrolase) is a central enzyme in histidine biosynthesis catalyzing the hydrolysis of the N1-C6 bond of the purine substrate, a reaction unique to this pathway. A source of the recombinant monofunctional Methanococc ... >> More

  N1-(5'-Phosphoribosyl)adenosine-5'-monophosphate cyclohydrolase (HisI, PR-AMP cyclohydrolase) is a central enzyme in histidine biosynthesis catalyzing the hydrolysis of the N1-C6 bond of the purine substrate, a reaction unique to this pathway. A source of the recombinant monofunctional Methanococcus vannielii PR-AMP cyclohydrolase has been developed, and the first characterization of a purified form of the enzyme is reported. The enzyme has a native molecular weight of 31 200 as determined by analytical ultracentrifugation that agrees with the molecular mass determined by gel filtration (34 kDa) and a subunit molecular weight of 15 486 based on MALDI-MS. An unusual characteristic of the protein is the complexity observed on SDS-PAGE, and N-terminal amino acid sequence analysis of all the isolated constituents confirms their origin as PR-AMP cyclohydrolase. A highly conserved region of the amino acid sequence is implicated in the self-cleavage events of the protein and provides an explanation for the complexity of this protein. Bound to the enzyme is 1 equiv of Zn2+ that can be removed only by extended dialysis with 1,10-phenanthroline (Kd </= 10(-)9 M). Removal of the Zn2+ correlates with the loss of enzyme activity. The enzyme is reversibly inhibited by inclusion of EDTA in the assay mixture, demonstrating that free Mg2+ (Ks = 4.9 +/-0.7 microM) is required for catalytic activity. Further evidence for a low-affinity binding site is indicated by the inhibitory effects of exogenous Zn2+ on enzyme activity. The pH dependence of the PR-AMP cyclohydrolase activity shows a single titration event in the kcat/Km profile with a pKa of 7.3 that is consistent with the functional role of a metal site in catalysis. These data are discussed in the context of the mechanism of other nucleotide hydrolases. << Less

  Biochemistry 38:1537-1546(1999) [PubMed] [EuropePMC]