title: Structural and biochemical characterization of N5-carboxyaminoimidazole ribonucleotide synthetase and N5-carboxyaminoimidazole ribonucleotide mutase from Staphylococcus aureus.
abstract: With the rapid rise of methicillin-resistant Staphylococcus aureus infections, new strategies against S. aureus are urgently needed. De novo purine biosynthesis is a promising yet unexploited target, insofar as abundant evidence has shown that bacteria with compromised purine biosynthesis are attenuated. Fundamental differences exist within the process by which humans and bacteria convert 5-aminoimidazole ribonucleotide (AIR) to 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). In bacteria, this transformation occurs through a two-step conversion catalyzed by PurK and PurE; in humans, it is mediated by a one-step conversion catalyzed by class II PurE. Thus, these bacterial enzymes are potential targets for selective antibiotic development. Here, the first comprehensive structural and biochemical characterization of PurK and PurE from S. aureus is presented. Structural analysis of S. aureus PurK reveals a nonconserved phenylalanine near the AIR-binding site that occupies the putative position of the imidazole ring of AIR. Mutation of this phenylalanine to isoleucine or tryptophan reduced the enzyme efficiency by around tenfold. The K(m) for bicarbonate was determined for the first time for a PurK enzyme and was found to be ∼18.8 mM. The structure of PurE is described in comparison to that of human class II PurE. It is confirmed biochemically that His38 is essential for function. These studies aim to provide foundations for future structure-based drug-discovery efforts against S. aureus purine biosynthesis.
authors.affiliation: Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, GCIS E321, Chicago, IL 60637, USA.
authors.forename: Pedro
authors.initials: P
authors.lastname: Brugarolas
authors.forename: Erica M
authors.initials: EM
authors.lastname: Duguid
authors.forename: Wen
authors.initials: W
authors.lastname: Zhang
authors.forename: Catherine B
authors.initials: CB
authors.lastname: Poor
authors.forename: Chuan
authors.initials: C
authors.lastname: He
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D055162:Biocatalysis; D002262:Carboxy-Lyases / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D013211:Staphylococcus aureus / Q000201:enzymology*; D040681:Structural Homology, Protein
chemical_list: D000244:Adenosine Diphosphate; D002262:Carboxy-Lyases