title: N5-CAIR mutase: role of a CO2 binding site and substrate movement in catalysis.
abstract: N5-Carboxyaminoimidazole ribonucleotide mutase (N5-CAIR mutase or PurE) from Escherichia coli catalyzes the reversible interconversion of N5-CAIR to carboxyaminoimidazole ribonucleotide (CAIR) with direct CO2 transfer. Site-directed mutagenesis, a pH-rate profile, DFT calculations, and X-ray crystallography together provide new insight into the mechanism of this unusual transformation. These studies suggest that a conserved, protonated histidine (His45) plays an essential role in catalysis. The importance of proton transfers is supported by DFT calculations on CAIR and N5-CAIR analogues in which the ribose 5'-phosphate is replaced with a methyl group. The calculations suggest that the nonaromatic tautomer of CAIR (isoCAIR) is only 3.1 kcal/mol higher in energy than its aromatic counterpart, implicating this species as a potential intermediate in the PurE-catalyzed reaction. A structure of wild-type PurE cocrystallized with 4-nitroaminoimidazole ribonucleotide (NO2-AIR, a CAIR analogue) and structures of H45N and H45Q PurEs soaked with CAIR have been determined and provide the first insight into the binding of an intact PurE substrate. A comparison of 19 available structures of PurE and PurE mutants in apo and nucleotide-bound forms reveals a common, buried carboxylate or CO2 binding site for CAIR and N5-CAIR in a hydrophobic pocket in which the carboxylate or CO2 interacts with backbone amides. This work has led to a mechanistic proposal in which the carboxylate orients the substrate for proton transfer from His45 to N5-CAIR to form an enzyme-bound aminoimidazole ribonucleotide (AIR) and CO2 intermediate. Subsequent movement of the aminoimidazole moiety of AIR reorients it for addition of CO2 at C4 to generate isoCAIR. His45 is now in a position to remove a C4 proton to produce CAIR.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Aaron A
authors.initials: AA
authors.lastname: Hoskins
authors.forename: Mariya
authors.initials: M
authors.lastname: Morar
authors.forename: T Joseph
authors.initials: TJ
authors.lastname: Kappock
authors.forename: Irimpan I
authors.initials: II
authors.lastname: Mathews
authors.forename: Judith B
authors.initials: JB
authors.lastname: Zaugg
authors.forename: Timothy E
authors.initials: TE
authors.lastname: Barder
authors.forename: Paul
authors.initials: P
authors.lastname: Peng
authors.forename: Akimitsu
authors.initials: A
authors.lastname: Okamoto
authors.forename: Steven E
authors.initials: SE
authors.lastname: Ealick
authors.forename: JoAnne
authors.initials: J
authors.lastname: Stubbe
mesh_terms: D001665:Binding Sites; D002245:Carbon Dioxide / Q000378:metabolism*; D002384:Catalysis; D003653:Decarboxylation; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D019751:Intramolecular Transferases / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D002245:Carbon Dioxide; D006639:Histidine; D019751:Intramolecular Transferases; C498062:N(5)-carboxyaminoimidazole ribonucletide mutase