title: Purine salvage in Methanocaldococcus jannaschii: Elucidating the role of a conserved cysteine in adenine deaminase.
abstract: Adenine deaminases (Ade) and hypoxanthine/guanine phosphoribosyltransferases (Hpt) are widely distributed enzymes involved in purine salvage. Characterization of the previously uncharacterized Ade (MJ1459 gene product) and Hpt (MJ1655 gene product) are discussed here and provide insight into purine salvage in Methanocaldococcus jannaschii. Ade was demonstrated to use either Fe(II) and/or Mn(II) as the catalytic metal. Hpt demonstrated no detectable activity with adenine, but was equally specific for hypoxanthine and guanine with a kcat /KM of 3.2 × 10(7) and 3.0 × 10(7) s(- 1) M(- 1) , respectively. These results demonstrate that hypoxanthine and IMP are the central metabolites in purine salvage in M. jannaschii for AMP and GMP production. A conserved cysteine (C127, M. jannaschii numbering) was examined due to its high conservation in bacterial and archaeal homologues. To assess the role of this highly conserved cysteine in M. jannaschii Ade, site-directed mutagenesis was performed. It was determined that mutation to serine (C127S) completely abolished Ade activity and mutation to alanine (C127A) exhibited 10-fold decrease in kcat over the wild type Ade. To further investigate the role of C127, detailed molecular docking and dynamics studies were performed and revealed adenine was unable to properly orient in the active site in the C127A and C127S Ade model structures due to distinct differences in active site conformation and rotation of D261. Together this work illuminates purine salvage in M. jannaschii and the critical role of a cysteine residue in maintaining active site conformation of Ade. Proteins 2016; 84:828-840. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, 24061.
authors.forename: Danielle V
authors.initials: DV
authors.lastname: Miller
authors.affiliation: Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, 24061.
authors.forename: Anne M
authors.initials: AM
authors.lastname: Brown
authors.affiliation: Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, 24061.
authors.forename: Huimin
authors.initials: H
authors.lastname: Xu
authors.affiliation: Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, 24061.
authors.forename: David R
authors.initials: DR
authors.lastname: Bevan
authors.affiliation: Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, 24061.
authors.forename: Robert H
authors.initials: RH
authors.lastname: White
mesh_terms: D000225:Adenine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D063747:Methanocaldococcus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D062105:Molecular Docking Simulation; D016415:Sequence Alignment
chemical_list: D000619:Aminohydrolases; C018774:adenine deaminase; D000225:Adenine; D003545:Cysteine
keywords: amino acid conservation; archaea; cysteine; methanogen; molecular docking; molecular dynamics