title: Characterization of a hyperthermophilic P-type ATPase from Methanococcus jannaschii expressed in yeast.
abstract: We report on the biochemical and structural properties of a putative P-type H(+)-ATPase, MJ1226p, from the anaerobic hyperthermophilic Archaea Methanococcus jannaschii. An efficient heterologous expression system was developed in Saccharomyces cerevisiae and a four-step purification protocol, using n-dodecyl beta-d-maltoside, led to a homogeneous detergent-solubilized protein fraction with a yield of over 2 mg of protein per liter of culture. The three-dimensional structure of the purified detergent-solubilized protein obtained at 2.4 nm resolution by electron microscopy showed a dimeric organization in which the size and the shape of each monomer was compatible with the reported structures of P-type ATPases. The purified MJ1226p ATPase was inactive at 40 degrees C and was active at elevated temperature reaching high specific activity, up to 180 micromol of P(i) x min(-1) x mg(-1) at 95 degrees C. Maximum ATPase activity was observed at pH 4.2 and required up to 200 mm monovalent salts. The ATPase activity was stable for several days upon storage at 65 degrees C and was highly resistant to urea and guanidine hydrochloride. The protein formed catalytic phosphoenzyme intermediates from MgATP or P(i), a functional characteristic specific of P-type ATPases. The highly purified, homogeneous, stable, and active MJ1226p ATPase provides a new model for further structure-function studies of P-type ATPases.
authors.affiliation: Unité de Biochimie Physiologique, Université Catholique de Louvain, Place Croix du Sud 2-20, B-1348 Louvain-la-Neuve, Belgium.
authors.forename: Pierre
authors.initials: P
authors.lastname: Morsomme
authors.forename: Mohamed
authors.initials: M
authors.lastname: Chami
authors.forename: Sergio
authors.initials: S
authors.lastname: Marco
authors.forename: Joseph
authors.initials: J
authors.lastname: Nader
authors.forename: Karen A
authors.initials: KA
authors.lastname: Ketchum
authors.forename: André
authors.initials: A
authors.lastname: Goffeau
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Rigaud
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D006863:Hydrogen-Ion Concentration; D017017:Methanococcus / Q000201:enzymology*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010802:Phylogeny; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D013696:Temperature
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D006180:Proton-Translocating ATPases