title: Structural insights into the low pH adaptation of a unique carboxylesterase from Ferroplasma: altering the pH optima of two carboxylesterases.
abstract: To investigate the mechanism for low pH adaptation by a carboxylesterase, structural and biochemical analyses of EstFa_R (a recombinant, slightly acidophilic carboxylesterase from Ferroplasma acidiphilum) and SshEstI (an alkaliphilic carboxylesterase from Sulfolobus shibatae DSM5389) were performed. Although a previous proteomics study by another group showed that the enzyme purified from F. acidiphilum contained an iron atom, EstFa_R did not bind to iron as analyzed by inductively coupled plasma MS and isothermal titration calorimetry. The crystal structures of EstFa_R and SshEstI were determined at 1.6- and 1.5-Å resolutions, respectively. EstFa_R had a catalytic triad with an extended hydrogen bond network that was not observed in SshEstI. Quadruple mutants of both proteins were created to remove or introduce the extended hydrogen bond network. The mutation on EstFa_R enhanced its catalytic efficiency and gave it an alkaline pH optimum, whereas the mutation on SshEstI resulted in opposite effects (i.e. a decrease in the catalytic efficiency and a downward shift in the optimum pH). Our experimental results suggest that the low pH optimum of EstFa_R activity was a result of the unique extended hydrogen bond network in the catalytic triad and the highly negatively charged surface around the active site. The change in the pH optimum of EstFa_R happened simultaneously with a change in the catalytic efficiency, suggesting that the local flexibility of the active site in EstFa_R could be modified by quadruple mutation. These observations may provide a novel strategy to elucidate the low pH adaptation of serine hydrolases.
authors.affiliation: From the Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aobayama 6-6-11, Sendai 980-8579, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Ohara
authors.affiliation: Graduate School of Engineering, Nagasaki University, Bunkyo-machi 1-14, Nagasaki 852-8521, Japan, and.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Unno
authors.affiliation: From the Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aobayama 6-6-11, Sendai 980-8579, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Oshima
authors.affiliation: From the Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aobayama 6-6-11, Sendai 980-8579, Japan.
authors.forename: Miho
authors.initials: M
authors.lastname: Hosoya
authors.affiliation: From the Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aobayama 6-6-11, Sendai 980-8579, Japan.
authors.forename: Naoto
authors.initials: N
authors.lastname: Fujino
authors.affiliation: From the Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aobayama 6-6-11, Sendai 980-8579, Japan.
authors.forename: Kazutake
authors.initials: K
authors.lastname: Hirooka
authors.affiliation: From the Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aobayama 6-6-11, Sendai 980-8579, Japan.
authors.forename: Seiji
authors.initials: S
authors.lastname: Takahashi
authors.affiliation: From the Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aobayama 6-6-11, Sendai 980-8579, Japan, satoshi0207@gmail.com.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Yamashita
authors.affiliation: Faculty of Life and Environmental Sciences, University of Yamanashi, 4-3-37 Takeda, Kofu 400-8511, Japan.
authors.forename: Masami
authors.initials: M
authors.lastname: Kusunoki
authors.affiliation: From the Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aobayama 6-6-11, Sendai 980-8579, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Nakayama
mesh_terms: D043182:Carboxylesterase / Q000737:chemistry / Q000378:metabolism*; D003460:Crystallization; D019605:Euryarchaeota / Q000201:enzymology*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics
chemical_list: D043182:Carboxylesterase
keywords: Archaea; Carboxylesterase; Enzyme Mechanism; Enzyme Structure; Ferroplasma acidiphilum; Hydrolase; Serine Hydrolase; pH Adaptation