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.
Highlights
Carboxylesterases typically have an alkaline pH optima, which limits their industrial application
We previously reported the molecular cloning and functional characterization of a carboxylesterase (SshEstI) from the thermoacidophilic archaeon Sulfolobus shibatae DSM5389
Archaeal Strains and Materials—F. acidiphilum JCM10970 was obtained from the Japan Collection of Microorganisms, RIKEN BioResource Center (Tsukuba, Japan). p-Nitrophenyl acetate and pNP butyrate were obtained from SigmaAldrich. pNP propionate and pNP caproate were obtained from Wako Pure Chemical Industries, Tokyo, Japan. pNP caprylate was obtained from Tokyo Chemical Industries, Tokyo, Japan
Summary
Carboxylesterases typically have an alkaline pH optima, which limits their industrial application. 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. The canonical catalytic triad is conserved in the primary amino acid sequences of EstFa, the reported optimum pH for catalytic activity of this enzyme is ϳ2.0 –3.5, which is exceptionally low for the values of serine hydrolases [13]. The experimental data suggested that the extension of a hydrogen bond network around the active site of EstFa_R may have contributed to the lower catalytic efficiency and low pH adaptation of this enzyme. Possible contributions of other structural factors of EstFa_R for catalytic efficiency and low pH adaptation are discussed
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