caa a22 4500 605502153 CHVBK 20210128100604.0 cr unu---uuuuu 210128e20150501xx s 000 0 eng 10.1007/s00253-014-6272-8 doi (NATIONALLICENCE)springer-10.1007/s00253-014-6272-8 Structural basis for the Ca2+-enhanced thermostability and activity of PET-degrading cutinase-like enzyme from Saccharomonospora viridis AHK190 [Elektronische Daten] [Takuya Miyakawa, Hiroki Mizushima, Jun Ohtsuka, Masayuki Oda, Fusako Kawai, Masaru Tanokura] A cutinase-like enzyme from Saccharomonospora viridis AHK190, Cut190, hydrolyzes the inner block of polyethylene terephthalate (PET); this enzyme is a member of the lipase family, which contains an α/β hydrolase fold and a Ser-His-Asp catalytic triad. The thermostability and activity of Cut190 are enhanced by high concentrations of calcium ions, which is essential for the efficient enzymatic hydrolysis of amorphous PET. Although Ca2+-induced thermostabilization and activation of enzymes have been well explored in α-amylases, the mechanism for PET-degrading cutinase-like enzymes remains poorly understood. We focused on the mechanisms by which Ca2+ enhances these properties, and we determined the crystal structures of a Cut190 S226P mutant (Cut190S226P) in the Ca2+-bound and free states at 1.75 and 1.45Å resolution, respectively. Based on the crystallographic data, a Ca2+ ion was coordinated by four residues within loop regions (the Ca2+ site) and two water molecules in a tetragonal bipyramidal array. Furthermore, the binding of Ca2+ to Cut190S226P induced large conformational changes in three loops, which were accompanied by the formation of additional interactions. The binding of Ca2+ not only stabilized a region that is flexible in the Ca2+-free state but also modified the substrate-binding groove by stabilizing an open conformation that allows the substrate to bind easily. Thus, our study explains the structural basis of Ca2+-enhanced thermostability and activity in PET-degrading cutinase-like enzyme for the first time and found that the inactive state of Cut190S226P is activated by a conformational change in the active-site sealing residue, F106. Springer-Verlag Berlin Heidelberg, 2014 PET hydrolase nationallicence Cutinase-type polyesterase nationallicence Ca2+ activation nationallicence Thermostability nationallicence Saccharomonospora viridis AHK190 nationallicence Miyakawa Takuya Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, 113-8657, Bunkyo-ku, Tokyo, Japan aut Mizushima Hiroki Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, 113-8657, Bunkyo-ku, Tokyo, Japan aut Ohtsuka Jun Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, 113-8657, Bunkyo-ku, Tokyo, Japan aut Oda Masayuki Laboratory of Biophysical Chemistry, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Shimogamo, 606-8522, Sakyo-ku, Kyoto, Japan aut Kawai Fusako Center for Fiber and Textile Science, Kyoto Institute of Technology, Matsugasaki, 606-8585, Sakyo-ku, Kyoto, Japan aut Tanokura Masaru Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, 113-8657, Bunkyo-ku, Tokyo, Japan aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/10(2015-05-01), 4297-4307 0175-7598 99:10<4297 2015 99 253 https://doi.org/10.1007/s00253-014-6272-8 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-014-6272-8 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Miyakawa Takuya Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, 113-8657, Bunkyo-ku, Tokyo, Japan aut NATIONALLICENCE 700 1- Mizushima Hiroki Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, 113-8657, Bunkyo-ku, Tokyo, Japan aut NATIONALLICENCE 700 1- Ohtsuka Jun Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, 113-8657, Bunkyo-ku, Tokyo, Japan aut NATIONALLICENCE 700 1- Oda Masayuki Laboratory of Biophysical Chemistry, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Shimogamo, 606-8522, Sakyo-ku, Kyoto, Japan aut NATIONALLICENCE 700 1- Kawai Fusako Center for Fiber and Textile Science, Kyoto Institute of Technology, Matsugasaki, 606-8585, Sakyo-ku, Kyoto, Japan aut NATIONALLICENCE 700 1- Tanokura Masaru Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, 113-8657, Bunkyo-ku, Tokyo, Japan aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/10(2015-05-01), 4297-4307 0175-7598 99:10<4297 2015 99 253