Structure-activity relationship of PET-degrading cutinase regulated by weak Ca2+ binding and temperature
Enzyme function is often regulated by weak metal-ion binding, which results from conformational changes while maintaining conformational fluctuations. We analyzed the structure and function of cutinase-like enzyme, Cut190, using biophysical methods such as X-ray crystallography and molecular dynamic...
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| Format: | Article |
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The Biophysical Society of Japan
2025-05-01
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| Series: | Biophysics and Physicobiology |
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| Online Access: | https://doi.org/10.2142/biophysico.bppb-v22.0009 |
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| author | Fumiya Kondo Narutoshi Kamiya Gert-Jan Bekker Satoshi Nagao Nobutaka Numoto Hiroshi Sekiguchi Nobutoshi Ito Masayuki Oda |
| author_facet | Fumiya Kondo Narutoshi Kamiya Gert-Jan Bekker Satoshi Nagao Nobutaka Numoto Hiroshi Sekiguchi Nobutoshi Ito Masayuki Oda |
| author_sort | Fumiya Kondo |
| collection | DOAJ |
| description | Enzyme function is often regulated by weak metal-ion binding, which results from conformational changes while maintaining conformational fluctuations. We analyzed the structure and function of cutinase-like enzyme, Cut190, using biophysical methods such as X-ray crystallography and molecular dynamics (MD) simulations, showing that its structure and function are finely regulated by weak Ca2+ binding and release. We succeeded to stabilize the enzyme by introducing a disulfide-bond which can degrade polyethylene terephthalate (PET) to PET monomers at the glass transition temperature of PET, ≈70°C. In this study, using the stabilized Cut190 mutants, Cut190**SS and Cut190**SS_F77L, we evaluated the requirement of Ca2+ for catalytic activity at 70°C, showing that the enzyme expressed the activity even in the absence of Ca2+, in contrast to that at 37°C. These results were supported by multicanonical MD analysis, which showed that the respective forms of the enzyme, such as closed, open, and engaged forms, were exchangeable, possibly because the potential energy barriers between the respective forms were lowered. Taken together, the conformational equilibrium to express the catalytic activity was regulated by weak Ca2+ binding at 37°C, and was also regulated by increasing temperature. The respective conformational states of Cut190**SS and Cut190**SS_F77L correlated well with their different catalytic activities for PET. |
| format | Article |
| id | doaj-art-af6cb00b01ba49738744e2cd48ce0856 |
| institution | Kabale University |
| issn | 2189-4779 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | The Biophysical Society of Japan |
| record_format | Article |
| series | Biophysics and Physicobiology |
| spelling | doaj-art-af6cb00b01ba49738744e2cd48ce08562025-08-20T03:47:49ZengThe Biophysical Society of JapanBiophysics and Physicobiology2189-47792025-05-012210.2142/biophysico.bppb-v22.0009Structure-activity relationship of PET-degrading cutinase regulated by weak Ca2+ binding and temperatureFumiya Kondo0Narutoshi Kamiya1Gert-Jan Bekker2Satoshi Nagao3Nobutaka Numoto4Hiroshi Sekiguchi5Nobutoshi Ito6Masayuki Oda7Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto 606-8522, JapanGraduate School of Information Science, University of Hyogo, Kobe, Hyogo 650-0047, JapanInstitute for Protein Research, Osaka University, Suita, Osaka 565-0871, JapanCenter for Synchrotron Radiation Research, Japan Synchrotron Radiation Research Institute, Hyogo 679-5198, JapanMedical Research Laboratory, Institute of Integrated Research, Institute of Science Tokyo, Tokyo 113-8510, JapanCenter for Synchrotron Radiation Research, Japan Synchrotron Radiation Research Institute, Hyogo 679-5198, JapanMedical Research Laboratory, Institute of Integrated Research, Institute of Science Tokyo, Tokyo 113-8510, JapanGraduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto 606-8522, JapanEnzyme function is often regulated by weak metal-ion binding, which results from conformational changes while maintaining conformational fluctuations. We analyzed the structure and function of cutinase-like enzyme, Cut190, using biophysical methods such as X-ray crystallography and molecular dynamics (MD) simulations, showing that its structure and function are finely regulated by weak Ca2+ binding and release. We succeeded to stabilize the enzyme by introducing a disulfide-bond which can degrade polyethylene terephthalate (PET) to PET monomers at the glass transition temperature of PET, ≈70°C. In this study, using the stabilized Cut190 mutants, Cut190**SS and Cut190**SS_F77L, we evaluated the requirement of Ca2+ for catalytic activity at 70°C, showing that the enzyme expressed the activity even in the absence of Ca2+, in contrast to that at 37°C. These results were supported by multicanonical MD analysis, which showed that the respective forms of the enzyme, such as closed, open, and engaged forms, were exchangeable, possibly because the potential energy barriers between the respective forms were lowered. Taken together, the conformational equilibrium to express the catalytic activity was regulated by weak Ca2+ binding at 37°C, and was also regulated by increasing temperature. The respective conformational states of Cut190**SS and Cut190**SS_F77L correlated well with their different catalytic activities for PET.https://doi.org/10.2142/biophysico.bppb-v22.0009catalytic activityconformational changeenzymemulticanonical molecular dynamics simulationsweak metal-ion binding |
| spellingShingle | Fumiya Kondo Narutoshi Kamiya Gert-Jan Bekker Satoshi Nagao Nobutaka Numoto Hiroshi Sekiguchi Nobutoshi Ito Masayuki Oda Structure-activity relationship of PET-degrading cutinase regulated by weak Ca2+ binding and temperature Biophysics and Physicobiology catalytic activity conformational change enzyme multicanonical molecular dynamics simulations weak metal-ion binding |
| title | Structure-activity relationship of PET-degrading cutinase regulated by weak Ca2+ binding and temperature |
| title_full | Structure-activity relationship of PET-degrading cutinase regulated by weak Ca2+ binding and temperature |
| title_fullStr | Structure-activity relationship of PET-degrading cutinase regulated by weak Ca2+ binding and temperature |
| title_full_unstemmed | Structure-activity relationship of PET-degrading cutinase regulated by weak Ca2+ binding and temperature |
| title_short | Structure-activity relationship of PET-degrading cutinase regulated by weak Ca2+ binding and temperature |
| title_sort | structure activity relationship of pet degrading cutinase regulated by weak ca2 binding and temperature |
| topic | catalytic activity conformational change enzyme multicanonical molecular dynamics simulations weak metal-ion binding |
| url | https://doi.org/10.2142/biophysico.bppb-v22.0009 |
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