期刊
BIOCHEMISTRY
卷 57, 期 36, 页码 5289-5300出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.biochem.8b00624
关键词
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资金
- Joint Studies Program of the Okazaki BIONEXT project of the Exploratory Research Center on Life and Living Systems (ExCELLS)
- Platform Project for Supporting Drug Discovery and Life Science Research [Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)] from AMED [JP17am0101001]
- Institute for Protein Research, Osaka University [CR-18-02]
- Japan Society for the Promotion of Science (JSPS) [JP16K07331]
- HPCI Research Project [hp170024]
- Institute for Fermentation, Osaka
A cutinase-type polyesterase from Saccharomonospora viridis AHK190 (Cut190) has been shown to degrade the inner block of polyethylene terephthalate. A unique feature of Cut190 is that its function and stability are regulated by Ca2+ binding. Our previous crystal structure analysis of Cut190S226P showed that one Ca2+ binds to the enzyme, which induces large conformational changes in several loop regions to stabilize an open conformation [Miyakawa, T., et al. (2015) Appl. Microbiol. Biotechnol. 99, 4297]. In this study, to analyze the substrate recognition mechanism of Cut190, we determined the crystal structure of the inactive form of a Cut190 mutant, Cut190*S176A, in complex with calcium ions and/or substrates. We found that three calcium ions bind to Cut190*S176A, which is supported by analysis using native mass spectrometry experiments and 3D Reference Interaction Site Model calculations. The complex structures with the two substrates, monoethyl succinate and monoethyl adipate (engaged and open forms), presumably correspond to the pre- and post-reaction states, as the ester bond is close to the active site and pointing outward from the active site, respectively, for the two complexes. Ca2+ binding induces the pocket to open, enabling the substrate to access the pocket more easily. Molecular dynamics simulations suggest that a post-reaction state in the engaged form presumably exists between the experimentally observed forms, indicating that the substrate would be cleaved in the engaged form and then requires the enzyme to change to the open form to release the product, a process that Ca2+ can greatly accelerate.
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