Crystal structure and enhanced activity of a cutinase‐like enzyme from Cryptococcus sp. strain S‐2 |
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| Authors: | Yuya Kodama Kazuo Masaki Hidemasa Kondo Mamoru Suzuki Sakae Tsuda Tomohiro Nagura Nobuhisa Shimba Ei‐ichiro Suzuki Haruyuki Iefuji |
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| Institution: | 1. Institute of Life Sciences, Ajinomoto Co., Inc., Kawasaki‐ku, Kawasaki‐shi 210‐8681, Japan;2. National Research Institute of Brewing, Higashi‐Hiroshima 739‐0046, Japan;3. Research Institute of Genome‐based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Toyohira‐ku, Sapporo 062‐8517, Japan;4. Research Center for Structural and Functional Proteomics, Institute for Protein Research, Osaka University, Suita, Osaka 565‐0871, Japan |
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| Abstract: | The structural and enzymatic characteristics of a cutinase‐like enzyme (CLE) from Cryptococcus sp. strain S‐2, which exhibits remote homology to a lipolytic enzyme and a cutinase from the fungus Fusarium solani (FS cutinase), were compared to investigate the unique substrate specificity of CLE. The crystal structure of CLE was solved to a 1.05 Å resolution. Moreover, hydrolysis assays demonstrated the broad specificity of CLE for short and long‐chain substrates, as well as the preferred specificity of FS cutinase for short‐chain substrates. In addition, site‐directed mutagenesis was performed to increase the hydrolysis activity on long‐chain substrates, indicating that the hydrophobic aromatic residues are important for the specificity to the long‐chain substrate. These results indicate that hydrophobic residues, especially the aromatic ones exposed to solvent, are important for retaining lipase activity. Proteins 2009. © 2009 Wiley‐Liss, Inc. |
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| Keywords: | CLE lipase cutinase substrate specificity hydrolysis site‐directed mutagenesis protein engineering structural biology |
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