Probing Structural Elements of Thermal Stability in Bacterial Oligomeric Alcohol Dehydrogenases. I. Construction and Characterization of Chimeras Consisting of Secondary ADHs from Thermoanaerobacter brockii and Clostridium beijerinckii |
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Authors: | Bogin Oren Peretz Moshe Burstein Yigal |
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Institution: | (1) Department of Organic Chemistry, The Weizmann Institute of Science, 76100 Rehovot, Israel |
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Abstract: | Summary Two tetrameric secondary alcohol dehydrogenases (ADHs), one from the mesophileClostridium beijerinckii (CBADH) and the other from the extreme thermophileThermoanaerobacter brockii (TBADH), share 75% sequence identity but differ by 26°C in thermal stability. To explore the role of linear segments of these
similar enzymes in maintaining the thermal stability of the thermostable TBADH, a series of 12 CBadh and TBadh chimeric genes and the two parental wild-type genes were expressed inEscherichia coli, and the enzymes were isolated, purified and characterized. The thermal stability of each chimeric enzyme was approximately
exponentially proportional to the content of the amino acid sequence of the thermophilic enzyme, indicating that the amino
acid residues contributing to the thermal stability of TBADH are distributed along the whole protein molecule. It is suggested
that major structural elements of thermal stability may reside among the nine discrepant amino acid residues between the N-terminal
50-amino acid residues of TBADH and CBADH. |
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Keywords: | ADH chimera mutagenesis protein engineering thermal stability |
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