Thermal stabilization of the protozoan Entamoeba histolytica alcohol dehydrogenase by a single proline substitution
Edi Goihberg
Department of Organic Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel
Search for more papers by this authorOrly Dym
Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel
Israel Structural Proteomics Center (ISPC), Weizmann Institute of Science, Rehovot 76100, Israel
Search for more papers by this authorShoshana Tel-Or
Department of Organic Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel
Search for more papers by this authorLinda Shimon
Chemical Research Support, Weizmann Institute of Science, Rehovot 76100, Israel
Search for more papers by this authorFelix Frolow
Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv 69978, Israel
The Daniella Rich Institute for Structural Biology, Tel-Aviv University, Ramat Aviv 69978, Israel
Search for more papers by this authorMoshe Peretz
Department of Organic Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel
Search for more papers by this authorCorresponding Author
Yigal Burstein
Department of Organic Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel
Department of Organic Chemistry, Weizmann Institute of Science, 76100 Rehovot, Israel===Search for more papers by this authorEdi Goihberg
Department of Organic Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel
Search for more papers by this authorOrly Dym
Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel
Israel Structural Proteomics Center (ISPC), Weizmann Institute of Science, Rehovot 76100, Israel
Search for more papers by this authorShoshana Tel-Or
Department of Organic Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel
Search for more papers by this authorLinda Shimon
Chemical Research Support, Weizmann Institute of Science, Rehovot 76100, Israel
Search for more papers by this authorFelix Frolow
Department of Molecular Microbiology and Biotechnology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat Aviv 69978, Israel
The Daniella Rich Institute for Structural Biology, Tel-Aviv University, Ramat Aviv 69978, Israel
Search for more papers by this authorMoshe Peretz
Department of Organic Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel
Search for more papers by this authorCorresponding Author
Yigal Burstein
Department of Organic Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel
Department of Organic Chemistry, Weizmann Institute of Science, 76100 Rehovot, Israel===Search for more papers by this authorAbstract
Analysis of the three-dimensional structures of two closely related thermophilic and hyperthermophilic alcohol dehydrogenases (ADHs) from the respective microorganisms Entamoeba histolytica (EhADH1) and Thermoanaerobacter brockii (TbADH) suggested that a unique, strategically located proline residue (Pro275) at the center of the dimerization interface might be crucial for maintaining the thermal stability of TbADH. To assess the contribution of Pro275 to the thermal stability of the ADHs, we applied site-directed mutagenesis to replace Asp275 of EhADH1 with Pro (D275P-EhADH1) and conversely Pro275 of TbADH with Asp (P275D-TbADH). The results indicate that replacing Asp275 with Pro significantly enhances the thermal stability of EhADH1 (ΔT1/2 ≤ +10°C), whereas the reverse mutation in the thermophilic TbADH (P275D-TbADH) reduces the thermostability of the enzyme (ΔT1/2 ≤ −18.8°C). Analysis of the crystal structures of the thermostabilized mutant D275P-EhADH1 and the thermocompromised mutant P275D-TbADH suggest that a proline residue at position 275 thermostabilized the enzymes by reducing flexibility and by reinforcing hydrophobic interactions at the dimer–dimer interface of the tetrameric ADHs. Proteins 2008. © 2008 Wiley-Liss, Inc.
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