Thermal stabilization of the protozoan Entamoeba histolytica alcohol dehydrogenase by a single proline substitution.
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 (DeltaT(1/2) <or= +10 degrees C), whereas the reverse mutation in the thermophilic TbADH (P275D-TbADH) reduces the thermostability of the enzyme (DeltaT(1/2) <or= -18.8 degrees 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.
Goihberg
Edi
E
Department of Organic Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Dym
Orly
O
Tel-Or
Shoshana
S
Shimon
Linda
L
Frolow
Felix
F
Peretz
Moshe
M
Burstein
Yigal
Y
D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D017931:DNA Primers; D004748:Entamoeba histolytica / Q000201:enzymology*; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000378:metabolism*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet
D017931:DNA Primers; D011392:Proline; D000426:Alcohol Dehydrogenase