A single proline substitution is critical for the thermostabilization of Clostridium beijerinckii alcohol dehydrogenase.
Analysis of the three-dimensional structures of three closely related mesophilic, thermophilic, and hyperthermophilic alcohol dehydrogenases (ADHs) from the respective microorganisms Clostridium beijerinckii (CbADH), Entamoeba histolytica (EhADH1), and Thermoanaerobacter brockii (TbADH) suggested that a unique, strategically located proline residue (Pro100) might be crucial for maintaining the thermal stability of EhADH1. To determine whether proline substitution at this position in TbADH and CbADH would affect thermal stability, we used site-directed mutagenesis to replace the complementary residues in both enzymes with proline. The results showed that replacing Gln100 with proline significantly enhanced the thermal stability of the mesophilic ADH: DeltaT(1/2) (60 min) = + 8 degrees C (temperature of 50% inactivation after incubation for 60 min), DeltaT(1/2) (CD) = +11.5 degrees C (temperature at which 50% of the original CD signal at 218 nm is lost upon heating between 30 degrees and 98 degrees C). A His100 --> Pro substitution in the thermophilic TbADH had no effect on its thermostability. An analysis of the three-dimensional structure of the crystallized thermostable mutant Q100P-CbADH suggested that the proline residue at position 100 stabilized the enzyme by reinforcing hydrophobic interactions and by reducing the flexibility of a loop at this strategic region.
Goihberg
Edi
E
Department of Organic Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Dym
Orly
O
Tel-Or
Shoshana
S
Levin
Inna
I
Peretz
Moshe
M
Burstein
Yigal
Y
D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002942:Circular Dichroism; D046970:Clostridium beijerinckii / Q000201:enzymology*; D004795:Enzyme Stability; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D011392:Proline / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D011994:Recombinant Proteins; D011392:Proline; D000426:Alcohol Dehydrogenase