Premium
A single proline substitution is critical for the thermostabilization of Clostridium beijerinckii alcohol dehydrogenase
Author(s) -
Goihberg Edi,
Dym Orly,
TelOr Shoshana,
Levin Inna,
Peretz Moshe,
Burstein Yigal
Publication year - 2007
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.21170
Subject(s) - thermostability , proline , thermophile , mesophile , biochemistry , chemistry , alcohol dehydrogenase , enzyme , biology , amino acid , bacteria , genetics
Abstract 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: Δ T 1/2 60 min= + 8°C (temperature of 50% inactivation after incubation for 60 min), Δ T 1/2 CD= +11.5°C (temperature at which 50% of the original CD signal at 218 nm is lost upon heating between 30° and 98°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. Proteins 2007. © 2006 Wiley‐Liss, Inc.