A single amino acid substitution in the Clostridium beijerinckii alcohol dehydrogenase is critical for thermostabilization
(see also Tetrameric alcohol dehydrogenases)
Publication Abstract from PubMed
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.
A single proline substitution is critical for the thermostabilization of Clostridium beijerinckii alcohol dehydrogenase., Goihberg E, Dym O, Tel-Or S, Levin I, Peretz M, Burstein Y, Proteins. 2007 Jan 1;66(1):196-204. PMID:17063493
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
The crystal structure of Clostridium beijerinckii alcohol dehydrogenase (CbADH; EC 1.1.1.2) with the substitution Q100P () was solved at 2.25 Å resolution. The of Gln100 with Pro did not cause significant structural changes in the protein structure. The residues of the wildtype protein are colored lime and the residues of the mutant one in cyan. Only 2 H-bonds were lost, one between Oε1 of Gln100 and the main chain N of Gly297, and the second between Nε2 of Gln100 and the main chain carbonyl O of Gly297. The mutation caused that an additional CH2 group (Cδ of Pro100) is surrounded by nonpolar residues: Pro88 (3.8 Å), Trp90 (3.5 Å), and Val95 (4 Å). These residues (P100, P88, W90, and V95) are situated on a protruding lobe of the protein. An additional 11 aliphatic and aromatic carbon atoms are situated within the distance of 6 Å from Cδ of Pro100 (two methyl groups of Val95; three carbon atoms of the Trp90 indole group; Cβ and Cγ methylene groups of Pro100; Cβ and Cγ of Gln101, and two carbons of the Phe99 phenyl ring).
About this Structure
2B83 is a Single protein structure of sequence from Clostridium beijerinckii. Full crystallographic information is available from OCA.
