Residues specific for class III alcohol dehydrogenase. Site-directed mutagenesis of the human enzyme

Biochemistry ◽  
1994 ◽  
Vol 33 (50) ◽  
pp. 15080-15085 ◽  
Author(s):  
Mats Estonius ◽  
Jan-Olov Höög ◽  
Olle Danielsson ◽  
Hans Jörnvall
Keyword(s):  
Alcohol Dehydrogenase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Class Iii ◽  
Human Enzyme

Site-directed mutagenesis of glycine-14 and two "critical" cysteinyl residues in Drosophila alcohol dehydrogenase

Biochemistry ◽  
1990 ◽  
Vol 29 (5) ◽  
pp. 1112-1118 ◽  
Author(s):  
Zhuo Chen ◽  
Ling Lu ◽  
Michelle Shirley ◽  
William R. Lee ◽  
Simon H. Chang
Keyword(s):  
Alcohol Dehydrogenase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals Features of Structural Zinc in Mammalian Alcohol Dehydrogenase. Site-Directed Mutagenesis of the Zinc Ligands

1994 ◽  
Vol 225 (3) ◽  
pp. 1015-1019 ◽  
Author(s):  
Jana Jelokova ◽  
Christina Karlsson ◽  
Mats Estonius ◽  
Hans Jornvall ◽  
Jan-Olov Hoog
Keyword(s):  
Alcohol Dehydrogenase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals Functional Analysis by Site-Directed Mutagenesis of the NAD+-Reducing Hydrogenase from Ralstonia eutropha

2002 ◽  
Vol 184 (22) ◽  
pp. 6280-6288 ◽  
Author(s):  
Tanja Burgdorf ◽  
Antonio L. De Lacey ◽  
Bärbel Friedrich
Keyword(s):  
Functional Analysis ◽  
Ralstonia Eutropha ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Class Iii ◽  
Hydrogen Activation ◽  
Major Class ◽  
Oxidation Of Hydrogen ◽  
Reducing Activity ◽  
Class Iv

ABSTRACT The tetrameric cytoplasmic [NiFe] hydrogenase (SH) of Ralstonia eutropha couples the oxidation of hydrogen to the reduction of NAD+ under aerobic conditions. In the catalytic subunit HoxH, all six conserved motifs surrounding the [NiFe] site are present. Five of these motifs were altered by site-directed mutagenesis in order to dissect the molecular mechanism of hydrogen activation. Based on phenotypic characterizations, 27 mutants were grouped into four different classes. Mutants of the major class, class I, failed to grow on hydrogen and were devoid of H2-oxidizing activity. In one of these isolates (HoxH I64A), H2 binding was impaired. Class II mutants revealed a high D2/H+ exchange rate relative to a low H2-oxidizing activity. A representative (HoxH H16L) displayed D2/H+ exchange but had lost electron acceptor-reducing activity. Both activities were equally affected in class III mutants. Mutants forming class IV showed a particularly interesting phenotype. They displayed O2-sensitive growth on hydrogen due to an O2-sensitive SH protein.

Effect of site-directed mutagenesis on conserved positions of Drosophila alcohol dehydrogenase

FEBS Letters ◽  
1993 ◽  
Vol 319 (1-2) ◽  
pp. 90-94 ◽  
Author(s):  
Neus Cols ◽  
Gemma Marfany ◽  
Sílvia Atrian ◽  
Roser Gonzàlez-Duarte
Keyword(s):  
Alcohol Dehydrogenase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

Tailoring the substrate specificity of secondary alcohol dehydrogenase

2002 ◽  
Vol 80 (6) ◽  
pp. 680-685 ◽  
Author(s):  
Robert S Phillips
Keyword(s):  
Alcohol Dehydrogenase ◽  
Substrate Specificity ◽  
Temperature Dependence ◽  
Secondary Alcohol ◽  
Reaction Medium ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Secondary Alcohol Dehydrogenase ◽  
Binding Pockets ◽  
Mutant Forms

Our research with the thermophilic secondary-alcohol dehydrogenase (SADH) from Thermoanaerobacter ethanolicus has provided novel information regarding the physical basis of enzyme substrate specificity and stereospecificity. We demonstrated that oxidation of secondary alcohols catalyzed by T. ethanolicus SADH exhibits temperature-dependent enantiospecificity. In other studies, we found that the structure of co-factor analogs also significantly affects the stereochemistry of the SADH reaction. More recently, we demonstrated that pH can also have a modest effect on SADH enantiospecificity. Organic solvents have also been shown by others to affect the stereochemistry of SADH reactions. We designed and prepared S39T and C295A mutant forms of SADH by site-directed mutagenesis, and we evaluated the effects of the mutations by analysis of the temperature dependence of the enantiomeric ratio (E) for simple chiral alcohols such as 2-butanol. This procedure allows for the determination of the differential Eyring parameters (ΔΔH‡ and ΔΔS‡) for the reaction. We demonstrated that this technique is a sensitive method for analysis of the effects of mutation on enzyme stereospecificity. S39T and C295A SADH exhibit significant changes in substrate specificity and stereospecificity consistent with the changes in the volume of the alkyl-binding pockets. Thus, it is possible to alter the substrate specificity and stereospecificity of alcohol dehydrogenase by changing either the reaction medium or the protein structure.Key words: alcohol dehydrogenase, substrate specificity, stereospecificity, temperature dependence, site-directed mutagenesis.

Sign in / Sign up

Export Citation Format

Share Document