scholarly journals Expanding the Substrate Specificity of Thermoanaerobacter pseudoethanolicus Secondary Alcohol Dehydrogenase by a Dual Site Mutation

2018 ◽  
Vol 2018 (6) ◽  
pp. 798-805 ◽  
Author(s):  
Musa M. Musa ◽  
Odey Bsharat ◽  
Ibrahim Karume ◽  
Claire Vieille ◽  
Masateru Takahashi ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Substrate Specificity ◽  
Secondary Alcohol ◽  
Site Mutation ◽  
Secondary Alcohol Dehydrogenase ◽  
Dual Site

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.

Cofactor specificity engineering of a long-chain secondary alcohol dehydrogenase from Micrococcus luteus for redox-neutral biotransformation of fatty acids

2019 ◽  
Vol 55 (96) ◽  
pp. 14462-14465 ◽  
Author(s):  
Eun-Ji Seo ◽  
Hye-Ji Kim ◽  
Myeong-Ju Kim ◽  
Jeong-Sun Kim ◽  
Jin-Byung Park
Keyword(s):  
Fatty Acids ◽  
Alcohol Dehydrogenase ◽  
Micrococcus Luteus ◽  
Secondary Alcohol ◽  
Cofactor Specificity ◽  
Secondary Alcohol Dehydrogenase ◽  
Long Chain

Structure-based cofactor specificity engineering of an alcohol dehydrogenase (mLSADH) enables a redox-neutral biotransformation of C18 fatty acids into C9 fatty acids.

scholarly journals Reconstruction of an Acetogenic 2,3-Butanediol Pathway Involving a Novel NADPH-Dependent Primary-Secondary Alcohol Dehydrogenase

2014 ◽  
Vol 80 (11) ◽  
pp. 3394-3403 ◽  
Author(s):  
Michael Köpke ◽  
Monica L. Gerth ◽  
Danielle J. Maddock ◽  
Alexander P. Mueller ◽  
FungMin Liew ◽  
...  
Keyword(s):  
Alcohol Dehydrogenase ◽  
Secondary Alcohol ◽  
Acetolactate Synthase ◽  
Chemical Production ◽  
Content Type ◽  
E Coli ◽  
Gas Fermentation ◽  
Secondary Alcohol Dehydrogenase ◽  
Clostridium Autoethanogenum ◽  
Butanediol Dehydrogenase

ABSTRACTAcetogenic bacteria use CO and/or CO2plus H2as their sole carbon and energy sources. Fermentation processes with these organisms hold promise for producing chemicals and biofuels from abundant waste gas feedstocks while simultaneously reducing industrial greenhouse gas emissions. The acetogenClostridium autoethanogenumis known to synthesize the pyruvate-derived metabolites lactate and 2,3-butanediol during gas fermentation. Industrially, 2,3-butanediol is valuable for chemical production. Here we identify and characterize theC. autoethanogenumenzymes for lactate and 2,3-butanediol biosynthesis. The putativeC. autoethanogenumlactate dehydrogenase was active when expressed inEscherichia coli. The 2,3-butanediol pathway was reconstituted inE. coliby cloning and expressing the candidate genes for acetolactate synthase, acetolactate decarboxylase, and 2,3-butanediol dehydrogenase. Under anaerobic conditions, the resultingE. colistrain produced 1.1 ± 0.2 mM 2R,3R-butanediol (23 μM h−1optical density unit−1), which is comparable to the level produced byC. autoethanogenumduring growth on CO-containing waste gases. In addition to the 2,3-butanediol dehydrogenase, we identified a strictly NADPH-dependent primary-secondary alcohol dehydrogenase (CaADH) that could reduce acetoin to 2,3-butanediol. Detailed kinetic analysis revealed that CaADH accepts a range of 2-, 3-, and 4-carbon substrates, including the nonphysiological ketones acetone and butanone. The high activity of CaADH toward acetone led us to predict, and confirm experimentally, thatC. autoethanogenumcan act as a whole-cell biocatalyst for converting exogenous acetone to isopropanol. Together, our results functionally validate the 2,3-butanediol pathway fromC. autoethanogenum, identify CaADH as a target for further engineering, and demonstrate the potential ofC. autoethanogenumas a platform for sustainable chemical production.

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