Alcohol dehydrogenase from Thermoanaerobacter ethanolicus

Musa, M.M., Ziegelmann-Fjeld, K.I., Vieille, C. et al. (2007) Asymmetric reduction and oxidation of aromatic ketones and alcohols using W110A secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus. The Journal of Organic Chemistry, 72 (1), 30-34. 

Musa, M., Ziegelman-Fjeld, K., Vieille, C., Zeikus, J. and Phillips, R., Xerogel-encapsulated WHOA secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus performs asymmetric reduction of hydrophobic ketones in organic solvents. Angew. Chem. Int. Ed., 2007, 46, 3091-3094. 

Pham, V.T., Phillips, R.S. and Ljungdahl, L.G. (1989) Temperature-dependent enantiospecificity of secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus. J. Am. Chem. Soc., 111, 1935-1936. 

C. Heiss, M. Laivenieks, G. J. Zeikus, and R. S. Phillips, The stereospecificity of secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus is partially determined by active site water,... 

Point mutation of enzymes has played an important role in determining those amino acid residues involved in catalytic activities. It has also been used to improve the enantioselectivity of dehydrogenases. For example, even a single point mutation of a secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus can change substantially the enantioselectivity for the reduction of 2-butanone and 2-pentanone as shown in Table 15-6 45l... 

Table 15-6. Control of enantioselectivity by a single mutation (serine-39 to threonine) of the secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus 5.

The ability of alcohol dehydrogenases to distinguish the methylene protons of ethanol is a paradigm of enzymic stereospecificity. Many alcohol dehydrogenases can function with both primary and secondary alcohols thus their ability to show selectivity toward the chirality of the secondary alcohols is remarkable. An example of the difficulties associated with stereospecificity of secondary alcohol dehydrogenases is illustrated by the thermophilic alcohol dehydrogenase from Thermoanaerobacter ethanolicus (57). At low temperatures the enzyme preferentially uses the 5 isomer of 2-pentanol or 2-butanol. The enantiomeric discrimination, however, is temperature dependent. Above a temperature 7, at which there is no enantiomeric discrimination, the enzyme shows the opposite preference and uses the R isomer. The value of 7, is a function of the substrate for... 

Bryant FO, Wiegel J, Ljungdahl LG (1988) Purification and properties of primary and secondary alcohol dehydrogenases from Thermoanaerobacter ethanolicus. Appl Environ Microbiol 54 460-465 Buchholz SE, Dooley MM, Eveleigh DE (1987) Zymomonas—an alcoholic enigma. Trends Biotechnol 5 199-204... 

Burdette D, Zeikus JG (1994) Purification of acetaldehyde dehydrogenase and alcohol dehydrogenases from Thermoanaerobacter ethanolicus 39E and... 

Rational Mutation to Widen Substrate Specificity Rational design in modification of enzyme by point mutation has been reported. The secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus 39E... 

Musa MM, Ziegelmann-Fjeld KI, Vieille C, Phillips RS. Activity and selectivity of WHOA secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus in organic solvents and ionic liquids mono-and biphasic media. Org. Biomol. Chem. 2008 6 887-892. 

C. R. Lowe, and N. C. Bruce, Cloning, sequencing and expression in Escherichia coli of the primary alcohol dehydrogenase gene from Thermoanaerobacter ethanolicus JW200, FEMS Microbiol. Lett. 2000, 190, 57-62. 

Reaction temperature is one of the parameters affecting the enantioselectivity of a reaction [16]. For the oxidation of an alcohol, the values of kcat/fQn were determined for the (R)- and (S)-stereodefining enantiomers E is the ratio between them. From the transition state theory, the free energy difference at the transition state between (R) and (S) enantiomers can be calculated from E (Equation 2), and AAG is in turn the function of temperature (Equation 3). The racemic temperature (% ) can be calculated as shown in (Equation 4). Using these equations, % for 2-butanol and 2-pentanol of the Thermoanaerobacter ethanolicus alcohol dehydrogenase were determined to be 26 and 77 °C, respectively. 

The results of the temperature dependence of the reaction rates of the enantiomers of secondary alcohols with a secondary alcohol dehydrogenase (SADE1) from the thermophilic bacterium Thermoanaerobacter ethanolicus demonstrated a temperature-dependent reversal of stereospecificity (Pham, 1990) (Figure 5.16). At T < 26°C, (S)-2-butanol was a better substrate than (i )-2-butanol on the basis of kCSLt/KM values however, at T> 26°C, (R)-2-butanol was a better substrate than (S)-2-butanol. (S)-2-Pentanol was the preferred substrate at T < 60°C however, the data predict that (i )-2-pentanol would be preferred at T > 70°C. (S)-2-Elexanol was predicted to be the preferred enantiomer only at T > 240°C. Therefore, the concept of isoinversion temperature is as valid for enzyme reactions as for others only the range of catalytically accessible temperatures is smaller. 

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