RESUMO
Secondary alcohol dehydrogenase (SADH) from Thermoanaerobacter ethanolicus reduces ketones to chiral alcohols, and generally obeys Prelog's Rule, with binding pockets for large and small alkyl substituents, giving (S)-alcohols. We have previously shown that mutations in both the large and small pockets can alter both substrate specificity and stereoselectivity. In the present work, Met-151 and Thr-153, residues located in the small pocket, were mutated to alanine. The M151A mutant SADH shows significantly lower activity and lower stereoselectivity for reduction of aliphatic ketones than wild-type SADH. Furthermore, M151A showed non-linear kinetics for reduction of acetone. T153A SADH shows lower activity but similar stereoselectivity for ketone reduction compared to wild-type SADH. The I86A/M151A/C295A and I86A/T153A/C295A triple mutant SADH show altered specificity for reduction of substituted acetophenones. These results confirm that these mutations are useful to combine with I86A/C295A SADH to expand the small pocket of SADH and broaden the substrate specificity.
Assuntos
Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Thermoanaerobacter/enzimologia , Thermoanaerobacter/genética , Oxirredutases do Álcool/química , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico/genética , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Especificidade por SubstratoRESUMO
Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase (SADH) reduces aliphatic ketones according to Prelog's Rule, with binding pockets for small and large substituents. It was shown previously that the I86A mutant SADH reduces acetophenone, which is not a substrate of wild-type SADH, to give the anti-Prelog R-product (Musa, M. M.; Lott, N.; Laivenieks, M.; Watanabe, L.; Vieille, C.; Phillips, R. S. ChemCatChem2009, 1, 89-93.). However, I86A SADH did not reduce aryl ketones with substituents larger than fluorine. We have now expanded the small pocket of the active site of I86A SADH by mutation of Cys-295 to alanine to allow reaction of substituted acetophenones. As predicted, the double mutant I86A/C295A SADH has broadened substrate specificity for meta-substituted, but not para-substituted, acetophenones. However, the increase of the substrate specificity of I86A/C295A SADH is accompanied by a decrease in the kcat/Km values of acetophenones, possibly due to the substrates fitting loosely inside the more open active site. Nevertheless, I86A/C295A SADH gives high conversions and very high enantiomeric excess of the anti-Prelog R-alcohols from the tested substrates.
