Protein engineering of alcohol dehydrogenases; effects of amino acid changes at positions 93 and 48 of yeast ADH1

By protein engineering we have investigated changes to two aminoacid residues (Trp93 and Ser48) in the substrate pocket of yeastalcohol dehydrogenase 1. Upon changing Thr48 to serine we producedan enzyme which has markedly greater activity towards aliphaticalcohols with chain length up to 8, together with a generalincrease in catalytic activity (V/K). Changes at position 93were less pronounced, with the Phe enzyme being more activethan the parent towards the range of alcohols but with the alanineenzyme showing very little difference from the wild-type. Enzymeswith the double changes at 48 and 93 showed increased activitytowards alcohols with 3–8 carbons but the increases werenot additive over the single changes. The enzymes with changesat the two positions would metabolize both stereoisomers of2-octanol whereas the parent ADH would attack only one of them.None of the engineered enzymes would attack cyclohexanol oraromatic alcohols. The results are in general agreement withthe prediction that reducing the size of amino acids in thesubstrate pocket would enhance the ability to oxidize alcoholslarger than ethanol.