Screening for variants of human liver cytosolic aldehyde dehydrogenase with altered properties

Random mutagenesis and screening for enzyme activity in the presence and absence of Mg2+ ions have been used for identifying variants of human liver cytosolic isozyme of aldehyde dehydrogenase (ALDH1) with altered properties. A number of the ALDH1 variants with double, triple or quadruple mutations were identified having a lower dehydrogenase activity than the native enzyme. Individual variants, however, showed an enhanced activity with Mgt, ions which normally have an inhibitory effect on the native enzyme. This Mg2+-activating effect together with the substrate specificity of individual variants suggests that the native enzyme and the variants have different rate limiting-steps. In addition, chloramphenicol inhibited differently the native enzyme and the variants. For the native enzyme, the activity on propionaldehyde or benzaldehyde was partially inhibited in the presence of chloramphenicol, and no activity was detected if Mg2+ ions were included in the reaction. For the variants, the activity on benzaldehyde was completely inhibited, whereas the activity on propionaldehyde was partially inhibited even if Mg2+ ions were added. A common mutation involving a change of Thr-244 to Ser was found in all the variants, suggesting that this mutation may be responsible for reversing the Mg2+ ion inhibitory effect on the native enzyme and the change of chloramphenicol inhibition.