Mutation of Serine-39 to threonine in thermostable secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus changes enantiospecificity

被引:37
作者
Tripp, AE
Burdette, DS
Zeikus, JG
Phillips, RS [1 ]
机构
[1] Univ Georgia, Dept Chem, Athens, GA 30602 USA
[2] Univ Georgia, Dept Biochem & Mol Biol, Athens, GA 30602 USA
[3] Univ Georgia, Ctr Metalloenzyme Studies, Athens, GA 30602 USA
[4] Michigan State Univ, Dept Biochem, E Lansing, MI 48824 USA
关键词
D O I
10.1021/ja974129t
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
The substrate specificity of wild-type and Ser39 --> Thr (S39T) secondary alcohol dehydrogenase (SADH) from Thermoanaerobacter ethanolicus was examined. The S39T mutation increases activity for 2-propanol without any significant effect on NADP(+) binding. There is no significant effect of the mutation on the primary and secondary alcohol specificity of SADH. However, an effect on the enantiospecificity of SADH by the S39T mutation is demonstrated. Throughout the temperature range from 15 to 55 degrees C, wild-type SADH exhibits a preference for (S)-2-pentanol. In contrast, a temperature-dependent reversal of enantiospecificity is observed for 2-butanol, with a racemic temperature of 297 K. Throughout the same range of temperatures, S39T SADH exhibits higher enantiospecificity for the (R)-enantiomers of both 2-butanol and 2-pentanol. Examination of individual k(cat)/K-m values for each enantiomer of the chiral alcohols reveals that the effect of the mutation is to decrease (S)-2-butanol specificity, and to preferentially enhance (R)-2-pentanol specificity relative to (S)-2-pentanol. These results are the first step toward expanding the synthetic utility of SADH to allow efficient preparation of a range of (R)-alcohols.
引用
收藏
页码:5137 / 5141
页数:5
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