PEDS Advance Access published online on February 5, 2007
Protein Engineering Design and Selection, doi:10.1093/protein/gzl052
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A Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase mutant derivative highly active and stereoselective on phenylacetone and benzylacetone
1 Department of Biochemistry and Molecular Biology, Michigan State University, 410 Biochemistry Building, East Lansing, MI 48824–1319 2 Department of Chemistry, University of Georgia, Athens, GA 30602–2556 3 Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602–2556, USA
4 To whom correspondence should be addressed. E-mail: vieille{at}msu.edu
The secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus 39E (TeSADH) is highly thermostable and solvent-stable, and it is active on a broad range of substrates. These properties make TeSADH an excellent template to engineer an industrial catalyst for chiral chemical synthesis. (S)-1-Phenyl-2-propanol was our target product because it is a precursor to major pharmaceuticals containing secondary alcohol groups. TeSADH has no detectable activity on this alcohol, but it is highly active on 2-butanol. The structural model we used to plan our mutagenesis strategy was based on the substrate's orientation in a horse liver alcohol dehydrogenase•p-bromobenzyl alcohol•NAD+ ternary complex (PDB entry 1HLD [PDB] ). The W110A TeSADH mutant now uses (S)-1-phenyl-2-propanol, (S)-4-phenyl-2-butanol and the corresponding ketones as substrates. W110A TeSADH's kinetic parameters on these substrates are in the same range as those of TeSADH on 2-butanol, making W110A TeSADH an excellent catalyst. In particular, W110A TeSADH is twice as efficient on benzylacetone as TeSADH is on 2-butanol, and it produces (S)-4-phenyl-2-butanol from benzylacetone with an enantiomeric excess above 99%. W110A TeSADH is optimally active at 87.5°C and remains highly thermostable. W110A TeSADH is active on aryl derivatives of phenylacetone and benzylacetone, making this enzyme a potentially useful catalyst for the chiral synthesis of aryl derivatives of alcohols. As a control in our engineering approach, we used the TbSADH•(S)-2-butanol binary complex (PDB entry 1BXZ) as the template to model a mutation that would make TeSADH active on (S)-1-phenyl-2-propanol. Mutant Y267G TeSADH did not have the substrate specificity predicted in this modeling study. Our results suggest that (S)-2-butanol's orientation in the TbSADH•(S)-2-butanol binary complex does not reflect its orientation in the ternary enzyme–substrate–cofactor complex.
Keywords: (S)-alcohol/benzylacetone/enantioselectivity/phenylacetone/secondary alcohol dehydrogenase
Received July 30, 2006; revised November 7, 2006; accepted November 13, 2006.