A Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase mutant derivative highly active and stereoselective on phenylacetone and benzylacetone.
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). 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 degrees 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.
Ziegelmann-Fjeld
Karla I
KI
Department of Biochemistry and Molecular Biology, Michigan State University, 410 Biochemistry Building, East Lansing, MI 48824-1319, USA.
Musa
Musa M
MM
Phillips
Robert S
RS
Zeikus
J Gregory
JG
Vieille
Claire
C
D000096:Acetone / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001593:Benzyl Compounds / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D015394:Molecular Structure; D009154:Mutation; D010084:Oxidation-Reduction; D010626:Phenylethyl Alcohol / Q000031:analogs & derivatives / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D046968:Thermoanaerobacter / Q000201:enzymology*
D001593:Benzyl Compounds; D000096:Acetone; C513216:1-phenyl-2-propanol; D000429:Alcohol Oxidoreductases; C025192:isopropanol dehydrogenase (NADP); D010626:Phenylethyl Alcohol; C008863:1-phenyl-2-propanone