Site-directed mutagenesis was employed to generate five different triple point mutations in the double mutant (C295A/I86A) of Thermoanaerobacter ethanolicus alcohol dehydrogenase (TeSADH) by computer-aided modeling with the aim of widening the small alkyl binding pocket. TeSADH engineering enables the enzyme to accept sterically hindered substrates which could not be accepted by the wild-type enzyme. The underline in the mutations highlights the additional point mutation on the double mutant TeSADH introduced in this work. The catalytic efficiency (kcat/KM) of the M151A/C295A/I86A triple TeSADH mutant for acetophenone increased about 4.8-fold higher than that of the double mutant. A 2.4-fold increase in conversion of 3'-methylacetophenone to (R)-1-(3-methylphenyl)-ethanol with a yield of 87% was obtained by using V115A/C295A/I86A mutant in asymmetric reduction. The A85G/C295A/I86A mutant also produced (R)-1-(3-methylphenyl)-ethanol (1.7-fold) from 3'-methylacetophenone and (R)-1-(3-methoxyphenyl)ethanol (1.2-fold) from 3'-methoxyacetophenone, with improved yield. In terms of thermal stability, the M151A/C295A/I86A and V115A/C295A/I86A mutants significantly increased ΔT1/2 by +6.8°C and +2.4°C, respectively, with thermal deactivation constant (kd) close to the wild-type enzyme. The M151A/C295A/I86A mutant reacts optimally at 70 °C with almost 4 times more residual activity than the wild-type. Considering broad substrate tolerance and thermal stability together, it would be promising to produce (R)-1-(3-methylphenyl)-ethanol from 3'-methylacetophenone by V115A/C295A/I86A, and (R)-1-phenylethanol from acetophenone by M151A/C295A/I86A mutant, in large-scale bioreduction processes.
ID: ARyouSQr
Submitter: Shu-Ching Ou
Submission Date: March 25, 2019, 1:21 p.m.
Version: 1
For V115A+C295A+I86A, A85G+C295A+I86A variants, protein concentration in the kinetic experiments is halved.
Colors: | D | E | R | H | K | S | T | N | Q | A | V | I | L | M | F | Y | W | C | G | P |
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Structure ID | Release Date | Resolution | Structure Title |
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1BXZ | 1998-10-09T00:00:00+0000 | 2.99 | CRYSTAL STRUCTURE OF A THERMOPHILIC ALCOHOL DEHYDROGENASE SUBSTRATE COMPLEX FROM THERMOANAEROBACTER BROCKII |
1YKF | 1996-03-25T00:00:00+0000 | 2.5 | NADP-DEPENDENT ALCOHOL DEHYDROGENASE FROM THERMOANAEROBIUM BROCKII |
2NVB | 2006-11-12T00:00:00+0000 | 2.8 | Contribution of Pro275 to the Thermostability of the Alcohol Dehydrogenases (ADHs) |
3FPC | 2009-01-05T00:00:00+0000 | 1.4 | Chimera of alcohol dehydrogenase by exchange of the cofactor binding domain res 153-294 of T. brockii ADH by E. histolytica ADH |
3FPL | 2009-01-05T00:00:00+0000 | 1.9 | Chimera of alcohol dehydrogenase by exchange of the cofactor binding domain res 153-295 of C. beijerinckii ADH by T. brockii ADH |
3FSR | 2009-01-11T00:00:00+0000 | 2.2 | Chimera of alcohol dehydrogenase by exchange of the cofactor binding domain res 153-295 of T. brockii ADH by C. beijerinckii ADH |
3FTN | 2009-01-13T00:00:00+0000 | 2.19 | Q165E/S254K Double Mutant Chimera of alcohol dehydrogenase by exchange of the cofactor binding domain res 153-295 of T. brockii ADH by C. beijerinckii ADH |
6SDM | 2019-07-28T00:00:00+0000 | 2.85 | NADH-dependent variant of TBADH |
7F3P | 2021-06-16T00:00:00+0000 | 2.6 | Crystal structure of a nadp-dependent alcohol dehydrogenase mutant in apo form |
7UTC | 2022-04-26T00:00:00+0000 | 1.85 | Crystal structure of secondary alcohol dehydrogenases from the Thermoanaerobacter ethanolicus with NADP and transition-state analogue inhibitor DMSO |
Percent Identity | Matching Chains | Protein | Accession | Entry Name |
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400.0 | A,B,C,D | NADP-dependent isopropanol dehydrogenase | P14941 | ADH_THEBR |