Directed Evolution of Alcohol Dehydrogenase for Improved Stereoselective Redox Transformations of 1-Phenylethane-1,2-diol and Its Corresponding Acyloin

Hamnevik, Emil; Maurer, Dirk; Enugala, Thilak Reddy; Chu, Thao; Löfgren, Robin; Dobritzsch, Doreen; Widersten, Mikael

doi:10.1021/acs.biochem.8b00055

Cited by 18 publications

(25 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The kinetic analysis in this study revealed that the enhanced catalytic capability of Q139G mainly originated from the high degree of productive binding rather than from the improved enzyme–substrate affinity. Moreover, the higher K m means lower affinity to the substrate, which might relieve the possible substrate inhibition to some degree [10a, 25a] …”

Section: Resultsmentioning

confidence: 99%

Single‐Point Mutant Inverts the Stereoselectivity of a Carbonyl Reductase toward β‐Ketoesters with Enhanced Activity

Wang

Tian

et al. 2021

Chemistry A European J

View full text Add to dashboard Cite

Enzyme stereoselectivity control is still a major challenge. To gain insight into the molecular basis of enzyme stereo‐recognition and expand the source of antiPrelog carbonyl reductase toward β‐ketoesters, rational enzyme design aiming at stereoselectivity inversion was performed. The designed variant Q139G switched the enzyme stereoselectivity toward β‐ketoesters from Prelog to antiPrelog, providing corresponding alcohols in high enantiomeric purity (89.1–99.1 % ee). More importantly, the well‐known trade‐off between stereoselectivity and activity was not found. Q139G exhibited higher catalytic activity than the wildtype enzyme, the enhancement of the catalytic efficiency (kcat/Km) varied from 1.1‐ to 27.1‐fold. Interestingly, the mutant Q139G did not lead to reversed stereoselectivity toward aromatic ketones. Analysis of enzyme–substrate complexes showed that the structural flexibility of β‐ketoesters and a newly formed cave together facilitated the formation of the antiPrelog‐preferred conformation. In contrast, the relatively large and rigid structure of the aromatic ketones prevents them from forming the antiPrelog‐preferred conformation.

show abstract

Section: Resultsmentioning

confidence: 99%

Single‐Point Mutant Inverts the Stereoselectivity of a Carbonyl Reductase toward β‐Ketoesters with Enhanced Activity

Wang

Tian

et al. 2021

Chemistry A European J

View full text Add to dashboard Cite

show abstract

“…Natural enzymes have been optimized by natural evolution to serve the host organisms' purpose, which does not necessarily coincide with the needs of an organic chemist aiming at the selective oxidation of a given target molecule. Next to screening natural diversity for more suitable enzymes, protein engineering has become a very powerful tool to tailor the properties of a given enzyme such as cofactor specificity, thermo and solvent stability, (enantio)selectivity, and more [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33].…”

Section: Substrate Scopementioning

confidence: 99%

Biocatalytic Oxidation of Alcohols

et al. 2020

View full text Add to dashboard Cite

Enzymatic methods for the oxidation of alcohols are critically reviewed. Dehydrogenases and oxidases are the most prominent biocatalysts, enabling the selective oxidation of primary alcohols into aldehydes or acids. In the case of secondary alcohols, region and/or enantioselective oxidation is possible. In this contribution, we outline the current state-of-the-art and discuss current limitations and promising solutions.

show abstract

“…Specifically, increases in the K m value was observed with a simultaneous increase in the k cat value for substrates 1 a when the mutant Q139S/V187S was used as a catalyst. This result revealed that the improved catalytic capability of Q139S/V187S toward substrate 1 a was mainly originated from the high degree of productive binding but not from the enhanced enzyme‐substrate affinity [13b,17] . It is obviously different from the determined kinetic parameters of the mutant Q139S/V187S toward halogenated α‐tetralones ( 2 a , 4 a and 5 a ), indicating the halogen substitution at aromatic ring of the substrates result in distinct effects on enzyme‐substrate binding.…”

Section: Resultsmentioning

confidence: 82%

Engineering a Carbonyl Reductase as a Potential Tool for the Synthesis of Chiral α‐Tetralinols

Wang

Yang

et al. 2021

ChemCatChem

View full text Add to dashboard Cite

Tailoring of enzyme toward α‐tetralones, a class of bulky‐bulky ketones, is still a challenge. In this work, the mutants of carbonyl reductase BaSDR1 with improved catalytic performance toward α‐tetralone 1 a were obtained by adjusting the steric hindrance and hydrophobicity of the residues that affect the approach of α‐tetralone with the catalytic residues. The designed mutants also showed enhanced catalytic performance toward halogenated α‐tetralones 2 a–6 a. Remarkably, the activity of the mutant Q237V/I291F toward 7‐fluoro‐α‐tetralone 5 a was 16.3‐fold higher than the wildtype enzyme with improved stereoselectivity (98.8 % ee). More notably, the mutants Q139S and Q139S/V187S exhibited decreased or reversed stereoselectivity toward α‐tetralone 1 a, 5‐bromo‐α‐tetralone 2 a, 7‐fluoro‐α‐tetralone 5 a and 7‐chloro‐α‐tetralone 6 a, while the relatively high ee values were obtained in the presence of 6‐chloro‐α‐tetralone 3 a and 6‐bromo‐α‐tetralone 4 a as substrates. Further analysis showed the larger size of the substrates was beneficial for the substrates binding to the active cavity with a more specific binding mode, which endows the reaction with higher stereoselectivity. Moreover, the recombinant E. coli expressing the variant Q237V/I291F successfully catalyzed the reduction of a high concentration 7‐fluoro‐α‐tetralone 5 a. These results not only offered a potential tool for chiral α‐tetralols, but also provided guiding information for the enzyme engineering toward bulky‐bulky ketones.

show abstract

Directed Evolution of Alcohol Dehydrogenase for Improved Stereoselective Redox Transformations of 1-Phenylethane-1,2-diol and Its Corresponding Acyloin

Abstract: Laboratory evolution of alcohol dehydrogenase produced enzyme variants with improved turnover numbers with a vicinal 1,2-diol and its corresponding hydroxyketone. Crystal structure and transient kinetics analysis aids in rationalizing the new functions of these variants.

Cited by 18 publications

References 29 publications

Single‐Point Mutant Inverts the Stereoselectivity of a Carbonyl Reductase toward β‐Ketoesters with Enhanced Activity

Single‐Point Mutant Inverts the Stereoselectivity of a Carbonyl Reductase toward β‐Ketoesters with Enhanced Activity

Biocatalytic Oxidation of Alcohols

Engineering a Carbonyl Reductase as a Potential Tool for the Synthesis of Chiral α‐Tetralinols

Contact Info

Product

Resources

About