Presentation Information
[1ENZ-11]Engineering of Geotrichum candidum acetophenone reductase expands the substrate scope of medium-chain dehydrogenase/reductase toward diaryl ketones
○Zhongyao Tang1, Yuuki Takagi1, Guillermo Germán Otárola Tejada1,2, Afifa Ayu Koesoema1, Tomoko Matsuda1 (1. Institute of Science Tokyo (Japan), 2. University of Alcalá (Spain))
Keywords:
Biocatalyst,Enzyme engineering,Medium-chain dehydrogenase/reductase,Diaryl ketone,Asymmetric reduction
Recently, using alcohol dehydrogenase (ADH) to catalyze the asymmetric reduction of ketones has gained increasing attention as a sustainable approach to produce important pharmaceutical intermediates, chiral alcohols. However, due to steric hindrance, diaryl ketones are challenging substrates for ADHs, particularly those from the medium-chain dehydrogenase/reductase (MDR) family. Moreover, the structural similarity between the two aromatic substituents flanking the carbonyl group makes it difficult to achieve high enantioselectivity.
To increase the diversity of ADHs capable of catalyzing the reduction of diaryl ketones, an MDR from Geotrichum candidum (GcAPRD) was selected as a suitable target, as it has exhibited excellent activity, enantioselectivity, and stability in the asymmetric reduction of aliphatic and aromatic ketones [1-3]. Particularly, in the asymmetric reductions of acetophenones, Phe56 mutants exhibited higher activity than the wild type while maintaining excellent (S)-enantioselectivity [4], whereas Trp288 mutants showed (R)-enantioselectivity opposite to that of the wild type [2].
Based on these previous studies, a double mutant of GcAPRD, Phe56Ile/Trp288Ala, was constructed in this study [4, 5]. Then, the mutant, together with the previously reported single mutant Trp288Ala, was employed to investigate the catalytic performance toward diaryl ketones, including 2-benzoylpyridines [4] and benzophenones [5]. Although the wild type exhibited low catalytic activity toward diaryl ketones, both mutants showed improved activity, up to a 22.5-fold increase in activity for 2-benzoylpyridines and more than an 80-fold increase in yield for benzophenones. This improvement was due to the mutation of Trp288 to Ala in the small binding pocket, which reduced steric hindrance and allowed accommodation of a phenyl ring.
Regarding enantioselectivity, (R)-enantioselectivity was mainly observed in the reduction of 2-benzoylpyridines, whereas interesting results were observed in the reduction of benzophenones. Notably, Trp288Ala exhibited excellent (R)-enantioselectivity toward 4-fluorobenzophenone (97% ee) due to the lower ligand strain energy of the pro-R binding pose compared with the pro-S pose. In contrast, Phe56Ile/Trp288Ala showed a clear enantiopreference toward substituted benzophenones, exhibiting (R)-enantioselectivity toward 3-substituted substrates (up to 89% ee) but reversing to (S)-enantioselectivity toward 4-substituted substrates (up to 92% ee). This enantioselectivity was determined by the combined effects of binding pocket remodeling, ligand-residue interactions, and reduced ligand strain energy.
References
[1] Koesoema AA, et al. Appl. Microbiol. Biotechnol. 2019, 103, 9543.
[2] Koesoema, AA, et al. Tetrahedron Lett. 2020, 61, 151820.
[3] Koesoema, AA, et al. Tetrahedron Lett. 2020, 61, 151682.
[4] Tang Z, et al. Appl. Microbiol. Biotechnol. 2024, 108, 545.
[5] Tang Z, et al. Appl. Microbiol. Biotechnol. 2026, 110, 48.
To increase the diversity of ADHs capable of catalyzing the reduction of diaryl ketones, an MDR from Geotrichum candidum (GcAPRD) was selected as a suitable target, as it has exhibited excellent activity, enantioselectivity, and stability in the asymmetric reduction of aliphatic and aromatic ketones [1-3]. Particularly, in the asymmetric reductions of acetophenones, Phe56 mutants exhibited higher activity than the wild type while maintaining excellent (S)-enantioselectivity [4], whereas Trp288 mutants showed (R)-enantioselectivity opposite to that of the wild type [2].
Based on these previous studies, a double mutant of GcAPRD, Phe56Ile/Trp288Ala, was constructed in this study [4, 5]. Then, the mutant, together with the previously reported single mutant Trp288Ala, was employed to investigate the catalytic performance toward diaryl ketones, including 2-benzoylpyridines [4] and benzophenones [5]. Although the wild type exhibited low catalytic activity toward diaryl ketones, both mutants showed improved activity, up to a 22.5-fold increase in activity for 2-benzoylpyridines and more than an 80-fold increase in yield for benzophenones. This improvement was due to the mutation of Trp288 to Ala in the small binding pocket, which reduced steric hindrance and allowed accommodation of a phenyl ring.
Regarding enantioselectivity, (R)-enantioselectivity was mainly observed in the reduction of 2-benzoylpyridines, whereas interesting results were observed in the reduction of benzophenones. Notably, Trp288Ala exhibited excellent (R)-enantioselectivity toward 4-fluorobenzophenone (97% ee) due to the lower ligand strain energy of the pro-R binding pose compared with the pro-S pose. In contrast, Phe56Ile/Trp288Ala showed a clear enantiopreference toward substituted benzophenones, exhibiting (R)-enantioselectivity toward 3-substituted substrates (up to 89% ee) but reversing to (S)-enantioselectivity toward 4-substituted substrates (up to 92% ee). This enantioselectivity was determined by the combined effects of binding pocket remodeling, ligand-residue interactions, and reduced ligand strain energy.
References
[1] Koesoema AA, et al. Appl. Microbiol. Biotechnol. 2019, 103, 9543.
[2] Koesoema, AA, et al. Tetrahedron Lett. 2020, 61, 151820.
[3] Koesoema, AA, et al. Tetrahedron Lett. 2020, 61, 151682.
[4] Tang Z, et al. Appl. Microbiol. Biotechnol. 2024, 108, 545.
[5] Tang Z, et al. Appl. Microbiol. Biotechnol. 2026, 110, 48.
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