TY - JOUR
T1 - Linkers in Action
T2 - Exploring Fusion Enzymes for Oxyfunctionlizations in Non-Conventional Media Through Experiments and Simulations
AU - Ma, Yu
AU - Bittner, Jan Philipp
AU - Vernet, Guillem
AU - Zhang, Ningning
AU - Kara, Selin
N1 - Publisher Copyright:
© 2025 The Author(s). ChemCatChem published by Wiley-VCH GmbH.
PY - 2025/5/8
Y1 - 2025/5/8
N2 - Baeyer–Villiger monooxygenases (BVMOs) are key for the selective oxidation of ketones into diverse (cyclic) esters. However, challenges like oxygen and cofactor dependence and substrate/product inhibition hinder their broader application. To address some of these issues, nonconventional media have been applied; still, they lack certain water required for enzyme hydration and cofactor regeneration, reducing activity and/or stability. Fusion approaches enable efficient cofactor recycling by shortening the diffusion distance between enzyme active sites in cascades, especially under low-water conditions. Trial-and-error linker design and time-intensive construction of fusion enzymes substantially slow down the development of fusion enzymes. In this study, we present the work on the fusions of cyclohexanone monooxygenase (CHMO) and alcohol dehydrogenase (ADH) with linkers owing varying lengths and flexibility in both orientations in nonconventional media, focusing on understanding the effects of linkers on the structural and catalytic properties of fusion enzymes. As such, 12 new fusion enzymes were constructed and evaluated regarding the kinetics, specific activity, and stability, identifying the optimal ones for the linear oxyfunctionlization cascade in aqueous–organic biphasic systems. The conformation and flexibility of linkers and the spatial arrangement of fusion enzymes were studied with simulations, which provides a deep understanding of linkers’ influence and offers insights into the rational design of fusion enzymes.
AB - Baeyer–Villiger monooxygenases (BVMOs) are key for the selective oxidation of ketones into diverse (cyclic) esters. However, challenges like oxygen and cofactor dependence and substrate/product inhibition hinder their broader application. To address some of these issues, nonconventional media have been applied; still, they lack certain water required for enzyme hydration and cofactor regeneration, reducing activity and/or stability. Fusion approaches enable efficient cofactor recycling by shortening the diffusion distance between enzyme active sites in cascades, especially under low-water conditions. Trial-and-error linker design and time-intensive construction of fusion enzymes substantially slow down the development of fusion enzymes. In this study, we present the work on the fusions of cyclohexanone monooxygenase (CHMO) and alcohol dehydrogenase (ADH) with linkers owing varying lengths and flexibility in both orientations in nonconventional media, focusing on understanding the effects of linkers on the structural and catalytic properties of fusion enzymes. As such, 12 new fusion enzymes were constructed and evaluated regarding the kinetics, specific activity, and stability, identifying the optimal ones for the linear oxyfunctionlization cascade in aqueous–organic biphasic systems. The conformation and flexibility of linkers and the spatial arrangement of fusion enzymes were studied with simulations, which provides a deep understanding of linkers’ influence and offers insights into the rational design of fusion enzymes.
KW - Baeyer–Villiger monooxygenases
KW - Biocatalytic cascades
KW - Fusion enzymes
KW - Linker design
KW - Molecular dynamics simulations
UR - http://www.scopus.com/inward/record.url?scp=105002226441&partnerID=8YFLogxK
U2 - 10.1002/cctc.202401893
DO - 10.1002/cctc.202401893
M3 - Journal article
AN - SCOPUS:105002226441
SN - 1867-3880
VL - 17
JO - ChemCatChem
JF - ChemCatChem
IS - 9
M1 - e202401893
ER -