Lipases That Activate at High Solvent Polarities

Abstract

Thermomyces lanuginosus lipase (TlL) and related lipases become activated in low-polarity environments that exist at the water-lipid interface where a structural change of the "lid" region occurs. In this work, we have investigated the activation of TlL (Lipase_W89) and certain lid mutants, containing either a single positive charge mutation, E87K (Lipase_K87_W89), within the lid region or a lid residue composition of both lipase and esterase character (Hybrid_W89) as a function of solvent polarity. Activation differences between the variants and TlL were studied by a combination of biophysical and theoretical methods. To investigate the structural changes taking place in the lid region upon lipase activation, we used a fluorescence-based method measuring the efficiency of Trp89 in the lid to quench the fluorescence of a bimane molecule attached in front (C255) and behind (C61) the lid. These structural changes were compared to the enzymatic activity of each variant at the water-substrate interface and to theoretical calculations of the energies associated with lid opening as a function of the dielectric constant (ε) of the environment. Our results show that the lid in Lipase_K87_W89 undergoes a pronounced structural transition toward an open conformation around ε = 50, whereas only small changes are detected for Lipase_W89 ascribed to the stabilizing effect of the positive charge mutation on the open lid conformation. Interestingly, Hybrid_W89, with the same charge as Lipase_W89, shows a stabilization of the open lid even more pronounced at high solvent polarities than that of Lipase_K87_W89, allowing activation at ε < 80. This is further indicated by measurement of the lipase activity for each variant showing that Hybrid_W89 is more quickly activated at the water-lipid interface of a true, natural substrate. Combined, we show that a correlation exists between structural changes and enzymatic activities detected on one hand and theoretical calculations on lid opening energies on the other. These results highlight the key role that the lid plays in determining the polarity-dependent activation of lipases.

The Lid Domain in Lipases: Structural and Functional Determinant of Enzymatic Properties

“…They showed that this intrinsic bond enables control of both lipase activity and interfacial binding. They also suggested the key role that the lid plays in determining the polarity-dependent activation of lipases using a combination of methods measuring enzymatic activity, detecting structural changes using the tryptophan-induced quenching method, and calculating the lid opening energies using an MD simulations, and suggested that mutagenesis of the lid can lower the energy barrier associated with lid opening (Skjold-Jorgensen et al, 2016). Tryptophan-induced quenching fluorescence method has been applied to successfully measure the lid movements in T. lanuginosus lipase and its variants in solvents with different dielectric constants (Skjold-Jorgensen et al, 2015).…”

Understanding the activation mechanism of Thermomyces lanuginosus lipase using rational design and tryptophan‐induced fluorescence quenching

“…Further studies have used similar protein engineering strategies in endothelial lipase (EL) , human pancreatic lipase (HPL) , C. rugusa lipase (CRL) , Rhizopus chinensis (RCL) lipase , Aspergillus niger lipase (ANL) . Recently, Skjold‐ Jørgensen et al altered the activation mechanism in Thermomyces lanugiunosus lipase (TLL) by rational design of the lid‐region . All these studies on structurally diverse lipases, suggest that the lid‐region plays a key role in hydrolytic activity, interfacial activation, and substrate specificity.…”

“…altered the activation mechanism in Thermomyces lanugiunosus lipase (TLL) by rational design of the lid-region [40,59,82,83]. All these studies on structurally diverse lipases, suggest that the lid-region plays a key role in hydrolytic activity, interfacial activation, and substrate specificity.…”

Carbon-dot–hydrogel for enzyme-mediated bacterial detection

“…40,41 Esterases constitute a diverse enzyme family secreted by both mammalian and bacterial cells, which function through hydrolysis of ester bonds in target substrates. 42 Notably, the hydrogel scaffold contains a multitude of ester bonds, both positioned in polar environments (e.g. ester bonds linking the glucose residues to the modied tartaric acid-based anhydride moieties, Fig.…”