Enzymes undergo a range of internal motions from local, active site fluctuations to large‐scale, global conformational changes. These motions are often important for enzyme function, including in ...ligand binding and dissociation and even preparing the active site for chemical catalysis. Protein engineering efforts have been directed towards manipulating enzyme structural dynamics and conformational changes, including targeting specific amino acid interactions and creation of chimeric enzymes with new regulatory functions. Post‐translational covalent modification can provide an additional level of enzyme control. These studies have not only provided insights into the functional role of protein motions, but they offer opportunities to create stimulus‐responsive enzymes. These enzymes can be engineered to respond to a number of external stimuli, including light, pH, and the presence of novel allosteric modulators. Altogether, the ability to engineer and control enzyme structural dynamics can provide new tools for biotechnology and medicine.
Computational design is a test of our understanding of enzyme catalysis and a means of engineering novel, tailor-made enzymes. While the de novo computational design of catalytically efficient ...enzymes remains a challenge, designed enzymes may comprise unique starting points for further optimization by directed evolution. Directed evolution of two computationally designed Kemp eliminases, KE07 and KE70, led to low to moderately efficient enzymes (k cₐₜ/ K ₘ values of ≤ 5 10 ⁴ M ⁻¹s ⁻¹). Here we describe the optimization of a third design, KE59. Although KE59 was the most catalytically efficient Kemp eliminase from this design series (by k cₐₜ/ K ₘ, and by catalyzing the elimination of nonactivated benzisoxazoles), its impaired stability prevented its evolutionary optimization. To boost KE59’s evolvability, stabilizing consensus mutations were included in the libraries throughout the directed evolution process. The libraries were also screened with less activated substrates. Sixteen rounds of mutation and selection led to > 2,000-fold increase in catalytic efficiency, mainly via higher k cₐₜ values. The best KE59 variants exhibited k cₐₜ/ K ₘ values up to 0.6 10 ⁶ M ⁻¹s ⁻¹, and k cₐₜ/ k ᵤₙcₐₜ values of ≤ 10 ⁷ almost regardless of substrate reactivity. Biochemical, structural, and molecular dynamics (MD) simulation studies provided insights regarding the optimization of KE59. Overall, the directed evolution of three different designed Kemp eliminases, KE07, KE70, and KE59, demonstrates that computational designs are highly evolvable and can be optimized to high catalytic efficiencies.
Compared with conventional tumor photothermal therapy (PTT), mild‐temperature PTT brings less damage to normal tissues, but also tumor thermoresistance, introduced by the overexpressed heat shock ...protein (HSP). A high dose of HSP inhibitor during mild‐temperature PTT might lead to toxic side effects. Glucose oxidase (GOx) consumes glucose, leading to adenosine triphosphate supply restriction and consequent HSP inhibition. Therefore, a combinational use of an HSP inhibitor and GOx not only enhances mild‐temperature PTT but also minimizes the toxicity of the inhibitor. However, a GOx and HSP inhibitor‐encapsulating nanostructure, designed for enhancing its mild‐temperature tumor PTT efficiency, has not been reported. Thermosensitive GOx/indocyanine green/gambogic acid (GA) liposomes (GOIGLs) are reported to enhance the efficiency of mild‐temperature PTT of tumors via synergistic inhibition of tumor HSP by the released GA and GOx, together with another enzyme‐enhanced phototherapy effect. In vitro and in vivo results indicate that this strategy of tumor starvation and phototherapy significantly enhances mild‐temperature tumor PTT efficiency. This strategy could inspire people to design more delicate platforms combining mild‐temperature PTT with other therapeutic methods for more efficient cancer treatment.
Thermosensitive liposomes made of DPPC and DSPE‐PEG2000 encapsulating GOx, ICG, and GA are presented. This system is used for synergistic starvation therapy, EEPT, and enhanced mild‐temperature PTT against tumors.
The book is intended to provide a sound basis for enzyme reactor design based on kinetic principles, and give an updated vision of the potentials and limitations of enzyme biocatalysis, especially ...with respect to recent applications in processes of organic synthesis. The book is structured in the form of a textbook that goes from basic principles of enzyme structure and function to reactor design for homogeneous systems with soluble enzymes, and heterogeneous systems with insolubilized enzymes. It contains an introductory chapter that gives an updated overview of enzyme technology, a chapter on enzyme production focused on large-scale operations, two chapters devoted to homogeneous and heterogeneous enzyme kinetics and a chapter on enzyme reactor design and operation based on enzyme kinetics principles, mass transfer limitations and enzyme inactivation. The book is complemented with case studies of biocatalytic processes of industrial relevance or potential, written by experts in those fields. Applications of proteases, acylases, lipases, aldolases and dehydrogenases in reactions of organic synthesis, and peroxidases in the degradation of recalcitrant organic compounds, are used as selected examples to illustrate the realities and potentials of enzymes as process catalysts.
Two new stilbenoids, cajanstilbenoid C (1) and cajanstilbenoid D (2), together with eight other known stilbenoids (3-10) and seventeen known flavonoids (11-27), were isolated from the petroleum ether ...and ethyl acetate portions of the 95% ethanol extract of leaves of Cajanus cajan (L.) Millsp. The planar structures of the new compounds were elucidated by NMR and high-resolution mass spectrometry, and their absolute configurations were determined by comparison of their experimental and calculated electronic circular dichroism (ECD) values. All the compounds were assayed for their inhibitory activities against yeast α-glucosidase. The results demonstrated that compounds 3, 8-9, 11, 13, 19-21, and 24-26 had strong inhibitory activities against α-glucosidase, with compound 11 (ICsub.50 = 0.87 ± 0.05 μM) exhibiting the strongest activity. The structure–activity relationships were preliminarily summarized. Moreover, enzyme kinetics showed that compound 8 was a noncompetitive inhibitor, compounds 11, 24-26 were anticompetitive, and compounds 9 and 13 were mixed-competitive.
Although Lecitase and the lipase from Thermomyces lanuginosus (TLL) could be coimmobilized on octyl-agarose, the stability of Lecitase was lower than that of TLL causing the user to discard active ...immobilized TLL when Lecitase was inactivated. Here, we propose the chemical amination of immobilized TLL to ionically exchange Lecitase on immobilized TLL, which should be released to the medium after its inactivation by incubation at high ionic strength. Using conditions where Lecitase was only adsorbed on immobilized TLL after its amination, the combibiocatalyst was produced. Unfortunately, the release of Lecitase was not possible using just high ionic strength solutions, and if detergent was added, TLL was also released from the support. This occurred when using 0.25 M ammonium sulfate, Lecitase did not immobilize on aminated TLL. That makes the use octyl-vinylsulfone supports necessary to irreversibly immobilize TLL, and after blocking with ethylendiamine, the immobilized TLL was aminated. Lecitase immobilized and released from this biocatalyst using 0.25 M ammonium sulfate and 0.1% Triton X-100. That way, a coimmobilized TLL and Lecitase biocatalyst could be produced, and after Lecitase inactivation, it could be released and the immobilized, aminated, and fully active TLL could be utilized to build a new combibiocatalyst.
•Lecitase and TLL can be coimmobilized on octyl agarose, but Lecitase is far less table than TLL.•The amination of immobilized TLL transform the enzyme in a anion exchanger.•Lecitase can be ionically exchanged in aminated TLL•Using octyl agarose, the reuse of TLL is not possible because Lecitase cannot be selectively desorbed,•The use of vinyl sulfone-octyl agarose permitted the reuse of immobilized and aminated TLL
Covalent ligand-target interactions offer significant pharmacological advantages. However, off-target reactivity of the reactive groups, which usually have electrophilic properties, must be ...minimized, and the selectivity of irreversible inhibitors is a crucial requirement. We therefore performed a systematic study to determine the selectivity of several electrophilic groups that can be used as building blocks for covalently binding ligands. Six reactive groups with modulated electrophilicity were combined with 11 nonreactive moieties, resulting in a small combinatorial library of 72 fragment-like compounds. These compounds were screened against a group of 11 enzyme targets to assess their selectivity and their potential for promiscuous binding to proteins. The assay results showed a considerably lower degree of promiscuity than initially expected, even for those members of the screening collection that contain supposedly highly reactive electrophiles.
UBA1 is the primary E1 ubiquitin-activating enzyme responsible for generation of activated ubiquitin required for ubiquitination, a process that regulates stability and function of numerous proteins. ...Decreased or insufficient ubiquitination can cause or drive aging and many diseases. Therefore, a small-molecule enhancing UBA1 activity could have broad therapeutic potential. Here we report that auranofin, a drug approved for the treatment of rheumatoid arthritis, is a potent UBA1 activity enhancer. Auranofin binds to the UBA1's ubiquitin fold domain and conjugates to Cys1039 residue. The binding enhances UBA1 interactions with at least 20 different E2 ubiquitin-conjugating enzymes, facilitating ubiquitin charging to E2 and increasing the activities of seven representative E3s in vitro. Auranofin promotes ubiquitination and degradation of misfolded ER proteins during ER-associated degradation in cells at low nanomolar concentrations. It also facilitates outer mitochondrial membrane-associated degradation. These findings suggest that auranofin can serve as a much-needed tool for UBA1 research and therapeutic exploration.
A proximity effect has been invoked to explain the enhanced activity of enzyme cascades on DNA scaffolds. Using the cascade reaction carried out by glucose oxidase and horseradish peroxidase as a ...model system, here we study the kinetics of the cascade reaction when the enzymes are free in solution, when they are conjugated to each other and when a competing enzyme is present. No proximity effect is found, which is in agreement with models predicting that the rapidly diffusing hydrogen peroxide intermediate is well mixed. We suggest that the reason for the activity enhancement of enzymes localized by DNA scaffolds is that the pH near the surface of the negatively charged DNA nanostructures is lower than that in the bulk solution, creating a more optimal pH environment for the anchored enzymes. Our findings challenge the notion of a proximity effect and provide new insights into the role of DNA scaffolds.
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