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Chiral CDots (c‐CDots) not only inherit those merits from CDots but also exhibit chiral effects in optical, electric, and bio‐properties. Therefore, c‐CDots have received significant interest from a wide range of research communities including chemistry, physics, biology, and device engineers. They have already made decent progress in terms of synthesis, together with the exploration of their optical properties and applications. In this review, the chiroptical properties and chirality origin in extinction circular dichroism (ECD) and circularly polarized luminescence (CPL) of c‐CDots is briefly discussed. Then, the synthetic strategies of c‐CDots is summarized, including one‐pot synthesis, post‐functionalization of CDots with chiral ligands, and assembly of CDots into chiral architectures with soft chiral templates. Afterward, the chiral effects on the applications of c‐CDots are elaborated. Research domains such as drug delivery, bio‐ or chemical sensing, regulation of enzyme‐like catalysis, and others are covered. Finally, the perspective on the challenges associated with the synthetic strategies, understanding the origin of chirality, and potential applications is provided. This review not only discusses the latest developments of c‐CDots but also helps toward a better understanding of the structure‐property relationship along with their respective applications. Chiral CDots (c‐CDots) not only inherit those merits from CDots, but also exhibit the chiral effects in optical, electric, and bio‐properties. Therefore, significant progress is achieved for c‐CDots as a result of numerous research efforts carried out. The synthetic strategies of c‐CDots, including postfunctionalization of CDots with chiral ligands, and one‐pot synthesis. and assembly of CDots with chiral soft templates. Afterward, a clear chiral effect of c‐CDots on their applications is elaborated.

F1·Fo-ATP synthases/ATPases (F1·Fo) are molecular machines that couple either ATP synthesis from ADP and phosphate or ATP hydrolysis to the consumption or production of a transmembrane electrochemical gradient of protons. Currently, in view of the spread of drug-resistant disease-causing strains, there is an increasing interest in F1·Fo as new targets for antimicrobial drugs, in particular, anti-tuberculosis drugs, and inhibitors of these membrane proteins are being considered in this capacity. However, the specific drug search is hampered by the complex mechanism of regulation of F1·Fo in bacteria, in particular, in mycobacteria: the enzyme efficiently synthesizes ATP, but is not capable of ATP hydrolysis. In this review, we consider the current state of the problem of “unidirectional” F1·Fo catalysis found in a wide range of bacterial F1·Fo and enzymes from other organisms, the understanding of which will be useful for developing a strategy for the search for new drugs that selectively disrupt the energy production of bacterial cells.

FTO demethylates internal N⁶-methyladenosine (m⁶A) and N⁶,2′-O-dimethyladenosine (m⁶Am; at the cap +1 position) in mRNA, m⁶A and m⁶Am in snRNA, and N¹-methyladenosine (m¹A) in tRNA in vivo, and in vitro evidence supports that it can also demethylate N⁶-methyldeoxyadenosine (6mA), 3-methylthymine (3mT), and 3-methyluracil (m³U). However, it remains unclear how FTO variously recognizes and catalyzes these diverse substrates. Here we demonstrate—in vitro and in vivo—that FTO has extensive demethylation enzymatic activity on both internal m⁶A and cap m⁶Am. Considering that 6mA, m⁶A, and m⁶Am all share the same nucleobase, we present a crystal structure of human FTO bound to 6mA-modified ssDNA, revealing the molecular basis of the catalytic demethylation of FTO toward multiple RNA substrates. We discovered that (i) N⁶-methyladenine is the most favorable nucleobase substrate of FTO, (ii) FTO displays the same demethylation activity toward internal m⁶A and m⁶Am in the same RNA sequence, suggesting that the substrate specificity of FTO primarily results from the interaction of residues in the catalytic pocket with the nucleobase (rather than the ribose ring), and (iii) the sequence and the tertiary structure of RNA can affect the catalytic activity of FTO. Our findings provide a structural basis for understanding the catalytic mechanism through which FTO demethylates its multiple substrates and pave the way forward for the structure-guided design of selective chemicals for functional studies and potential therapeutic applications.

Homologous enzymes with identical folds often exhibit different thermal and kinetic behaviors. Understanding how an enzyme sequence encodes catalytic activity at functionally optimal temperatures is a fundamental problem in biophysics. Recently it was shown that the residues that tune catalytic activities of thermophilic/mesophilic variants of the C-terminal domain of bacterial enzyme I (EIC) are largely localized within disordered loops, offering a model system with which to investigate this phenomenon. In this work, we use molecular dynamics simulations and mutagenesis experiments to reveal a mechanism of sequence-dependent activity tuning of EIC homologs. We find that a network of contacts in the catalytic loops is particularly sensitive to changes in temperature, with some contacts exhibiting distinct linear or nonlinear temperaturedependent trends. Moreover, these trends define structurally clustered dynamical modes and can distinguish regions that tend toward order or disorder at higher temperatures. Assaying several thermophilic EIC mutants, we show that complementary mesophilic mutations to the most temperature-sensitive positions exhibit the most enhanced activity, while mutations to relatively temperature insensitive positions exhibit the least enhanced activities. These results provide a mechanistic explanation of sequencedependent temperature tuning and offer a computational method for rational enzyme modification.

The Michaelis–Menten equation is usually expressed in terms of k cat and K m values: v = k cat [S]/( K m + [S]). However, it is impossible to interpret K m in the absence of additional information, while the ratio of k cat / K m provides a measure of enzyme specificity and is proportional to enzyme efficiency and proficiency. Moreover, k cat / K m provides a lower limit on the second order rate constant for substrate binding. For these reasons it is better to redefine the Michaelis–Menten equation in terms of k cat and k cat / K m values: v = k SP [S]/(1 + k SP [S]/ k cat ), where the specificity constant, k SP = k cat / K m . In this short review, the rationale for this assertion is explained and it is shown that more accurate measurements of k cat / K m can be derived directly using the modified form of the Michaelis–Menten equation rather than calculated from the ratio of k cat and K m values measured separately. Even greater accuracy is achieved with fitting the raw data directly by numerical integration of the rate equations rather than using analytically derived equations. The importance of fitting to derive k cat and k cat / K m is illustrated by considering the role of conformational changes in enzyme specificity where k cat and k cat / K m can reflect different steps in the pathway. This highlights the pitfalls in attempting to interpret K m , which is best understood as the ratio of k cat divided by k cat / K m .

Microfluidics has received extensive attention due to its ability to rapidly prepare a large number of microdroplets with controlled sizes and defined morphologies. In addition to having large surface areas and controllable confinement environments, these prepared microdroplets can be used as analytical detection devices to screen and optimize various kinetic parameters. This review summarizes recent advances in the microfluidic control of droplet‐based catalytic reactions and discusses the role of these droplets in both homogeneous and heterogeneous catalyzes and in the catalysis of macromolecular biological enzymes in water‐in‐oil and oil‐in‐oil environments. Additionally, the existing problems and future development directions of droplets in catalysis are highlighted to promote the development of catalytic reactions in droplet media and provide guidance for the high‐throughput screening of catalysts and the directed evolution of biological enzymes. Micro/nanofluidics provides a powerful method for controlling the size and shape of synthesized droplets. Thus, it opens a pathway for facilitating microscale chemistry. Here, we summarize recent developments in catalytic reactions occurring in droplets generated by micro/nanofluidics, and highlight the application of droplets in catalytic processes.

During gliotoxin biosynthesis in fungi, the cytochrome P450 GliF enzyme catalyzes an unusual C–N ring-closure step while also an aromatic ring is hydroxylated in the same reaction cycle, which may have relevance to drug synthesis reactions in biotechnology. However, as the details of the reaction mechanism are still controversial, no applications have been developed yet. To resolve the mechanism of gliotoxin biosynthesis and gain insight into the steps leading to ring-closure, we ran a combination of molecular dynamics and density functional theory calculations on the structure and reactivity of P450 GliF and tested a range of possible reaction mechanisms, pathways and models. The calculations show that, rather than hydrogen atom transfer from the substrate to Compound I, an initial proton transfer transition state is followed by a fast electron transfer en route to the radical intermediate, and hence a non-synchronous hydrogen atom abstraction takes place. The radical intermediate then reacts by OH rebound to the aromatic ring to form a biradical in the substrate that, through ring-closure between the radical centers, gives gliotoxin products. Interestingly, the structure and energetics of the reaction mechanisms appear little affected by the addition of polar groups to the model and hence we predict that the reaction can be catalyzed by other P450 isozymes that also bind the same substrate. Alternative pathways, such as a pathway starting with an electrophilic attack on the arene to form an epoxide, are high in energy and are ruled out.

How enzymes achieve their enormous rate enhancements remains a central question in biology, and our understanding to date has impacted drug development, influenced enzyme design, and deepened our appreciation of evolutionary processes. While enzymes position catalytic and reactant groups in active sites, physics requires that atoms undergo constant motion. Numerous proposals have invoked positioning or motions as central for enzyme function, but a scarcity of experimental data has limited our understanding of positioning and motion, their relative importance, and their changes through the enzyme’s reaction cycle. To examine positioning and motions and test catalytic proposals, we collected “room temperature” X-ray crystallography data for Pseudomonas putida ketosteroid isomerase (KSI), and we obtained conformational ensembles for this and a homologous KSI from multiple PDB crystal structures. Ensemble analyses indicated limited change through KSI’s reaction cycle. Active site positioning was on the 1- to 1.5-Å scale, and was not exceptional compared to noncatalytic groups. The KSI ensembles provided evidence against catalytic proposals invoking oxyanion hole geometric discrimination between the ground state and transition state or highly precise general base positioning. Instead, increasing or decreasing positioning of KSI’s general base reduced catalysis, suggesting optimized Ångstrom-scale conformational heterogeneity that allows KSI to efficiently catalyze multiple reaction steps. Ensemble analyses of surrounding groups for WT and mutant KSIs provided insights into the forces and interactions that allow and limit active-site motions. Most generally, this ensemble perspective extends traditional structure–function relationships, providing the basis for a new era of “ensemble–function” interrogation of enzymes.

Abstract How does enzymatic activity emerge? To shed light on this fundamental question, we study type B dihydrofolate reductases (DfrB), which were discovered for their role in antibiotic resistance. These rudimentary enzymes are evolutionarily distinct from the ubiquitous, monomeric FolA dihydrofolate reductases targeted by the antibiotic trimethoprim. DfrB is unique: it homotetramerizes to form a highly symmetrical central tunnel that accommodates its substrates in close proximity and the right orientation, thus promoting the metabolically essential production of tetrahydrofolate. It is the only known enzyme built from the ancient Src Homology 3 fold, typically a binding module. Strikingly, by studying the evolution of this enzyme family, we observe that no active-site residues are conserved across catalytically active homologs. Integrating experimental and computational analyses, we identify an intricate relationship between homotetramerization and catalytic activity, where formation of a tunnel featuring positive electrostatic potential proves to be a powerful predictor of activity. We demonstrate that the DfrB enzymes have not evolved in response to the synthetic antibiotic to which they confer strong resistance, and propose that DfrB domains evolved the capacity for rudimentary catalysis from a binding capacity. That (rudimentary) catalysis can emerge from the homotetramerization of a binding domain, and that it has been recently recruited by pathogenic bacteria, manifests the opportunistic nature of evolution.

Polyurethanes (PU) are one of the most-used classes of synthetic polymers in Europe, having a considerable impact on the plastic waste management in the European Union. Therefore, they represent a major challenge for the recycling industry, which requires environmentally friendly strategies to be able to re-utilize their monomers without applying hazardous and polluting substances in the process. In this work, enzymatic hydrolysis of a polyurethane-polyester (PU-PE) copolymer using Humicola insolens cutinase (HiC) has been investigated in order to achieve decomposition at milder conditions and avoiding harsh chemicals. PU-PE films have been incubated with the enzyme at 50 °C for 168 h, and hydrolysis has been followed throughout the incubation. HiC effectively hydrolysed the polymer, reducing the number average molecular weight (Mn) and the weight average molecular weight (Mw) by 84% and 42%, respectively, as shown by gel permeation chromatography (GPC), while scanning electron microscopy showed cracks at the surface of the PU-PE films as a result of enzymatic surface erosion. Furthermore, Fourier Transform Infrared (FTIR) analysis showed a reduction in the peaks at 1725 cm−1, 1164 cm−1 and 1139 cm−1, indicating that the enzyme preferentially hydrolysed ester bonds, as also supported by the nuclear magnetic resonance spectroscopy (NMR) results. Liquid chromatography time-of-flight/mass spectrometry (LC-MS-Tof) analysis revealed the presence in the incubation supernatant of all of the monomeric constituents of the polymer, thus suggesting that the enzyme was able to hydrolyse both the ester and the urethane bonds of the polymer.