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Carbohydrate degradation is crucial for living organisms due to their essential functions in providing energy and composing various metabolic pathways. Nevertheless, in the catalytic cycle of polysaccharide degradation, the details of how the substrates bind and how the products release need more case studies. Here, we choose an inulin fructotransferase (SpIFTase) as a model system, which can degrade inulin into functionally difructose anhydride I. At first, the crystal structures of SpIFTase in the absence of carbohydrates and complex with fructosyl-nystose (GF4), difructose anhydride I, and fructose are obtained, giving the substrate trajectory and product path of SpIFTase, which are further supported by steered molecular dynamics simulations (MDSs) along with mutagenesis. Furthermore, structural topology variations at the active centers of inulin fructotransferases are suggested as the structural base for product release, subsequently proven by substitution mutagenesis and MDSs. Therefore, this study provides a case in point for a deep understanding of the catalytic cycle with substrate trajectory and product path.

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Type IIA DNA topoisomerases are molecular nanomachines responsible for controlling topological states of DNA molecules. Here, we explore the dynamic landscape of yeast topoisomerase IIA during key stages of its catalytic cycle, focusing in particular on the events preceding the passage of the T-segment. To this end, we generated six configurations of fully catalytic yeast topo IIA, strategically inserted a T-segment into the N-gate in relevant configurations, and performed all-atom simulations. The essential motion of topo IIA protein dimer was characterized by rotational gyrating-like movement together with sliding motion within the DNA-gate. Both appear to be inherent properties of the enzyme and an inbuilt feature that allows passage of the T-segment through the cleaved G-segment. Coupled dynamics of the N-gate and DNA-gate residues may be particularly important for controlled and smooth passage of the T-segment and consequently the prevention of DNA double-strand breaks. QTK loop residue Lys367, which interacts with ATP and ADP molecules, is involved in regulating the size and stability of the N-gate. The unveiled features of the simulated configurations provide insights into the catalytic cycle of type IIA topoisomerases and elucidate the molecular choreography governing their ability to modulate the topological states of DNA topology.

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Three decades of studies on the multifunctional 6-deoxyerythronolide B synthase have laid a foundation for understanding the chemistry and evolution of polyketide antibiotic biosynthesis by a large family of versatile enzymatic assembly lines. Recent progress in applying chemical and structural biology tools to this prototypical assembly-line polyketide synthase (PKS) and related systems has highlighted several features of their catalytic cycles and associated protein dynamics. There is compelling evidence that multiple mechanisms have evolved in this enzyme family to channel growing polyketide chains along uniquely defined sequences of 10-100 active sites, each of which is used only once in the overall catalytic cycle of an assembly-line PKS. Looking forward, one anticipates major advances in our understanding of the mechanisms by which the free energy of a repetitive Claisen-like reaction is harnessed to guide the growing polyketide chain along the assembly line in a manner that is kinetically robust yet evolutionarily adaptable.

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Previous studies have revealed the extraordinarily large catalytic efficiency of some enzymes. High catalytic proficiency is an essential accomplishment of biological evolution. Natural selection led to the increased turnover number, kcat, and enzyme efficiency, kcat/KM, of uni-uni enzymes, which convert a single substrate into a single product. We added or multiplied random noise with chosen rate constants to explore the correlation between dissipation and catalytic efficiency for ten enzymes: beta-galactosidase, glucose isomerase, ß-lactamases from three bacterial strains, ketosteroid isomerase, triosephosphate isomerase, and carbonic anhydrase I, II, and T200H. Our results highlight the role of biological evolution in accelerating thermodynamic evolution. The catalytic performance of these enzymes is proportional to overall entropy production-the main parameter from irreversible thermodynamics. That parameter is also proportional to the evolutionary distance of ß-lactamases PC1, RTEM, and Lac-1 when natural or artificial evolution produces the optimal or maximal possible catalytic efficiency. De novo enzyme design and attempts to speed up the rate-limiting catalytic steps may profit from the described connection between kinetics and thermodynamics.

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ATP-binding cassette (ABC) transporters are ubiquitous membrane proteins responsible for the translocation of a wide diversity of substrates across biological membranes. Some of them confer multidrug or antimicrobial resistance to cancer cells and pathogenic microorganisms, respectively. Despite a wealth of structural data gained in the last two decades, the molecular mechanism of these multidrug efflux pumps remains elusive, including the extent of separation between the two nucleotide-binding domains (NBDs) during the transport cycle. Based on recent outward-facing structures of BmrA, a homodimeric multidrug ABC transporter from Bacillus subtilis, we introduced a cysteine mutation near the C-terminal end of the NBDs to analyze the impact of disulfide-bond formation on BmrA function. Interestingly, the presence of the disulfide bond between the NBDs did not prevent the ATPase, nor did it affect the transport of Hoechst 33342 and doxorubicin. Yet, the 7-amino-actinomycin D was less efficiently transported, suggesting that a further opening of the transporter might improve its ability to translocate this larger compound. We solved by cryo-EM the apo structures of the cross-linked mutant and the WT protein. Both structures are highly similar, showing an intermediate opening between their NBDs while their C-terminal extremities remain in close proximity. Distance measurements obtained by electron paramagnetic resonance spectroscopy support the intermediate opening found in these 3D structures. Overall, our data suggest that the NBDs of BmrA function with a tweezers-like mechanism distinct from the related lipid A exporter MsbA.

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We have studied the reduction reactions of two cytosolic human peroxiredoxins (Prx) in their disulfide form by three thioredoxins (Trx; two human and one bacterial), with the aim of better understanding the rate and mechanism of those reactions, and their relevance in the context of the catalytic cycle of Prx. We have developed a new methodology based on stopped-flow and intrinsic fluorescence to study the bimolecular reactions, and found rate constants in the range of 105 -106 m-1 s-1 in all cases, showing that there is no marked kinetic preference for the expected Trx partner. By combining experimental findings and molecular dynamics studies, we found that the reactivity of the nucleophilic cysteine (CN ) in the Trx is greatly affected by the formation of the Prx-Trx complex. The protein-protein interaction forces the CN thiolate into an unfavorable hydrophobic microenvironment that reduces its hydration and results in a remarkable acceleration of the thiol-disulfide exchange reactions by more than three orders of magnitude and also produces a measurable shift in the pKa of the CN . This mechanism of activation of the thiol disulfide exchange may help understand the reduction of Prx by alternative reductants involved in redox signaling.

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Catalytic hydrogenation of CO2 to methanol has attracted lots of attention as it makes CO2 useable as a sustainable carbon source. This study combines theoretical calculations based on the dummy catalytic cycle model with experimental studies on the performance and degradation of indium-based model catalysts for methanol synthesis. In detail, the reversibility of phase transitions in the In2 O3 /In(OH)3 system under industrial methanol synthesis conditions are investigated depending on conversion, temperature and feed ratio. The dummy catalytic cycle model predicts a peculiar degradation behavior of In(OH)3 at 275 °C depending on the water formed either by methanol synthesis or the competing reverse water-gas-shift reaction. These results were validated by dedicated experimental studies confirming the predicted trends. Moreover, X-ray diffraction and thermogravimetric analysis proved the ensuing phase transition between the indium species. Finally, the validated model is used to predict how hydrogen drop out will affect the stability of the catalyst and derive practical strategies to prevent irreversible catalyst degradation.

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Type IIA DNA topoisomerases are complex molecular nanomachines that manage topological states of the DNA molecule in the cell and play a crucial role in cellular processes such as cell division and transcription. They are also established targets of cancer chemotherapy. Starting from the available crystal structure of a fully catalytic topoisomerase IIA homodimer from Saccharomyces cerevisiae, we constructed three states of this molecular motor primarily changing the configurations of the DNA segment bound in the DNA gate and performed µs-long all-atom molecular simulations. A comprehensive analysis revealed a sliding motion within the DNA gate and a teamwork between the N-gate and DNA gate that may be associated with the necessary molecular events that allow passage of the T-segment of DNA. The observed movement of the ATPase dimer relative to the DNA domain was reflected in different interaction patterns between the K-loops of the transducer domain and the B-A-B form of the bound DNA. Based on the obtained results, we mapped simulated configurations to the structures in the proposed catalytic cycle through which type IIA topoisomerases exert their function and discussed the possible transition events. The results extend our understanding of the mechanism of action of type IIA topoisomerases and provide an atomistic interpretation of some of the observed features of these molecular motors.

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Neisseria gonorrhoeae is an obligate human pathogenic bacterium responsible for gonorrhea, a sexually transmitted disease. The bacterial peroxidase, an enzyme present in the periplasm of this bacterium, detoxifies the cells against hydrogen peroxide and constitutes one of the primary defenses against exogenous and endogenous oxidative stress in this organism. The 38 kDa heterologously produced bacterial peroxidase was crystallized in the mixed-valence state, the active state, at pH 6.0, and the crystals were soaked with azide, producing the first azide-inhibited structure of this family of enzymes. The enzyme binds exogenous ligands such as cyanide and azide, which also inhibit the catalytic activity by coordinating the P heme iron, the active site, and competing with its substrate, hydrogen peroxide. The inhibition constants were estimated to be 0.4 ± 0.1 µM and 41 ± 5 mM for cyanide and azide, respectively. Imidazole also binds and inhibits the enzyme in a more complex mechanism by binding to P and E hemes, which changes the reduction potential of the latest heme. Based on the structures now reported, the catalytic cycle of bacterial peroxidases is revisited. The inhibition studies and the crystal structure of the inhibited enzyme comprise the first platform to search and develop inhibitors that target this enzyme as a possible new strategy against N. gonorrhoeae.

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P-glycoprotein (ABCB1) is the first discovered mammalian member of the large family of ATP binding cassette (ABC) transporters. It facilitates the movement of compounds (called allocrites) across membranes, using the energy of ATP binding and hydrolysis. Here, we review the thermodynamics of allocrite binding and the kinetics of ATP hydrolysis by ABCB1. In combination with our previous molecular dynamics simulations, these data lead to a new model for allocrite transport by ABCB1. In contrast to previous models, we take into account that the transporter was evolutionarily optimized to operate within a membrane, which dictates the nature of interactions. Hydrophobic interactions drive lipid-water partitioning of allocrites, the transport process's first step. Weak dipolar interactions (including hydrogen bonding, π-π stacking, and π-cation interactions) drive allocrite recognition, binding, and transport by ABCB1 within the membrane. Increasing the lateral membrane packing density reduces allocrite partitioning but enhances dipolar interactions between allocrites and ABCB1. Allocrite flopping (or reorientation of the polar part towards the extracellular aqueous phase) occurs after hydrolysis of one ATP molecule and opening of ABCB1 at the extracellular side. Rebinding of ATP re-closes the transporter at the extracellular side and expels the potentially remaining allocrite into the membrane. The high sensitivity of the steady-state ATP hydrolysis rate to the nature and number of dipolar interactions, as well as to the dielectric constant of the membrane, points to a flopping process, which occurs to a large extent at the membrane-transporter interface. The proposed unidirectional ABCB1 transport cycle, driven by weak dipolar interactions, is consistent with membrane biophysics.

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ABCG2 is an exporter-type ABC protein that can expel numerous chemically unrelated xeno- and endobiotics from cells. When expressed in tumor cells or tumor stem cells, ABCG2 confers multidrug resistance, contributing to the failure of chemotherapy. Molecular details orchestrating substrate translocation and ATP hydrolysis remain elusive. Here, we present methods to concomitantly investigate substrate and nucleotide binding by ABCG2 in cells. Using the conformation-sensitive antibody 5D3, we show that the switch from the inward-facing (IF) to the outward-facing (OF) conformation of ABCG2 is induced by nucleotide binding. IF-OF transition is facilitated by substrates, and hindered by the inhibitor Ko143. Direct measurements of 5D3 and substrate binding to ABCG2 indicate that the high-to-low affinity switch of the drug binding site coincides with the transition from the IF to the OF conformation. Low substrate binding persists in the post-hydrolysis state, supporting that dissociation of the ATP hydrolysis products is required to reset the high substrate affinity IF conformation of ABCG2.

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OBJECTIVE: Natural product berberine was reported to inhibit platelet activation and thrombosis by suppressing the class Ⅰ PI3Kß/Rasa3/Rap1 pathway. This study aims to investigate the effects and mechanisms of berberrubine, a main metabolite of berberine, to inhibit thrombus formation. METHODS: Carrageenan-induced mouse tail thrombosis model was used to evaluate the effects of berberrubine hydrochloride (BBB) on thrombus formation in vivo. Non-targeted metabolomics was performed with UPLC-Q-TOF/MS to explore the potential mechanisms of BBB in inhibiting thrombosis. The effects of BBB on bleeding risk and prothrombin time were determined. And molecular docking was used to identify the possible target of BBB. RESULTS: After oral administration, BBB significantly inhibited carrageenan-induced thrombosis in mice without prolonging bleeding time. The results of non-targeted metabolomics showed that oral BBB could regulate 'Phenylalanine, tyrosine and tryptophan biosynthesis' and 'Ubiquinone and other terpenoid-quinone biosynthesis', which is closely related to the vitamin K catalytic cycle. Molecular docking revealed BBB could combine and interact with vitamin K epoxide reductase (VKOR) and γ-Glutamyl carboxylase (GGCX), which was mutually confirmed with the experimental results that oral BBB could significantly prolong prothrombin time. CONCLUSIONS: Integrated metabolomics and molecular docking reveal BBB inhibited thrombosis by regulating the vitamin K catalytic cycle. Our research is helpful in deeply understanding the antithrombotic material basis of oral berberine, and also could provide scientific evidence for developing new antithrombotic drugs based on BBB in the future.

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Distinct isoforms of FAD-containing ferredoxin-NADP+ oxidoreductase (FNR) and ferredoxin (Fd) are involved in photosynthetic and non-photosynthetic electron transfer systems. The FNR (FAD)-Fd [2Fe-2S] redox pair complex switches between one- and two-electron transfer reactions in steps involving FAD semiquinone intermediates. In cyanobacteria and some algae, one-electron carrier Fd serves as a substitute for low-potential FMN-containing flavodoxin (Fld) during growth under low-iron conditions. This complex evolves into the covalent FNR (FAD)-Fld (FMN) pair, which participates in a wide variety of NAD(P)H-dependent metabolic pathways as an electron donor, including bacterial sulfite reductase, cytochrome P450 BM3, plant or mammalian cytochrome P450 reductase and nitric oxide synthase isoforms. These electron transfer systems share the conserved Ser-Glu/Asp pair in the active site of the FAD module. In addition to physiological electron acceptors, the NAD(P)H-dependent diflavin reductase family catalyzes a one-electron reduction of artificial electron acceptors such as quinone-containing anticancer drugs. Conversely, NAD(P)H: quinone oxidoreductase (NQO1), which shares a Fld-like active site, functions as a typical two-electron transfer antioxidant enzyme, and the NQO1 and UDP-glucuronosyltransfease/sulfotransferase pairs function as an antioxidant detoxification system. In this review, the roles of the plant FNR-Fd and FNR-Fld complex pairs were compared to those of the diflavin reductase (FAD-FMN) family. In the final section, evolutionary aspects of NAD(P)H-dependent multi-domain electron transfer systems are discussed.

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ATP binding cassette (ABC) proteins are present in all phyla of life and form one of the largest protein families. The Bacillus subtilis ABC transporter BmrA is a functional homodimer that can extrude many different harmful compounds out of the cell. Each BmrA monomer is composed of a transmembrane domain (TMD) and a nucleotide binding domain (NBD). While the TMDs of ABC transporters are sequentially diverse, the highly conserved NBDs harbor distinctive conserved motifs that enable nucleotide binding and hydrolysis, interdomain communication and that mark a protein as a member of the ABC superfamily. In the catalytic cycle of an ABC transporter, the NBDs function as the molecular motor that fuels substrate translocation across the membrane via the TMDs and are thus pivotal for the entire transport process. For a better understanding of the structural and dynamic consequences of nucleotide interactions within the NBD at atomic resolution, we determined the 1H, 13C and 15N backbone chemical shift assignments of the 259 amino acid wildtype BmrA-NBD in its post-hydrolytic, ADP-bound state.

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To date, the scope of single-atom catalysts (SAC) in liquid-phase transformations is rather limited owing to stability issues and the inability to activate complex substances. This calls for a better design of the catalyst support that can provide a dynamic coordination environment needed for catalytic action, and yet retain robustness against leaching or aggregation. In addition, the chemical orthogonality of SAC is useful for designing tandem or multicomponent reactions, in which side reactions common to metal nanoparticles are suppressed. In this review, the intrinsic mechanism will be highlighted that controls reaction efficiency and selectivity in SAC-catalyzed pathways, as well as the structural dynamism of SAC under complex liquid-phase conditions. These mechanistic insights are helpful for the development of next-generation SAC systems for the synthesis of high-value pharmaceuticals through late-stage functionalization, sequential and multicomponent strategies.

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The cytochrome P450 enzymes (CYPs) are the most important enzymes in the oxidative metabolism of hydrophobic drugs and other foreign compounds (xenobiotics). The versatility of these enzymes results in some unusual kinetic properties, stemming from the simultaneous interaction of multiple substrates with the CYP active site. Often, the CYPs display kinetics that deviate from standard hyperbolic saturation or inhibition kinetics. Non-Michaelis-Menten or "atypical" saturation kinetics include sigmoidal, biphasic, and substrate inhibition kinetics (see Chapter 2 ). Interactions between substrates include competitive inhibition, noncompetitive inhibition, mixed inhibition, partial inhibition, activation, and activation followed by inhibition (see Chapters 4 and 6 ). Models and equations that can result in these kinetic profiles will be presented and discussed.

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Cytochrome ba3 from Thermus thermophilus belongs to the B family of heme-copper oxidases and pumps protons across the membrane with an as yet unknown mechanism. The K channel of the A family heme-copper oxidases provides delivery of a substrate proton from the internal water phase to the binuclear heme-copper center (BNC) during the reductive phase of the catalytic cycle, while the D channel is responsible for transferring both substrate and pumped protons. By contrast, in the B family oxidases there is no D-channel and the structural equivalent of the K channel seems to be responsible for the transfer of both categories of protons. Here we have studied the effect of the T315V substitution in the K channel on the kinetics of membrane potential generation coupled to the oxidative half-reaction of the catalytic cycle of cytochrome ba3. The results suggest that the mutated enzyme does not pump protons during the reaction of the fully reduced form with molecular oxygen in a single turnover. Specific inhibition of proton pumping in the T315V mutant appears to be a consequence of inability to provide rapid (τ ~ 100 µs) reprotonation of the internal transient proton donor(s) of the K channel. In contrast to the A family, the K channel of the B-type oxidases is necessary for the electrogenic transfer of both pumped and substrate protons during the oxidative half-reaction of the catalytic cycle.

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P-glycoprotein (P-gp, ABCB1) is an ABC transporter associated with the development of multidrug resistance to chemotherapy. During its catalytic cycle, P-gp undergoes significant conformational changes. Recently, atomic structures of some of these conformations have been resolved using cryo-electron microscopy. The ATP hydrolysis-defective mutant of the catalytic glutamate residue of the Walker B motif (E556Q/E1201Q) has been used to determine the structure of the ATP-bound inward-closed conformation of P-gp. Here, we show that this mutant does not appear to undergo the same steps as wild-type P-gp. We discuss conformational differences in the EQ mutant that may lead to a better understanding of the catalytic cycle of P-gp and propose that additional structural studies with wild-type P-gp are required.

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Drug transporters, classified in various ways like efflux transporters and influx transporters; secretory transporters and absorptive transporters; ATP-driven transporters and Solute Linked Carrier (SLC) transporters are of great importance while studying pharmacokinetics. They have impeccable roles in the drug discovery process of infectious diseases. Many of these find a pivotal role in synthetic antimicrobial peptides. The chapter briefly elucidates the varied types and their significance.

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