Gustation-Inspired Dual-Responsive Hydrogels for Taste Sensing Enabled by Machine Learning.

Human gustatory system recognizes salty/sour or sweet tastants based on their different ionic or nonionic natures using two different signaling pathways. This suggests that evolution has selected this detection dualism favorably. Analogically, this work constructs herein bioinspired stimulus-responsive hydrogels to recognize model salty/sour or sweet tastes based on two different responses, that is, electrical and volumetric responsivities. Different compositions of zwitter-ionic sulfobetainic N-(3-sulfopropyl)-N-(methacryloxyethyl)-N,N-dimethylammonium betaine (DMAPS) and nonionic 2-hydroxyethyl methacrylate (HEMA) are co-polymerized to explore conditions for gelation. The hydrogel responses upon adding model tastant molecules are explored using electrical and visual de-swelling observations. Beyond challenging electrochemical impedance spectroscopy measurements, naive multimeter electrical characterizations are performed, toward facile applicability. Ionic model molecules, for example, sodium chloride and acetic acid, interact electrostatically with DMAPS groups, whereas nonionic molecules, for example, D(-)fructose, interact by hydrogen bonding with HEMA. The model tastants induce complex combinations of electrical and volumetric responses, which are then introduced as inputs for machine learning algorithms. The fidelity of such a trained dual response approach is tested for a more general taste identification. This work envisages that the facile dual electric/volumetric hydrogel responses combined with machine learning proposes a generic bioinspired avenue for future bionic designs of artificial taste recognition, amply needed in applications.

IL-6 is a predictor and potential therapeutic target for coronavirus disease 2019-related heart failure: A single-center retrospective study.

BACKGROUND: Inflammation is linked to coronavirus disease 2019 (COVID-19)-related heart failure (HF), but the specific mechanisms are unclear. This study aimed to assess the relationship between specific inflammatory factors, such as interleukin (IL)-1ß, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-17, interferon (IFN)-α, and IFN-γ, and COVID-19-related HF. METHODS: We retrospectively identified 212 adult patients with COVID-19 who were hospitalized at Shanghai Public Health Center from March 1 to May 30, 2022 (including 80 patients with HF and 132 without HF). High-sensitivity C-reactive protein (hs-CRP), procalcitonin (PCT), and inflammatory factors, including IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-17, IFN-α, and IFN-γ, were compared between patients with COVID-19 with and without HF. RESULTS: Patients with COVID-19 having and not having HF differed with regard to sex, age, hs-CRP, PCT, and IL-6 levels (p < 0.05). Logistic regression analysis indicated a significant positive association between IL and 6 and HF (odds ratio = 1.055; 95 % confidence interval: 1.019-1.093, p < 0.005). Sex, age, and hs-CRP were also associated with HF. Women had a greater risk of HF than men. Older age, higher levels of hs-CRP, and IL-6 were associated with a greater risk of HF. CONCLUSIONS: In patients with COVID-19, increased IL-6 levels are significantly associated with COVID-19-related HF.

Theoretical investigation of the reaction mechanism of THP oxidative rearrangement catalysed by BBOX.

γ-Butyrobetaine hydroxylase (BBOX) is a non-heme FeII/2OG dependent enzyme that is able to perform two different kinds of catalytic reactions on 3-(2,2,2-trimethylhydrazinium) propionate (THP) to produce distinct catalytic products. Although the structure of BBOX complexed with THP has been resolved, the details of its catalytic mechanism are still elusive. In this study, by employing molecular dynamics (MD) simulations and density functional theory (DFT) calculations, the mechanism of the THP oxidative rearrangement reactions catalysed by BBOX was investigated. Our calculations revealed how the enzyme undergoes a conformational conversion to initiate the catalytic reactions. In the first catalytic step, BBOX performs hydrogen abstraction from the substrate THP as a common non-heme iron enzyme. Due to the structure of the substrate stabilizing the radical species and polarizing the adjacent N-N bond, in the next step, THP takes the pathway for N-N bond homolysis but not regular hydroxyl rebounding. The cleaved ammonium radical could either react with the hydroxyl group on the iron centre of the enzyme or recombine with the other cleaved fragment of the substrate to generate the rearranged product. This study revealed the catalytic mechanism of BBOX, detailing how the enzyme and the substrate regulated the hydroxyl rebound process to generate various products.

Computation-Assisted Design of ssDNA Framework Nanorobots for Cancer Logical Recognition, Toehold Disintegration, Visual Dual-Diagnosis, and Synergistic Therapy.

Single-stranded DNA (ssDNA) allows flexible and directional modifications for multiple biological applications, while being greatly limited by their poor stability, increased folding errors, and complicated sequence optimizations. This greatly challenges the design and optimization of ssDNA sequences to fold stable 3D structures for diversified bioapplications. Herein, the stable pentahedral ssDNA framework nanorobots (ssDNA nanorobots) were intelligently designed, assisted by examining dynamic folding of ssDNA in self-assemblies via all-atom molecular dynamics simulations. Assisted by two functional siRNAs (S1 and S2), two ssDNA strands were successfully assembled into ssDNA nanorobots, which include five functional modules (skeleton fixation, logical dual recognition of tumor cell membrane proteins, enzyme loading, dual-miRNA detection and synergy siRNA loading) for multiple applications. By both theoretical calculations and experiments, ssDNA nanorobots were demonstrated to be stable, flexible, highly utilized with low folding errors. Thereafter, ssDNA nanorobots were successfully applied to logical dual-recognition targeting, efficient and cancer-selective internalization, visual dual-detection of miRNAs, selective siRNA delivery and synergistic gene silencing. This work has provided a computational pathway for constructing flexible and multifunctional ssDNA frameworks, enlarging biological application of nucleic acid nanostructures.

Why different sugarcane cultivars show different resistant abilities to smut? : Comparisons of endophytic microbial compositions and metabolic functions in stems of sugarcane cultivars with different abilities to resist smut.

To elucidate the mechanisms underlying the resistance to smut of different sugarcane cultivars, endophytic bacterial and fungal compositions, functions and metabolites in the stems of the sugarcane cultivars were analyzed using high-throughput sequencing techniques and nontargeted metabolomics. The results showed that the levels of ethylene, salicylic acid and jasmonic acid in sugarcane varieties that were not sensitive to smut were all higher than those in sensitive sugarcane varieties. Moreover, endophytic fungi, such as Ramichloridium, Alternaria, Sarocladium, Epicoccum, and Exophiala species, could be considered antagonistic to sugarcane smut. Additionally, the highly active arginine and proline metabolism, pentose phosphate pathway, phenylpropanoid biosynthesis, and tyrosine metabolism in sugarcane varieties that were not sensitive to smut indicated that these pathways contribute to resistance to smut. All of the above results suggested that the relatively highly abundant antagonistic microbes and highly active metabolic functions of endophytes in non-smut-sensitive sugarcane cultivars were important for their relatively high resistance to smut.

The merging of dual umbilical port-incisions for contained morcellation in laparoscopic myomectomy.

Uncontained power morcellation during laparoscopic myomectomy may spread tissue fragments or malignant cells into the abdominal cavity. Recently, various approaches to contained morcellation, have been adopted to retrieve the specimen. However, each of these methods has its own drawbacks. Intraabdominal bag-contained power morcellation adopts a complex isolation system, which prolongs the operation and increases medical costs. Contained manual morcellation via colpotomy or mini-laparotomy increases the trauma and the risk of infection. Contained manual morcellation via umbilical incision during single-port laparoscopic myomectomy may be the most minimally invasive and cosmetic approach. But the popularization of single-port laparoscopy is challenging because of technical difficulties and high costs. We have therefore, developed a surgical technique using 2 umbilical port-incisions (5 mm and 10 mm), which are merged into 1 large umbilical incision (25-30mm) for contained manual morcellation during specimen retrieval, and one 5mm incision in the lower left abdomen for an ancillary instrument. As demonstrated in the video, this technique significantly facilitates surgical manipulation using conventional laparoscopic instruments while still keeping the incisions minimal. It is also economical because the use of an expensive single-port platform and special surgical instruments is avoided. In conclusion, the merging of dual umbilical port-incisions for contained morcellation adds a minimally invasive, cosmetic, and economical option to laparoscopic specimen retrieval that would enrich a gynecologist's skill set, which is particularly relevant in a low-resource settings.

Confined surface-enhanced indole cation-radical cyclization studied by mass spectrometry.

Reactions in confined spaces exhibit unique reactivity, while how the confinement effect enhances reactions remains unclear. Herein, the reaction in the confined space of a nanopipette reactor was examined by in situ nano-electrospray mass spectrometry (nanoESI-MS). The indole cation-radical cyclization was selected as the model reaction, catalyzed by a common visible-light-harvesting complex Ru(bpz)3(PF6)2 (1% eq.) rather than traditional harsh reaction conditions (high temperature or pressure, etc.). As demonstrated by in situ nanoESI-MS, this reaction was readily promoted in the nanopipette under mild conditions, while it was inefficient in both normal flasks and microdroplets. Both experimental and theoretical evidence demonstrated the formation of concentrated Ru(II)-complexes on the inner surface of the nanopipette, which facilitated the accelerated reactions. As a result, dissociative reactive cation radicals with lower HOMO-LUMO gap were generated from the Ru(II)-complexes by ligand-to-metal charge transfer (LMCT). Furthermore, the crucial cation radical intermediates were captured and dynamically monitored via in situ nanoESI-MS, responsible for the electronically matched [4 + 2] cycloaddition and subsequent intramolecular dehydrogenation. This work inspires a deeper understanding of the unique reactions in confined spaces.

Conformation- and Coordination Mode-Dependent Stimuli-Responsive Salicylaldehyde Hydrazone Zn(II) Complexes.

Luminescent Zn(II) complexes that respond to external stimuli are of wide interest due to their potential applications. Schiff base with O,N,O-hydrazone shows excellent luminescence properties with multi-coordination sites for different coordination modes. In this work, three salicylaldehyde hydrazone Zn(II) complexes (1, 2a, 2b) were synthesized and their stimuli-responsive behaviors in different states were explored. Only complex 1 exhibits reversible and self-recoverable photochromic and photoluminescence properties in solution. This may be due to the configuration eversion and the excited-state intramolecular proton transfer (ESIPT) process. In the solid state, 2a has obvious mechanochromic luminescence property, which is caused by the destruction of intermolecular interactions and the transformation from crystalline state to amorphous state. 2a and 2b have delayed fluorescence properties due to effective halogen bond interactions in structures. 2a could undergo crystal-phase transformation into its polymorphous 2b by force/vapor stimulation. Interestingly, 2b shows photochromic property, which can be attributed to the electron transfer and generation of radicals induced by UV irradiation. Due to different conformations and coordination modes, the three Zn(II) complexes show different stimuli-responsive properties. This work presents the multi-stimuli-responsive behaviors of salicylaldehyde hydrazone Zn(II) complexes in different states and discusses the response mechanism in detail, which may provide new insights into the design of multi-stimuli-responsive materials.

Theoretical investigation on the reaction mechanism of UTP cyclohydrolase.

Nucleoside triphosphate cyclohydrolase (UrcA) is a critical enzyme of the uracil catabolism pathway that catalyses the two-step hydrolysis of uridine triphosphate (UTP). Although the recently resolved X-ray structure of UrcA in complex with substrate analogue dUTP provided insights into the structural characteristics of the enzyme, the detailed catalytic mechanism, including how the reaction intermediate accomplishes conformational conversion in the active centre, remains unclear. In this study, extensive DFT calculations and MD simulations were performed to investigate the catalytic reaction process of UrcA. This study shows that the first hydrolytic reactions in UrcA follow a three-step mechanism, while the second hydrolytic reaction follows a two-step mechanism. Glu392 plays a critical role in deprotonating the lytic water in both hydrolytic reactions. The rate-limiting step of the first hydrolytic reaction lies in the cleavage of the uracil ring, in which an extraneous water molecule bridges the proton transfer from C6-OH to N1 to enable the reaction to go through a six-membered transition state with relatively low steric tension. In the second hydrolytic reaction, Glu392 abstracts protons from the lytic water and directly transfers them to the nitrogen atom of the cleaved C4-N3 bond so that the hydrolytic reaction is no longer rate-limited by the C-N bond cleavage step. MD simulations show that the reaction intermediate experiences spontaneous conformation overturn in the active site of UrcA under the assistance of the hydrogen bond interaction from Tyr307 to place its C4-N3 bond alongside the Zn2+ centre of the enzyme to trigger the second hydrolytic reaction.

Reorienting Mechanism of Harderoheme in Coproheme Decarboxylase-A Computational Study.

Coproheme decarboxylase (ChdC) is an important enzyme in the coproporphyrin-dependent pathway (CPD) of Gram-positive bacteria that decarboxylates coproheme on two propionates at position 2 and position 4 sequentially to generate heme b by using H2O2 as an oxidant. This work focused on the ChdC from Geobacillus stearothermophilus (GsChdC) to elucidate the mechanism of its sequential two-step decarboxylation of coproheme. The models of GsChdC in a complex with substrate and reaction intermediate were built to investigate the reorienting mechanism of harderoheme. Targeted molecular dynamics simulations on these models validated that harderoheme is able to rotate in the active site of GsChdC with a 19.06-kcal·mol-1 energy barrier after the first step of decarboxylation to bring the propionate at position 4 in proximity of Tyr145 to continue the second decarboxylation step. The harderoheme rotation mechanism is confirmed to be much easier than the release-rebinding mechanism. In the active site of GsChdC, Trp157 and Trp198 comprise a "gate" construction to regulate the clockwise rotation of the harderoheme. Lys149 plays a critical role in the rotation mechanism, which not only keeps the Trp157-Trp198 "gate" from being closed but also guides the propionate at position 4 through the gap between Trp157 and Trp198 through a salt bridge interaction.

Effects of Through-Bond and Through-Space Conjugations on the Photoluminescence of Small Aromatic and Aliphatic Aldimines.

Through-bond conjugation (TBC) and/or through-space conjugation (TSC) determine the photophysical properties of organic luminescent compounds. No systematic studies have been carried out to understand the transition from aromatic TBC to non-aromatic TSC on the photoluminescence of organic luminescent compounds. In this work, a series of small aromatic and aliphatic aldimines were synthesized. For the aromatic imines, surprisingly, N,1-diphenylmethanimine with the highest TBC is non-emissive, while N-benzyl-1-phenylmethanimine and N-cyclohexyl-1-phenylmethanimine emit bright fluorescence in aggregate states. The aliphatic imines are all emissive, and their maximum emission wavelength decreases while the quantum yield increases with a decrease in steric hindrance. The imines show concentration-dependent and excitation-dependent emissions. Theoretical calculations show that the TBC extents in the aromatic imines are not strong enough to induce photoluminescence in a single molecule state, while the intermolecular TSC becomes dominant for the fluorescence emissions of both aromatic and aliphatic imines in aggregate states, and the configurations and spatial conformations of the molecules in aggregate states play a key role in the formation of effective TSC. This study provides an understanding of how chemical and spatial structures affect the formation of TBC and TSC and their functions on the photoluminescence of organic luminescent materials.

Drum-like Metallacages with Size-Dependent Fluorescence: Exploring the Photophysics of Tetraphenylethylene under Locked Conformations.

Materials with aggregation-induced emission (AIE) properties are of growing interest due to their widespread applications. AIEgens, such as tetraphenylethylene units, display varying emission behaviors during their conformational changes. However, the structure-property relationships of the intermediate conformations have rarely been explored. Herein, we show that the conformational restriction on TPE units can affect the structural relaxation in the excited state and the resulting photophysical behaviors. Specifically, three metallacages of different sizes were prepared via the coordination-driven self-assembly of a TPE-based tetrapyridyl donor with length-increasing Pt(II) acceptors. While the metallacages share similar scaffolds, they exhibit a trend of red-shifted fluorescence and attenuated quantum yield with the increase of their sizes. Furthermore, spectroscopic and computational studies together with a control experiment were conducted, revealing that the degree of cage tension imposed on the excited-state conformational relaxation of TPE moieties resulted in their distinct photophysical properties. The precise control of conformation holds promise as a strategy for understanding the AIE mechanism as well as optimizing the photophysical behaviors of materials on the platform of supramolecular coordination complexes.

One-Step Prepared Water-Resistant Organic-Inorganic-Hybrid Perovskite Quantum Dots with Zn-Oxygen Vacancies for Attempts at Nitrogen Fixation.

Applying organic-inorganic hybrid perovskite quantum dots (PQDs) to photocatalytic nitrogen fixation is hindered long-term by the inherent instability in water and tedious preparations. Here, to realize PQD-catalyzed photocatalytic N2 reduction reaction (NRR), water-resistant PQDs are simply prepared through one-step electrospray synthesis in microseconds. During the fast electrospray, PQDs of Zn/PbO-doped methylammonium lead bromide (Zn/PbO/PC-Zn/MAPbBr3 , MA: CH3 NH3 ) are prepared and part-encapsulated by polycarbonate. The synthesis maintains good water resistance, whose restriction on charge transport is overcome skillfully. Simultaneously, substitution of Zn with Pb on water-resistant surface is also achieved, which fabricates new Zn-oxygen vacancies (Zn-OVs) with Zn/PbO-Zn/MAPbBr3 type I heterojunction. This facilitates efficient electron transfer from internal heterojunction interface of Zn/MAPbBr3 PQDs to the surface of Zn/PbO. Demonstrated by theoretical calculations, Zn-OVs promote chemisorption and polarization of N2 . In addition, s-electrons in exposed Zn become active due to changes of electron filling of Zn orbitals under OVs' co-doping. Thus, photocatalytic N2 reduction reaction catalyzed by organic-inorganic hybrid PQDs is first achieved in aqueous phase without sacrificial agents being added. This initiates possibilities for photocatalytic applications of organic-inorganic hybrid PQDs in aqueous phase.

Interactive Regulation between Aliphatic Hydroxylation and Aromatic Hydroxylation of Thaxtomin D in TxtC: A Theoretical Investigation.

TxtC is an unusual bifunctional cytochrome P450 that is able to perform sequential aliphatic and aromatic hydroxylation of the diketopiperazine substrate thaxtomin D in two distinct sites to produce thaxtomin A. Though the X-ray structure of TxtC complexed with thaxtomin D revealed a binding mode for its aromatic hydroxylation, the preferential hydroxylation site is aliphatic C14. It is thus intriguing to unravel how TxtC accomplishes such two-step catalytic hydroxylation on distinct aliphatic and aromatic carbons and why the aliphatic site is preferred in the hydroxylation step. In this work, by employing molecular docking and molecular dynamics (MD) simulation, we revealed that thaxtomin D could adopt two different conformations in the TxtC active site, which were equal in energy with either the aromatic C20-H or aliphatic C14-H pointing toward the active Cpd I oxyferryl moiety. Further ONIOM calculations indicated that the energy barrier for the rate-limiting hydroxylation step on the aliphatic C14 site was 9.6 kcal/mol more favorable than that on the aromatic C20 site. The hydroxyl group on the monohydroxylated intermediate thaxtomin B C14 site formed hydrogen bonds with Ser280 and Thr385, which induced the l-Phe moiety to rotate around the Cß-Cγ bond of the 4-nitrotryptophan moiety. Thus, it adopted an energetically favorable conformation with aromatic C20 adjacent to the oxyferryl moiety. In addition, the hydroxyl group induced solvent water molecules to enter the active site, which propelled thaxtomin B toward the heme plane and resulted in heme distortion. Based on this geometrical layout, the rate-limiting aromatic hydroxylation energy barrier decreased to 15.4 kcal/mol, which was comparable to that of the thaxtomin D aliphatic hydroxylation process. Our calculations indicated that heme distortion lowered the energy level of the lowest Cpd I α-vacant orbital, which promoted electron transfer in the rate-limiting thaxtomin B aromatic hydroxylation step in TxtC.

N-Nitrosation Mechanism Catalyzed by Non-heme Iron-Containing Enzyme SznF Involving Intramolecular Oxidative Rearrangement.

The non-heme iron-dependent enzyme SznF catalyzes a critical N-nitrosation step during the N-nitrosourea pharmacophore biosynthesis in streptozotocin. The intramolecular oxidative rearrangement process is known to proceed at the FeII-containing active site in the cupin domain of SznF, but its mechanism has not been elucidated to date. In this study, based on the density functional theory calculations, a unique mechanism was proposed for the N-nitrosation reaction catalyzed by SznF in which a four-electron oxidation process is accomplished through a series of complicated electron transferring between the iron center and substrate to bypass the high-valent FeIV═O species. In the catalytic reaction pathway, the O2 binds to the iron center and attacks on the substrate to form the peroxo bridge intermediate by obtaining two electrons from the substrate exclusively. Then, instead of cleaving the peroxo bridge, the Cε-Nω bond of the substrate is homolytically cleaved first to form a carbocation intermediate, which polarizes the peroxo bridge and promotes its heterolysis. After O-O bond cleavage, the following reaction steps proceed effortlessly so that the N-nitrosation is accomplished without NO exchange among reaction species.

Multistimulus Response of Two Tautomeric Zn(II) Complexes and Their White-Light Emission Based on Different Mechanisms.

A triphenylamine (TPA)-based 2H-quinazoline Zn(II) complex (Q-TPA-Zn) exhibiting dual fluorescence and phosphorescence emission in the solid state was designed and prepared. It possesses mechanochromic luminescence and thermochromic luminescence properties. In the solid state, the white afterglow luminescence could be observed at 77 K (CIExy: 0.27, 0.33) while cyan luminescence could be observed at 297 K. After thermolysis at 300 °C, Q-TPA-Zn could be transformed into Schiff base complex S-TPA-Zn with white fluorescence in the powder state (CIExy: 0.32, 0.38), in methanol (CIExy: 0.32, 0.39), and in dimethylformamide (CIExy: 0.26, 0.32) at room temperature. This arises from dual emission of normal* emission and tautomeric* emission induced by excited-state intramolecular proton transfer (ESIPT) from the benzimidazole NH group to the Schiff base N atom. Q-TPA-Zn could also be transformed into its isomeric form, S-TPA-Zn, through photochemical ring-opening reaction upon irradiation under 365 nm in the solution, exhibiting high-contrast photochromic luminescence. Interestingly, S-TPA-Zn could further be transformed into its zwitterionic isomer after continuous irradiation. The same ring-opening reaction could also take place for the orgainc compound Q-TPA via heating or 365 nm irradiation. The ring-opening reaction mechanism and ESIPT emission were interpreted via theoretical calculation.

Ligand-Triggered Platinum(II) Metallacycle with Mechanochromic and Vapochromic Responses.

Supramolecular coordination complexes with solid-state stimuli-responsive characteristics are highly desirable but are rarely reported. Herein, we describe two coordination-driven self-assembled monoanthracene or dianthracene-based hexagonal metallacycles by subtle structure modification. Notably, the dianthracene-containing hexagon 1 exhibits tricolor mechanochromic and vapochromic characteristics, while the monoanthracene-containing hexagon 4 does not show obvious changes toward mechanical force. Further studies have indicated that changes in hexagon 1, especially the ulterior anthracene of hexagon 1 in the molecular stacking through intermolecular interactions toward external stimuli, are responsible for the above behavioral differences. Furthermore, the present work also demonstrates a novel light-harvesting strategy for achieving high-contrast mechanochromic fluorescence involving solid-state energy transfer from hexagon 1 to an organic carbazole derivant 6 without mechanofluorochromism or tetraphenylethylene derivant 7 exhibiting inconspicuous mechanofluorochromism.

Associations of indoor carbon dioxide concentrations, air temperature, and humidity with perceived air quality and sick building syndrome symptoms in Chinese homes.

The indoor environment influences occupants' health. From March 1, 2018, to February 28, 2019, we continuously monitored indoor temperature (T), relative humidity (RH), and CO2 concentration in bedrooms via an online system in 165 residences that covered all five climate zones of China. Meanwhile, we asked one specific occupant in each home to complete questionnaires about perceived air quality and sick building syndrome (SBS) symptoms at the end of each month. Higher CO2 concentration was significantly associated with a higher percentage of perceived stuffy odor and skin SBS symptoms. Higher relative humidity was associated with higher percentage of perceived moldy odor and humid air, while lower RH was associated with a higher percentage of perceived dry air. Occupants who lived in residences with high RH were less likely to have mucosal and skin SBS symptoms (adjusted odds ratio (AOR): 0.73-0.78). However, the benefit of high humidity for perceived dry air and skin dryness symptoms is weaker if there is a high CO2 concentration level.

Investigation of the Detailed AMPylated Reaction Mechanism for the Huntingtin Yeast-Interacting Protein E Enzyme HYPE.

AMPylation is a prevalent posttranslational modification that involves the addition of adenosine monophosphate (AMP) to proteins. Exactly how Huntingtin-associated yeast-interacting protein E (HYPE), as the first human protein, is involved in the transformation of the AMP moiety to its substrate target protein (the endoplasmic reticulum chaperone binding to immunoglobulin protein (BiP)) is still an open question. Additionally, a conserved glutamine plays a vital key role in the AMPylation reaction in most filamentation processes induced by the cAMP (Fic) protein. In the present work, the detailed catalytic AMPylation mechanisms in HYPE were determined based on the density functional theory (DFT) method. Molecular dynamics (MD) simulations were further used to investigate the exact role of the inhibitory glutamate. The metal center, Mg2+, in HYPE has been examined in various coordination configurations, including 4-coordrinated, 5-coordinated and 6-coordinated. DFT calculations revealed that the transformation of the AMP moiety of HYPE with BiP followed a sequential pathway. The model with a 4-coordinated metal center had a barrier of 14.7 kcal/mol, which was consistent with the experimental value and lower than the 38.7 kcal/mol barrier of the model with a 6-coordinated metal center and the 31.1 kcal/mol barrier of the model with a 5-coordinated metal center. Furthermore, DFT results indicated that Thr518 residue oxygen directly attacks the phosphorus, while the His363 residue acts as H-bond acceptor. At the same time, an MD study indicated that Glu234 played an inhibitory role in the α-inhibition helix by regulating the hydrogen bond interaction between Arg374 and the Pγ of the ATP molecule. The revealed sequential pathway and the inhibitory role of Glu234 in HYPE were inspirational for understanding the catalytic and inhibitory mechanisms of Fic-mediated AMP transfer, paving the way for further studies on the physiological role of Fic enzymes.

Theoretical Studies on the Binding Mode and Reaction Mechanism of TLP Hydrolase kpHIUH.

In this work, we have investigated the binding conformations of the substrate in the active site of 5-HIU hydrolase kpHIUH and its catalytic hydrolysis mechanism. Docking calculations revealed that the substrate adopts a conformation in the active site with its molecular plane laying parallel to the binding interface of the protein dimer of kpHIUH, in which His7 and His92 are located adjacent to the hydrolysis site C6 and have hydrogen bond interactions with the lytic water. Based on this binding conformation, density functional theory calculations indicated that the optimal catalytic mechanism consists of two stages: (1) the lytic water molecule is deprotonated by His92 and carries out nucleophilic attack on C6=O of 5-HIU, resulting in an oxyanion intermediate; (2) by accepting a proton transferred from His92, C6-N5 bond is cleaved to completes the catalytic cycle. The roles of His7, His92, Ser108 and Arg49 in the catalytic reaction were revealed and discussed in detail.