Rate-limiting Step Research Articles

Cytochromes P450 evolution in the plant terrestrialization context.

Plants started to colonize land around 500 million years ago. It meant dealing with new challenges like absence of buoyancy, water and nutrients shortage, increased light radiation, reproduction on land, and interaction with new microorganisms. This obviously required the acquisition of novel functions and metabolic capacities. Cytochrome P450 (CYP) monooxygenases form the largest superfamily of enzymes and are present to catalyse critical and rate-limiting steps in most plant-specific pathways. The different families of CYP enzymes are typically associated with specific functions. CYP family emergence and evolution in the green lineage thus offer the opportunity to obtain a glimpse into the timing of the evolution of the critical functions that were required (or became dispensable) for the plant transition to land. Based on the analysis of currently available genomic data, this review provides an evolutionary history of plant CYPs in the context of plant terrestrialization and describes the associated functions in the different lineages. Without surprise it highlights the relevance of the biosynthesis of antioxidants and UV screens, biopolymers, and critical signalling pathways. It also points to important unsolved questions that would deserve to be answered to improve our understanding of plant adaptation to challenging environments and the management of agricultural traits. This article is part of the theme issue 'The evolution of plant metabolism'.

Microkinetic insights into temperature pulsing for accelerating ammonia decomposition

We investigate ammonia decomposition under temperature pulsing toward enhancing H2 production. We observed up to a 10x increase in conversion with rapid pulsing under certain conditions. Our kinetic analysis identified the association of N2 as the rate-limiting step, showing significant rate enhancement by pulsing at high frequencies and temperature amplitudes. These findings highlight that rapid heat addition and removal, through joule heating, can boost performance and assist with decarbonization.

Tailoring the d-Band Center of WS2 by Metal and Nonmetal Dual-Doping for Enhanced Electrocatalytic Nitrogen Reduction.

Tuning the adsorption energy of nitrogen intermediates and lowering the reaction energy barrier is essential to accelerate the kinetics of nitrogen reduction reaction (NRR), yet remains a great challenge. Herein, the electronic structure of WS2 is tailored based on a metal and nonmetal dual-doping strategy (denoted Fe, F-WS2) to lower the d-band center of W in order to optimize the adsorption of nitrogen intermediates. The obtained Fe, F-WS2 nanosheet catalyst presents a high Faradic efficiency (FE) of 22.42% with a NH3 yield rate of 91.46µg h-1 mgcat. -1. The in situ characterizations and DFT simulations consistently show the enhanced activity is attributed to the downshift of the d-band center, which contributes to the rate-determining step of the second protonation to form N2H2 * key intermediates, thereby boosting the overall nitrogen electrocatalysis reaction kinetics. This work opens a new avenue to enhanced electrocatalysis by modulating the electronic structure and surrounding microenvironment of the catalytic metal centers.

Axial modulation of catalysis mechanism via charge-asymmetric for enhanced electrocatalytic performance toward hydrogen and oxygen evolution reactions

Designing efficient, stable, and nonprecious metal bifunctional catalysts is warranted for realizing the industrial application of electrocatalytic overall water splitting. Herein, we report a new catalyst (CoFe/SNC) for the HER and OER. The active site of catalysis composed of two charge-asymmetric Co/Fe atoms could axial modulation of the catalysis mechanism, thus improving the kinetics of the HER and OER. Moreover, the use of hollow porous carbon spheres as catalyst support not only enhances the distribution of Co/Fe atoms but also improves the mass transfer during the HER and OER processes. Therefore, the CoFe/SNC displays a good alkaline HER and OER activity with a small overpotential of 96.2 mV and 285.8 mV at 10 mA cm−2, respectively. Theoretical calculations reveal that the Fe atoms with optimal absorption energy for the hydrogen intermediate and the Co centers with a reduced energy barrier for the rate-determining step are the active sites for HER and OER, respectively. Additionally, the synergistic effect of Co/Fe dual-atoms could effectively optimize the intermediate adsorption of Co/Fe-N4 sites and improve the barriers of HER and OER activity and stability. This study provides valuable insight for designing bifunctional alkaline water-splitting electrocatalysts.

Peptidoglycan Endopeptidase PBP7 Facilitates the Recruitment of FtsN to the Divisome and Promotes Peptidoglycan Synthesis in Escherichia coli.

Escherichia coli has many periplasmic hydrolases to degrade and modify peptidoglycan (PG). However, the redundancy of eight PG endopeptidases makes it challenging to define specific roles to individual enzymes. Therefore, the cellular role of PBP7 (encoded by pbpG) is not clearly defined. In this work, we show that PBP7 localizes in the lateral cell envelope and at midcell. The C-terminal α-helix of PBP7 is crucial for midcell localization but not for its activity, which is dispensable for this localization. Additionally, midcell localization of PBP7 relies on the assembly of FtsZ up to FtsN in the divisome, and on the activity of PBP3. PBP7 was found to affect the assembly timing of FtsZ and FtsN in the divisome. The absence of PBP7 slows down the assembly of FtsN at midcell. The ΔpbpG mutant exhibited a weaker incorporation of the fluorescent D-amino acid HADA, reporting on transpeptidase activity, compared to wild-type cells. This could indicate reduced PG synthesis at the septum of the ΔpbpG strain, explaining the slower accumulation of FtsN and suggesting that endopeptidase-mediated PG cleavage may be a rate-limiting step for septal PG synthesis.

Enhanced Hydrophobicity and Oleophilicity of Modified Activated Carbons Derived from Agro-Wastes Biomass for the Removal of Crude Oil from Aqueous Medium

Crude oil spillage has tremendous environmental impacts and poses severe pollution problems worldwide due to the continuous activities and operations in the oil and gas sector. This has resulted in the urgent need for clean-up techniques such as the use of natural adsorbents which is considered a relatively low-cost, readily-available, efficient, eco-friendly, and easy-to-deploy mode of addressing oil spillage due to its high oil sorption capacity/efficiency, high oil selectivity, oleophilic, enduring, reusability and biodegradable properties. Empty palm fruit bunch and coconut coir were used as precursors to produce activated carbons for oil spill remediation. The influence of varying parameters was investigated using a batch experimental procedure resulting in the crude oil adsorption capacity increasing with a corresponding increase in contact time, initial oil concentration, temperature, agitation speed, and particle size but decreasing in adsorbent dosage. The combination of surface morphological modification and hydrophobicity enhancement resulted in significantly improved adsorption capacity for crude oil removal (2710.0 mg/g and 4859.5 mg/g for EPFBAC<SUB>LA</SUB> and CCAC<SUB>L.A</SUB> respectively), as evidenced by both FTIR and SEM analyses. The experimental isotherm data were analysed using various isotherm models and the best-fitted isotherm model was the Freundlich model with a correlation coefficient (R<sup>2 </sup>= 0.991 and R<sup>2 </sup>= 0.999) for EPFB<SUB>L.A</SUB> and CCAC<SUB>L.A</SUB> respectively. The kinetic behaviour of the adsorption process was best described by pseudo-second order with R<sup>2</sup> values of 0.970 and 0.999 for EPFBAC<SUB>LA</SUB> and CCAC<SUB>L.A</SUB> respectively while Boyd model revealed that the adsorption was controlled by an internal transport mechanism and film diffusion was the rate-limiting step. The crude oil adsorption was chemisorption and endothermic owing to the positive enthalpy values (ΔH<sup>o</sup> = 183.890 KJ/mol for EPFBAC<SUB>L.A </SUB>and ΔH<sup>o</sup> = 69.656 KJ/mol for CCAC<SUB>L.A</SUB>), the positive value of entropy suggested that the adsorption process was accompanied by an increase in the degree of randomness or disorder at the interface between the adsorbent and the adsorbate. A temperature rise led to a decline in Gibbs energy (ΔG<sup>o</sup>), suggesting that adsorption became more feasible and spontaneous at higher temperatures and the significant activation energies indicated the existence of a substantial energy barrier that must be overcome to initiate the reaction. The results showed the significant capability of the prepared adsorbents to be used as a low-cost, re-generable and eco-friendly adsorbent in oil spill clean-up and is recommended to exploit its usage on a large scale.

A novel Co-free and efficient Pr0.5Ba0.5Fe0.8Cu0.2O3-δ nanofiber cathode material for intermediate temperature solid oxide fuel cells

Fe-based perovskite without Co has a low thermal expansion coefficient and can be used as a solid oxide fuel cell (SOFC) cathode to ensure the thermal compatibility with the electrolyte; however, its further advancement is impeded by sluggish oxygen reduction reaction (ORR) dynamics. In this work, the electrospinning method and Cu doping strategy are employed to improve the microstructure and oxygen reduction kinetics of Pr0.5Ba0.5FeO3-δ cathodes. The results show that Pr0.5Ba0.5Fe0.8Cu0.2O3-δ has a unique fiber structure and high specific surface area, and the introduction of Cu facilitates the release of lattice oxygen, effectively increasing the oxygen vacancy content. The relaxation time distribution analysis and oxygen reduction reaction dynamics indicate that the oxygen adsorption-dissociation process is the main rate-limiting step in the oxygen reduction reaction of Pr0.5Ba0.5Fe0.8Cu0.2O3-δ electrode materials. At 700 °C, the area-specific resistance (ASR) of the symmetric cell of Pr0.5Ba0.5Fe0.8Cu0.2O3-δ fiber material is 0.071 Ω cm2, and the power density of the single cell reaches 988 mW cm−2, which shows good electrochemical output performance and long-term operational stability. The preparation of nanofiber cathodes with high specific surface area and porosity by electrospinning provides an effective strategy to enhance the electrocatalytic activity of the IT-SOFC cathode.

Main-group element-boosted oxygen electrocatalysis of Cu-N-C sites for zinc-air battery with cycling over 5000 h

Developing highly active and durable air cathode catalysts is crucial yet challenging for rechargeable zinc-air batteries. Herein, a size-adjustable, flexible, and self-standing carbon membrane catalyst encapsulating adjacent Cu/Na dual-atom sites is prepared using a solution blow spinning technique combined with a pyrolysis strategy. The intrinsic activity of the Cu-N4 site is boosted by the neighboring Na-containing functional group, which enhances O2 adsorption and optimizes the rate-determining step of O2 activation (*O2 → *OOH) during the oxygen reduction reaction process. Meanwhile, the Cu-N4 sites are encapsulated within carbon nanofibers and anchored by the carbon matrix to form a C2-Cu-N4 configuration, thereby reinforcing the stability of the Cu centers. Moreover, the introduction of Na-containing functional groups on the carbon atoms significantly reduces the positive charge on their outer shell C atoms, rendering the carbon skeletons less susceptible to corrosion by oxygen species and further preventing the dissolution of Cu centers. Under these multi-type regulations, the zinc-air battery with Cu/Na-carbon membrane catalyst as the air cathode demonstrates long-term discharge/charge cycle stability of over 5000 h. This considerable stability improvement represents a critical step towards developing Cu-N4 active sites modified with the neighboring main-group metal-containing functional groups to overcome the durability barriers of zinc-air batteries for future practical applications.

The Particle Drifting Effect: A Combined Function of Colloidal and Drug Properties.

The particle drifting effect, where nanosized colloidal drug particles overcome the diffusional resistance of the aqueous boundary layer adjacent to the intestinal wall and increase drug absorption rates, is drawing increasing attention in pharmaceutical research. However, mechanistic understanding and accurate prediction of the particle drifting effect remain lacking. In this study, we systematically evaluated the extent of the particle drifting effect affected by drug and colloidal properties, including the size, number, and type of the moving species using biphasic diffusion experiments combined with computational fluid dynamics simulations and mass transport analyses. The results showed that the particle drifting effect is a sequential reaction of particle dissolution/dissociation in the diffusional boundary layer, followed by absorption of the free drug. Therefore, factors affecting the rate-limiting step, which can be either process or both under different circumstances, alter the particle drifting effect. Experimental results also agree with the theory that the particle dissolution rate is dependent on particle size, concentration, and drug solubility. In addition, rapid bile micelle dissociation and bile salt absorption facilitated drug absorption by the particle drifting effect. Our findings explain the highly dynamic nature of the particle drifting effect and will contribute to rational formulation development and better bioavailability prediction for formulations containing colloidal particles.

Comparing low-temperature NH3-SCR activity, operating temperature window and kinetic properties of the Mn-Fe-Nb/TiO2 catalysts prepared by different methods

In order to overcome the shortcomings of traditional V-based catalysts, such as poor low-temperature activity, narrow temperature window, and toxicity. Therefore, the Mn-Fe-Nb/TiO2 catalysts were prepared using impregnation (IM), citric acid complexation (CA), co-precipitation (CP), hydrothermal synthesis (HY), and sol–gel (SG) methods to develop novel NH3 selective catalytic reduction (NH3-SCR) catalysts with excellent low-temperature activity, a wide temperature window, and environmental friendliness. Their NH3-SCR catalytic activity was evaluated, followed by a detailed analysis of their morphology, structure, specific surface area, chemical state, redox properties, acidity, and interactions using X-ray diffraction (XRD), Bruaner-Emmet-Teller (BET), Scanning Electron Microscopy (SEM), High Resolution Transmission Electron Microscopy (HR-TEM), X-ray photoelectron spectroscopy (XPS), H2 temperature-programmed reduction (H2-TPR), NH3 temperature-programmed desorption (NH3-TPD), and Fourier transform infrared spectroscopy (FTIR) techniques. The 5Mn-7Fe-4Nb/TiO2-SG catalyst, prepared via sol–gel, exhibited the highest denitrification (de-NOx) activity and the widest operating temperature window, achieving over 80% Nitric oxide (NO) conversion at 180–350 °C and a peak conversion of 97% at 250 °C. The XRD, BET, SEM, and HR-TEM characterization results showed that the catalyst prepared using the sol–gel method had the smallest particles, the highest specific surface area, and the greatest dispersion. XPS and NH3-TPD results revealed that the catalyst prepared using the sol–gel method possessed the highest surface adsorbed oxygen (Oα) content and the largest number of acidic sites. H2-TPR and FTIR analyses indicated that the catalyst prepared using the sol–gel method enhanced the reduction capacity and possessed the strongest interaction forces among support-active-promoter components. Steady-state kinetic tests confirmed that in the NH3-SCR reaction, the rate-determining step was the adsorption activation of NO on the catalyst surface. The sol–gel method reduced the activation energy for de-NOx reactions, enhancing the low-temperature activity of the catalyst.

Atomically Isolated Pd Sites Promote Electrochemical CO Reduction to Acetate through a Protonation-Regulated Mechanism.

Electrochemical CO reduction reaction (CORR) offers a promising approach for sustainable acetate production, the promotion of which requires the control of multiple protonation steps. This paper describes the synthesis of atomically isolated Pd sites onto Cu nanoflakes to regulate the protonation of key intermediates. The Pd sites with moderate water activation capability are found to enhance the protonation of *CO at the neighboring Cu site to *COH, which is confirmed to be the rate-determining step through kinetic isotope effect studies. The formation of *COH-*CO is therefore promoted. Additionally, the Pd sites would preferentially protonate the C-OH group in *COH-*CO due to the spatial approximability and electronic modulation effects, generating *CCO for the selective formation of acetate. An acetate Faradaic efficiency of 59.5% is achieved at -0.78 V vs reversible hydrogen electrode (RHE), with a maximum partial current density of 286 mA cm-2 at -0.86 V vs RHE. The optimized catalyst also exhibits long-term stability for 500 h at 100 mA cm-2 in a membrane electrode assembly. This work reveals a new promoting mechanism for selective CORR with simultaneous tuning of the structural and electronic properties of the proton-supplying sites.

Comparative analysis of the interaction mechanism of γ-globulin and hemoglobin with spherical and rod-shaped gold nanoparticles.

The interaction mechanism of spherical gold nanoparticles (AuNPs) and rod-shaped gold nanoparticles (AuNRs) with γ-globulin and hemoglobin was comprehensively and comparatively analyzed. γ-Globulin and hemoglobin have high affinity with AuNPs, and with two different types of binding sites on AuNRs surface. Except hemoglobin interaction with the first binding site of AuNRs, the interaction between γ-globulin/hemoglobin and AuNPs/AuNRs is the spontaneous, endothermic and entropy-driven process, and hydrophobic interaction plays a dominant role. The molecular adsorption mechanism of γ-globulin/hemoglobin on AuNPs and AuNRs surface conforms to Langmuir model and Freundlich model, respectively. The kinetic molecular mechanism between them conforms to the pseudo-second-order model, and chemisorption is the rate-limiting step. AuNPs result in the loosening and unfolding of γ-globulin backbone. AuNRs have no significant effect on γ-globulin backbone. AuNPs/AuNRs result in no significant changes in hemoglobin structure and heme group microenvironment. AuNPs/AuNRs decrease the hydrophobicity of Trp microenvironment of γ-globulin, but there is an intramolecular energy transfer from Trp residue to Tyr residue of hemoglobin. The β-sheet of γ-globulin and the α-helix of hemoglobin reduce by increasing concentrations of AuNPs/AuNRs. Molecular docking is suggesting that the specific amino acid residues of γ-globulin and hemoglobin interaction with AuNPs/AuNRs, and validates the experimental results.

Oxidative desulfurization of dibenzothiophene over V-Mo co-doped akaganeite

In this paper, V-Mo co-doped akaganeite (β-FeOOH) was prepared by one-pot hydrothermal method and the oxidative desulfurization (ODS) of dibenzothiophene over the catalyst was carefully investigated. 0.07g (ca. 0.35wt.% of model oil) of catalyst induced 100% of DBT removal in 60min with a O/S of 20:1 under 60 °C for the model oils with any of the sulfur contents, 100 ppm, 500ppm, or 1000 ppm. Moreover, the DBT removal after recycling 4 times is still 100% and it only declines to 98.04% in the fifth cycle, which indicates that the catalyst has a good stability and recycling performance. Doping V species in Mo doped β-FeOOH not only initiates a micro-variation in catalyst structure and the resultant more active •OH species but also forms a more stable structure of the catalyst, enabling the catalyst to exhibit a higher ODS reaction rate and a better reusability. The GC-MS analyses demonstrated that the DBT was converted to DBTO2 by two steps: (1) DBT→ DBTO; (2) DBTO→ DBTO2, and the former is the rate-limiting step.

Boosting Oxygen Evolution Reaction Performance on NiFe-Based Catalysts Through d-Orbital Hybridization

HighlightsThe NiFeLa catalyst with 3d-5d orbital coupling exhibits remarkable oxygen evolution reaction (OER) activity and stability, enabling an anion-exchange membrane water electrolyzers device to achieve a cell voltage of only 1.58 V at 1 A cm−2 as well as long-term stability over 600 h.The introduction of La disrupts the symmetry of Ni-Fe units and optimize d band center, which affects the d-p orbital hybridization between the metal sites on the surface of the catalyst and oxygen-containing intermediates during the OER process.The 5d-introduced NiFeLa has enhanced adsorption strength of oxygen intermediates, which can reduce the rate-determining step energy barrier and prevent catalyst dissolution.

Understanding in-vivo refolding of antibody fragments (Fab): Biosimilar Ranibizumab a case study

In-vitro protein refolding is a major bottleneck step in the large-scale manufacturing of antibody fragments expressed in a microbial host system. The formation of an inter-chain disulfide bond is a key rate-limiting step during in-vitro refolding of antibody fragment therapeutics. In this investigation, we report the use of two redox mutant strains of E. coli viz., SHuffle® T7 (DE3) and SHuffle® T7 Express (DE3) possessing an oxidizing cytoplasm for the soluble expression of a refolded biosimilar antibody fragment. The effect of various critical process parameters on antibody fragment refolding yield was studied using a Design of Experiment (DoE) approach. The optimized upstream processing led to the expression of 167 ± 2.53 mg/L and 95 ± 1.44 mg/L of soluble refolded biosimilar Ranibizumab using the SHuffle® T7 (DE3) and SHuffle® T7 Express (DE3) strains, respectively, at the bioreactor scale. Physicochemical characteristics of the in-vivo refolded antibody fragment were studied and compared with an innovator molecule using various orthogonal analytical methods. A biological activity study of in-vivo refolded Ranibizumab using HUVEC cell-based bioassay proved that cell proliferation inhibition is comparable to the innovator Ranibizumab. The proposed strategy offers a time and cost-effective manufacturing platform for antibody fragments.

Efficient Water Reforming of Biomass to H2 via Well-Organized Redox-Neutral Cleavage of C-C, O-H and C-H Bonds.

Hydrogen (H2) is a clean and environmentally friendly energy carrier. The depletion of fossil fuels makes renewable H2 production highly desirable. Water reforming of renewable biomass to hydrogen, with a relay of natural photosynthesis to biomass, would be an indirect pathway to realize the ideal but extremely challenging photocatalytic overall water splitting to hydrogen, with favorable thermodynamics. Since the seminal work of water reforming of biomass in 1980, great endeavors have been made. Nevertheless, hitherto, the entire kinetic pathway has been elusive, which seriously limits the reforming processes. Using a designed well-organized redox-neutral cleavage of C-C, O-H and C-H bonds enabled by photoelectrocatalysis, here, we show the efficient water reforming of biomass to hydrogen at room temperature, with a yield up to 93%. The clear insights into the kinetic pathway with oxidation of carbon radicals to carbon cations as the indicated rate-determining step, would cast brightness for efficient and sustainable hydrogen production to accelerate the hydrogen economy.

(CAAC)CuCl: A Competent Precatalyst for Carbonyl and Ester Hydrosilylation.

Cu-catalyzed carbonyl hydrosilylation involves a ligated "[(L)CuH]" as the active catalyst, where the ligand L has a crucial role toward the stability, stereoselectivity, and enhancement of the hydridicity. Strongly σ-donating N-heterocyclic carbenes (NHCs), their ring-expanded form, and an abnormal NHC as ligands have yielded robust and efficient Cu catalysts. However, cyclic(alkyl)(amino)carbenes (CAACs), despite being stronger σ-donors than NHCs and already having a salient Cu(I) chemistry, are yet to be reported as a similar ligand platform for this purpose. We establish here the familiar [(Me2CAAC)CuCl] as a powerful precatalyst in this regard. Additionally, it also catalyzes the more challenging ester hydrosilylation, which is a rare feat for a Cu catalyst. Apart from the stronger σ-donating ability, the more steric "openness" of CAACs than bulky NHCs also seems to be advantageous. To corroborate, three new (CAAC)CuCl complexes [(ArCH2,MeCAAC)CuCl] (Ar = Ph, 1-naphthyl, and 1-prenyl) are devised, where the effective steric around the copper is practically unaltered from the case of [(Me2CAAC)CuCl]. All three are equally active in carbonyl and ester hydrosilylation as [(Me2CAAC)CuCl]. Computation suggests the carbonyl insertion into a "(CAAC)Cu-H" as the rate-limiting step. To elucidate the involvement of a "(CAAC)CuH", "(PhCH2,MeCAAC)CuH" is generated in situ and is trapped as its BH3 adduct (PhCH2,MeCAAC)CuBH4.

Atomically dispersed Fe-N4 sites in activation of peroxymonosulfate for wastewater purification: Mechanism of non-radical pathways and their quantification

Single-atom catalysts are promising alternatives to homogeneous and heterogeneous catalysts in the advanced oxidation processes for contaminant degradation; however, quantifying the exact contribution of nonradical reactive oxygen species has remained challenging. Herein, Fe single atoms anchored on nitrogen-doped carbonaceous substrates (Fe SAs-N-C) were synthesized for peroxymonosulfate (PMS) activation toward pollutant degradation. Fe SAs-N-C achieved high activity within 30 min, which is 17.20 and 5.08 times higher than those of N-C catalysts without or with Fe nanoparticles, respectively. A synergistic mechanism of singlet oxygen (1O2) and electron transfer for dominating pollutant degradation was revealed. The former contributed 78.50 % while the latter contributed 21.50 % in pollutant elimination. Density functional theory calculations uncovered that the O atom in −SO4 moiety of PMS was adsorbed on the Fe atoms, leading to the OH bond elongation and generating 1O2. While the absorbed O atom in OO bond near to −SO4 moiety of PMS tended to enlarge the OO bond and accelerate the electron transfer. The adsorbed PMS was prone to being decomposed into 1O2 due to a lower energy barrier (0.43 eV) of 1O2 rate-determining step than that of electron transfer (0.95 eV). Fe-N4 sites are both electron transfer channels and 1O2 formation sites. This work provides insights into the quantitative contributions of non-radical active species to pollutant degradation.

Enzyme Tunnel Dynamics and Catalytic Mechanism of Norcoclaurine Synthase: Insights from a Combined LiGaMD and DFT Study.

This study conducts a systematic investigation into the catalytic mechanism of norcoclaurine synthase (NCS), a key enzyme in the biosynthesis of tetrahydroisoquinolines (THIQs) with therapeutic applications. By integration of LiGaMD and DFT calculations, the reaction pathway of NCS is mapped, providing detailed insights into its catalytic activity and selectivity. Our findings underscore the critical role of E103 in substrate capture and reveal the hitherto unappreciated influence of nonpolar residues M183 and L76 on tunnel dynamics. A prominent discovery is the identification of a high-energy barrier (44.2 kcal/mol) associated with the aromatic electrophilic attack, which pinpoints the rate-limiting step. Moreover, we disclose the existence of dual transition states leading to different products with the energetically favored six-membered ring formation consistent with experimental evidence. These mechanistic revelations not only refine our understanding of NCS but also advocate for a renewed emphasis on enzyme tunnel engineering for optimizing THIQs biosynthesis. The research sets the stage for translating these findings into practical enzyme modifications. Our results highlight the potential of NCS as a biocatalyst to overcome the limitations of current synthetic methodologies, such as low yields and environmental impacts, and provide a theoretical contribution to the efficient, eco-friendly production of THIQs-based pharmaceuticals.

Glycogen synthase activity in Candida albicans is partly controlled by the functional ortholog of Saccharomyces cerevisiae Gac1p.

To adapt to various host microenvironments, the human fungal pathogen Candida albicans possesses the capacity to accumulate and store glycogen as an internal carbohydrate source. In the model yeast Saccharomyces cerevisiae, ScGlc7p and ScGac1p are the serine/threonine type 1 protein phosphatase catalytic and regulatory subunits that control glycogen synthesis by altering the phosphorylation state of the glycogen synthase Gsy2p. Despite recent delineation of the glycogen synthesis pathway in C. albicans, the molecular events driving synthase activation are currently undefined. In this study, using a combination of microbiologic and genetic techniques, we determined that the protein encoded by uncharacterized gene C1_01140C, and not the currently annotated C. albicans Gac1p, is the major regulatory subunit involved in glycogen synthesis. C1_01140Cp contains a conserved GVNK motif observed across multiple starch/glycogen-binding proteins in various species, and alanine substitution of each residue in this motif significantly impaired glycogen accumulation in C. albicans. Fluorescent protein tagging and microscopy indicated that C1_01140Cp-GFPy colocalized with CaGlc7p-tdTomato and CaGsy1p-tdTomato accordingly. Co-immunoprecipitation assays further confirmed that C1_01140Cp associates with CaGlc7p and CaGsy1p during glycogen synthesis. Lastly, c1_01140cΔ/Δ exhibited colonization defects in a murine model of vulvovaginal candidiasis. Collectively, our data indicate that uncharacterized C1_01140Cp is the functional ortholog of the PPP1R subunit ScGac1p in C. albicans.IMPORTANCEThe capacity to synthesize glycogen offers microbes metabolic flexibility, including the fungal pathogen Candida albicans. In Saccharomyces cerevisiae, dephosphorylation of glycogen synthase by the ScGlc7p-containing phosphatase is a critical rate-limiting step in glycogen synthesis. Subunits, including ScGac1p, target ScGlc7p to α-1,4-glucosyl primers for efficient ScGsy2p synthase activation. However, this process in C. albicans had not been delineated. Here, we show that the C. albicans genome encodes for two homologous phosphatase-binding subunits, annotated CaGac1p and uncharacterized C1_01140Cp, both containing a GVNK motif required for polysaccharide affinity. Surprisingly, loss of CaGac1p only moderately reduced glycogen accumulation, whereas loss of C1_01140Cp ablated it. Fluorescence microscopy and co-immunoprecipitation approaches revealed that C1_01140Cp associates with CaGlc7p and CaGsy1p during glycogen synthesis. Moreover, C1_01140Cp contributed to fungal fitness at the vaginal mucosa during murine vaginitis. Therefore, this work demonstrates that glycogen synthase regulation is conserved in C. albicans and C1_01140Cp is the functional ortholog of ScGac1p.