High-Entropy Oxide of (BiZrMoWCeLa)O2 as a Novel Catalyst for Vanadium Redox Flow Batteries.

In this study, new fluorite high-entropy oxide (HEO), (BiZrMoWCeLa)O2, nanoparticles were produced using a surfactant-assisted hydrothermal technique followed by calcination and were used as novel catalytic materials for vanadium redox flow batteries (VRFBs). The HEO calcined at 750 °C (HEO-750) demonstrates superior electrocatalytic activity toward V3+/V2+ and VO2+/VO2+ redox couples compared to those of cells assembled with other samples. The charge-discharge tests further confirm that VRFBs using the HEO-750 catalyst demonstrate excellent Coulombic efficiency, voltage efficiency, and energy efficiency of 97.22, 87.47, and 85.04% at a current density of 80 mA cm-2 and 98.10, 74.76, and 73.34% at a higher current density of 160 mA cm-2, respectively. Moreover, with 500 charge-discharge cycles, there is no discernible degradation. These results are attributed to the calcination heat treatment, which induces the formation of a new single-phase fluorite structure, which facilitates the redox reactions of the vanadium redox couples. Furthermore, a high surface area, wettability, and plenty of oxygen vacancies can give more surface electroactive sites, improving the electrochemical performance, the charge transfer of the redox processes, and the stability of the VRFBs' electrode. This is the first report on the development of fluorite structure HEO nanoparticles in VRFBs, and it opens the door to further research into other HEOs.

Increased intraoperative vein ligation in microsurgical varicocelectomy is associated with pain improvement.

Varicocele is a major cause of male infertility. However, few studies have discussed the potential associations between the pain caused by varicocele and preoperative and intraoperative factors. The aim of this study was to evaluate factors potentially associated with changes in pain score after microsurgical varicocelectomy. This retrospective study was conducted between August 2020 and August 2022 at China Medical University Hospital in Taichung, Taiwan. Patient characteristics including age, body mass index, semen analysis, testicular volume, and the number of veins ligated were collected. Preoperative and intraoperative factors were analyzed to determine if they were correlated with changes in numeric rating scale (NRS) after microsurgical varicocelectomy. A total of 44 patients with clinical varicocele underwent subinguinal microsurgical varicocelectomy and were analyzed. The overall pain resolution rate was 91%, and the average satisfaction score after surgery was 9.2 according to their subjective feelings. Multivariate analysis revealed that severe varicocele grade (odds ratio [OR] 16.5, 95% confidence interval [CI] 3.01-90.47; P = .018) and the number of veins ligated (OR 6, 95% CI 1.6-22.48; P = .013), were significantly associated with changes in NRS after surgery. In addition, the area under the receiver operating characteristic curve for changes in NRS and the total number of veins ligated was 0.869. Microsurgical varicocelectomy had a high success rate for scrotal pain and satisfaction. Severe varicocele grade and the number of veins ligated in microsurgical varicocelectomy were associated with postoperative pain improvement.

Simultaneous determination of 24 congeners of 2- and 3-monochloropropanediol esters and 7 congeners of glycidyl esters using direct multi-residue analytical LC-MS/MS methods in various food matrices.

Glycidyl esters (GEs) and 2- and 3-monochloropropanediol esters (MCPDEs) are emerging process-generated food contaminants known as possible carcinogens. Herein, a direct method is developed and validated for the first time to simultaneously quantify seven GEs and twenty-four MCPDE congeners of processed foods using liquid chromatography-tandem mass spectrometry in a single sequence without ester cleavage or derivatisation, thereby allowing for the simultaneous analysis of numerous food matrices with high accuracy and precision. Our results show levels of GEs varying from

N-Formimidoylation/-iminoacetylation modification in aminoglycosides requires FAD-dependent and ligand-protein NOS bridge dual chemistry.

Oxidized cysteine residues are highly reactive and can form functional covalent conjugates, of which the allosteric redox switch formed by the lysine-cysteine NOS bridge is an example. Here, we report a noncanonical FAD-dependent enzyme Orf1 that adds a glycine-derived N-formimidoyl group to glycinothricin to form the antibiotic BD-12. X-ray crystallography was used to investigate this complex enzymatic process, which showed Orf1 has two substrate-binding sites that sit 13.5 Å apart unlike canonical FAD-dependent oxidoreductases. One site could accommodate glycine and the other glycinothricin or glycylthricin. Moreover, an intermediate-enzyme adduct with a NOS-covalent linkage was observed in the later site, where it acts as a two-scissile-bond linkage facilitating nucleophilic addition and cofactor-free decarboxylation. The chain length of nucleophilic acceptors vies with bond cleavage sites at either N-O or O-S accounting for N-formimidoylation or N-iminoacetylation. The resultant product is no longer sensitive to aminoglycoside-modifying enzymes, a strategy that antibiotic-producing species employ to counter drug resistance in competing species.

Control of Frequency Combs with Passive Resonators.

Tailored optical frequency combs are generated by nesting passive etalons within mode-locked oscillators. In this work, the oscillator generates a comb of 6.8 GHz with 106 MHz side-bands. This tailored comb results from the self-synchronized locking of two cavities with precision optical frequency tuning. In this manuscript, it is demonstrated that these combs can be precisely predicted utilizing a temporal ABCD matrix method and precise comb frequency tuning by scanning over the D1 transition line of 87Rb and observing the fluorescence.

Emotional Intelligence and Transformational Leadership: Meta-Analysis and Explanatory Model of Female Leadership Advantage.

Emotional intelligence is a second-stratum factor of general intelligence (MacCann et al. 2014) that: (a) has been popularly touted as an essential individual difference for effective leadership (Goleman 1998), but also (b) exhibits large gender group differences favoring women (Joseph and Newman 2010). Combining these insights, we propose that emotional intelligence is a key mechanism in the so-called female leadership advantage (Eagly and Carli 2003-which emphasizes the finding that women are rated slightly higher in transformational leadership compared to men). The current study seeks to explain this gender leadership gap by specifying three personality-based theoretical mechanisms that enhance transformational leadership: (a) emotional intelligence (favoring women), (b) communion (stereotypical femininity; favoring women; Hsu et al. 2021), as well as an offsetting effect of (c) agency (stereotypical masculinity; favoring men). Meta-analytic data (including original meta-analyses among the leader's ability-based emotional intelligence, transformational leadership, communion, and agency) are used to test our theorized model. Results confirm the full mediation model of female leadership advantage. Because the three unique mechanisms operate in different directions, their individual indirect effects are notable, but their cumulative indirect effect is small and near-zero. In conclusion, we emphasize incorporating emotional intelligence with other personality-based explanations of gender effects in leadership perceptions.

Intermolecular Interactions of Nucleoside Antibiotic Tunicamycin with On-Target MraYCB-TUN and Off-Target DPAGT1-TUN in the Active Sites Delineated by Quantum Mechanics/Molecular Mechanics Calculations.

Tunicamycin (TUN) is a nucleoside antibiotic with a complex structure comprising uracil, tunicamine sugar, N-acetylglucosamine (GlcNAc), and fatty acyl tail moieties. TUN, known as a canonical inhibitor, blocks vital functions of certain transmembrane protein families, for example, the insect enzyme dolichyl phosphate α-N-acetylglucosaminylphosphotransferase (DPAGT1) of Spodoptera frugiperda and the bacterial enzyme phospho-N-acetylmuramoylpentapeptide translocase (MraYCB) of Clostridium bolteae. Accurate description of protein-drug interactions has an immense impact on structure-based drug design, while the main challenge is to create proper topology and parameter entries for TUN in modeling protein-TUN interactions given the structural complexity. Starting from DPAGT1-TUN and MraYCB-TUN crystal structures, we first sketched these structural complexes on the basis of the CHARMM36 force field and optimized each of them using quantum mechanics/molecular mechanics (QM/MM) calculations. By continuing calculations on the active site (QM region) of each optimized structure, we specified the characteristics of intermolecular interactions contributing to the binding of TUN to each active site by quantum theory of atoms in molecules (QTAIM) and natural bond orbital (NBO) analyses at the M06-2X/6-31G** level. The results outlined that TUN insertion into each active site requires multiple weak, moderate, and strong hydrogen bonds accompanying charge-dipole, dipole-dipole, and hydrophobic interactions among different TUN moieties and adjacent residues. The water-mediated interactions also play central roles in situating the uracil and tunicamine moieties of TUN within the DPAGT1 active site as well as in preserving the uracil-binding pocket in the MraYCB active site. The TUN binds more strongly to DPAGT1 than to MraYCB. The information garnered here is valuable particularly for better understanding mode of action at the molecular level, as it is conducive to developing next generations of nucleoside antibiotics.

Insights into the long-term fates and impacts of polybrominated diphenyl ethers in sediment samples in Taiwan: The national project for background monitoring of the environmental distribution of chemical substances (BMECs).

This study aimed to evaluate the impact factors and effectiveness of management policies on the presence of polybrominated diphenyl ethers (PBDEs) in sediment samples in Taiwan from the last 10 years. Twenty-four PBDE congeners were detected in 838 sediment samples collected from 4 stages (2006-2019) in 30 principal rivers, based on the national project for background monitoring of the environmental distribution of chemical substances. The ΣPBDE concentrations in the 4 stages ranged from 30.00 to 147.10 ng/g dw, 6.03-15.30 ng/g dw, 4.99-7.00 ng/g dw, and 1.20-2.10 ng/g dw in the northern, southern, central, and eastern areas, respectively. The concentrations of PBDEs (e.g., penta-BDE and octa-BDE) in sediment samples notably decreased (-6 to -73%) as the Taiwan Environmental Protection Administration implemented policies banning PBDEs (except deca-BDE). The PBDEs levels of the sediment samples collected in the dry season were higher than those collected in the wet season. The levels of ΣPBDEs in sediment samples were affected by season, the amount of general waste present, and nearby PBDE-related factories and e-waste recycling facilities. Reducing the release of PBDEs, especially deca-BDE, through sound waste management and recycling practices is still needed to improve environmental sustainability in Taiwan.

KasQ an Epimerase Primes the Biosynthesis of Aminoglycoside Antibiotic Kasugamycin and KasF/H Acetyltransferases Inactivate Its Activity.

Kasugamycin (KSM), an aminoglycoside antibiotic, is composed of three chemical moieties: D-chiro-inositol, kasugamine and glycine imine. Despite being discovered more than 50 years ago, the biosynthetic pathway of KSM remains an unresolved puzzle. Here we report a structural and functional analysis for an epimerase, KasQ, that primes KSM biosynthesis rather than the previously proposed KasF/H, which instead acts as an acetyltransferase, inactivating KSM. Our biochemical and biophysical analysis determined that KasQ converts UDP-GlcNAc to UDP-ManNAc as the initial step in the biosynthetic pathway. The isotope-feeding study further confirmed that 13C, 15N-glucosamine/UDP-GlcNH2 rather than glucose/UDP-Glc serves as the direct precursor for the formation of KSM. Both KasF and KasH were proposed, respectively, converting UDP-GlcNH2 and KSM to UDP-GlcNAc and 2-N'-acetyl KSM. Experimentally, KasF is unable to do so; both KasF and KasH are instead KSM-modifying enzymes, while the latter is more specific and reactive than the former in terms of the extent of resistance. The information gained here lays the foundation for mapping out the complete KSM biosynthetic pathway.

Dual-Mechanism Confers Self-Resistance to the Antituberculosis Antibiotic Capreomycin.

Capreomycin (CMN) is an important second-line antituberculosis antibiotic isolated from Saccharothrix mutabilis subspecies capreolus. The gene cluster for CMN biosynthesis has been identified and sequenced, wherein the cph gene was annotated as a phosphotransferase likely engaging in self-resistance. Previous studies reported that Cph inactivates two CMNs, CMN IA and IIA, by phosphorylation. We, herein, report that (1) Escherichia coli harboring the cph gene becomes resistant to both CMN IIA and IIB, (2) phylogenetic analysis regroups Cph to a new clade in the phosphotransferase protein family, (3) Cph shares a three-dimensional structure akin to the aminoglycoside phosphotransferases with a high binding affinity (KD) to both CMN IIA and IIB at micromolar levels, and (4) Cph utilizes either ATP or GTP as a phosphate group donor transferring its γ-phosphate to the hydroxyl group of CMN IIA. Until now, Cph and Vph (viomycin phosphotransferase) are the only two known enzymes inactivating peptide-based antibiotics through phosphorylation. Our biochemical characterization and structural determination conclude that Cph confers the gene-carrying species resistance to CMN by means of either chemical modification or physical sequestration, a naturally manifested belt and braces strategy. These findings add a new chapter into the self-resistance of bioactive natural products, which is often overlooked while designing new bioactive molecules.

ß-Hydroxylation of α-amino-ß-hydroxylbutanoyl-glycyluridine catalyzed by a nonheme hydroxylase ensures the maturation of caprazamycin.

Caprazamycin is a nucleoside antibiotic that inhibits phospho-N-acetylmuramyl-pentapeptide translocase (MraY). The biosynthesis of nucleoside antibiotics has been studied but is still far from completion. The present study characterized enzymes Cpz10, Cpz15, Cpz27, Mur17, Mur23 out of caprazamycin/muraymycin biosynthetic gene cluster, particularly the nonheme αKG-dependent enzyme Cpz10. Cpz15 is a ß-hydroxylase converting uridine mono-phosphate to uridine 5' aldehyde, then incorporating with threonine by Mur17 (Cpz14) to form 5'-C-glycyluridine. Cpz10 hydroxylates synthetic 11 to 12 in vitro. Major product 13 derived from mutant Δcpz10 is phosphorylated by Cpz27. ß-Hydroxylation of 11 by Cpz10 permits the maturation of caprazamycin, but decarboxylation of 11 by Mur23 oriented to muraymycin formation. Cpz10 recruits two iron atoms to activate dioxygen with regio-/stereo-specificity and commit electron/charge transfer, respectively. The chemo-physical interrogations should greatly advance our understanding of caprazamycin biosynthesis, which is conducive to pathway/protein engineering for developing more effective nucleoside antibiotics.

Structural and chemical trapping of flavin-oxide intermediates reveals substrate-directed reaction multiplicity.

Though reactive flavin-N5/C4α-oxide intermediates can be spectroscopically profiled for some flavin-assisted enzymatic reactions, their exact chemical configurations are hardly visualized. Structural systems biology and stable isotopic labelling techniques were exploited to correct this stereotypical view. Three transition-like complexes, the α-ketoacid…N5-FMNox complex (I), the FMNox -N5-aloxyl-C'α- -C4α+ zwitterion (II), and the FMN-N5-ethenol-N5-C4α-epoxide (III), were determined from mandelate oxidase (Hmo) or its mutant Y128F (monooxygenase) crystals soaked with monofluoropyruvate (a product mimic), establishing that N5 of FMNox an alternative reaction center can polarize to an ylide-like mesomer in the active site. In contrast, four distinct flavin-C4α-oxide adducts (IV-VII) from Y128F crystals soaked with selected substrates materialize C4α of FMN an intrinsic reaction center, witnessing oxidation, Baeyer-Villiger/peroxide-assisted decarboxylation, and epoxidation reactions. In conjunction with stopped-flow kinetics, the multifaceted flavin-dependent reaction continuum is physically dissected at molecular level for the first time.

Microplastics and their affiliated PAHs in the sea surface connected to the southwest coast of Taiwan.

Contamination by microplastics (MPs) and the associated organic pollutants has caused potential threats to the ecological environment of global waters. In this study, MPs were sampled by trawling from the surface waters of the estuary, fishing port entrance and harbor entrance areas connected to the southwestern coast of Taiwan. Moreover, the abundance, morphological characteristics, composition, and associated polycyclic aromatic hydrocarbons (PAHs) of MPs were analyzed. The abundance of MPs was 0.36 ± 0.21 items/m3, which was 6.4 ± 10.7% of the abundance of zooplanktons. The average abundance of MPs was the highest in the estuary area, indicating that river transport was the primary way for MPs to enter the ocean. The most dominant MPs were small (0.33-2 mm; 78.8 ± 8.1%), colored (60.0 ± 12.8%), fragments (66.1 ± 10.6%), comprising PE (52.6 ± 7.6%), and PP (38.7 ± 9.4%). The decomposition of various plastic products and disposable plastic packaging may be the most significant source. The total concentration of PAHs in MPs ranged from 104 to 3595 ng/g dw, with an average of 818 ± 874 ng/g dw. The diagnostic ratios and the results of principal component analysis (PCA) and multiple linear regression of the absolute principal component scores (MLR-APCS) indicated that the PAHs were mainly contributed from sources related to petrogenic (71.4%) and vehicles (28.6%). Most likely due to MPs on the sea surface coming into contact with floating oil spills from ships or floating tar particles.

Sonogenetic Modulation of Cellular Activities Using an Engineered Auditory-Sensing Protein.

Biomolecules that respond to different external stimuli enable the remote control of genetically modified cells. We report herein a sonogenetic approach that can manipulate target cell activities by focused ultrasound stimulation. This system requires an ultrasound-responsive protein derived from an engineered auditory-sensing protein prestin. Heterologous expression of mouse prestin containing two parallel amino acid substitutions, N7T and N308S, that frequently exist in prestins from echolocating species endowed transfected mammalian cells with the ability to sense ultrasound. An ultrasound pulse of low frequency and low pressure efficiently evoked cellular calcium responses after transfecting with prestin(N7T, N308S). Moreover, pulsed ultrasound can also noninvasively stimulate target neurons expressing prestin(N7T, N308S) in deep regions of mouse brains. Our study delineates how an engineered auditory-sensing protein can cause mammalian cells to sense ultrasound stimulation. Moreover, our sonogenetic tools will serve as new strategies for noninvasive therapy in deep tissues.

Puf6 and Loc1 Are the Dedicated Chaperones of Ribosomal Protein Rpl43 in Saccharomyces cerevisiae.

Ribosomal proteins are highly expressed, and the quality of ribosomal proteins must be rigorously controlled to build up a functional ribosome. Rpl43, ribosomal protein large subunit 43, is located nearby the E-site of ribosomes. In our previous study, we found that Puf6, Loc1, and Rpl43 form a trimeric complex in Saccharomyces cerevisiae. Rpl43 protein levels are under-accumulated in the absence of PUF6 or LOC1. However, why the loss of Puf6 or Loc1 decreased the protein levels of Rpl43 remained unclear. In the present study, we further dissected the connections among these three proteins and found that the processing defects of pre-ribosomal RNA in puf6Δ and loc1Δ are similar to those of the mutant with depletion of Rpl43. The stability of newly synthesized Rpl43 protein decreased slightly in puf6Δ and significantly in loc1Δ. We also found that Puf6 and Loc1 could interact with nascent Rpl43 co-translationally via the N-terminus of Rpl43. While the association and dissociation of Rpl43 with karyopherins did not depend on Puf6 and Loc1, Puf6 and Loc1 interacted with nascent Rpl43 in collaboration. While the N-terminus of Puf6 contained nuclear localization signals for transport, the PUF (Pumilio) domain was essential to interaction with Loc1, Rpl43, and 60S subunits. The C-terminus of Loc1 is more important for interaction with Puf6 and Rpl43. In this study, we found that Puf6 and Loc1 are the dedicated chaperones of ribosomal protein Rpl43 and also analyzed the potential interaction domains among the three proteins. Correct formation of the Puf6, Loc1, and Rpl43 ternary complex is required to properly proceed to the next step in 60S biogenesis.

The flavin mononucleotide cofactor in α-hydroxyacid oxidases exerts its electrophilic/nucleophilic duality in control of the substrate-oxidation level.

The Y128F single mutant of p-hydroxymandelate oxidase (Hmo) is capable of oxidizing mandelate to benzoate via a four-electron oxidative decarboxylation reaction. When benzoylformate (the product of the first two-electron oxidation) and hydrogen peroxide (an oxidant) were used as substrates the reaction did not proceed, suggesting that free hydrogen peroxide is not the committed oxidant in the second two-electron oxidation. How the flavin mononucleotide (FMN)-dependent four-electron oxidation reaction takes place remains elusive. Structural and biochemical explorations have shed new light on this issue. 15 high-resolution crystal structures of Hmo and its mutants liganded with or without a substrate reveal that oxidized FMN (FMNox) possesses a previously unknown electrophilic/nucleophilic duality. In the Y128F mutant the active-site perturbation ensemble facilitates the polarization of FMNox to a nucleophilic ylide, which is in a position to act on an α-ketoacid, forming an N5-acyl-FMNred dead-end adduct. In four-electron oxidation, an intramolecular disproportionation reaction via an N5-alkanol-FMNred C'α carbanion intermediate may account for the ThDP/PLP/NADPH-independent oxidative decarboxylation reaction. A synthetic 5-deaza-FMNox cofactor in combination with an α-hydroxyamide or α-ketoamide biochemically and structurally supports the proposed mechanism.

Biochemical and structural explorations of α-hydroxyacid oxidases reveal a four-electron oxidative decarboxylation reaction.

p-Hydroxymandelate oxidase (Hmo) is a flavin mononucleotide (FMN)-dependent enzyme that oxidizes mandelate to benzoylformate. How the FMN-dependent oxidation is executed by Hmo remains unclear at the molecular level. A continuum of snapshots from crystal structures of Hmo and its mutants in complex with physiological/nonphysiological substrates, products and inhibitors provides a rationale for its substrate enantioselectivity/promiscuity, its active-site geometry/reactivity and its direct hydride-transfer mechanism. A single mutant, Y128F, that extends the two-electron oxidation reaction to a four-electron oxidative decarboxylation reaction was unexpectedly observed. Biochemical and structural approaches, including biochemistry, kinetics, stable isotope labeling and X-ray crystallography, were exploited to reach these conclusions and provide additional insights.

Effects of an iPad-based Speech-Generating Device Infused into Instruction with the Picture Exchange Communication System for Adolescents and Young Adults with Severe Autism Spectrum Disorder.

This study used a multiple baseline, single-subject research design to investigate the efficacy of an iPad®-based speech-generating device (SGD). The iPad was equipped with the SPEAKall!® application to function as a SGD. SGDs are a form of aided augmentative and alternative communication (AAC) allowing a user to communicate using digitized and/or synthesized speech. Instruction followed a modified version of the intervention phases from the Picture Exchange Communication System (PECS). This modified PECS protocol was implemented with two adolescents and one young adult between the ages of 14 and 23. All three participants were diagnosed with severe autism spectrum disorder and little to no functional speech. Dependent measures included the ability to request for edible and tangible items as the primary measure, and the ability to engage in natural speech production as an ancillary measure to determine simultaneous, additive effects on speech acquisition. Results indicated increases in requesting behaviors for all three participants across intervention and maintenance phases. Once participants mastered requesting of edible items, they were able to generalize the skill to tangible items. However, mixed results were found when targeting natural speech production. Based on the current findings, the infusion of an iPad-based SGD into PECS instruction may be effective in increasing initial requesting skills; however, a facilitative effect on increasing speech acquisition cannot necessarily be expected for every participant.

Teicoplanin Reprogrammed with the N-Acyl-Glucosamine Pharmacophore at the Penultimate Residue of Aglycone Acquires Broad-Spectrum Antimicrobial Activities Effectively Killing Gram-Positive and -Negative Pathogens.

Lipoglycopeptide antibiotics, for example, teicoplanin (Tei) and A40926, are more potent than vancomycin against Gram-positive (Gram-(+)) drug-resistant pathogens, for example, methicillin-resistant Staphylococcus aureus (MRSA). To extend their therapeutic effectiveness on vancomycin-resistant S. aureus (VRSA), the biosynthetic pathway of the N-acyl glucosamine (Glc) pharmacophore at residue 4 (r4) of teicoplanin pseudoaglycone redirection to residue 6 (r6) was attempted. On the basis of crystal structures, two regioselective biocatalysts Orf2*T (a triple-mutation mutant S98A/V121A/F193Y) and Orf11*S (a single-mutation mutant W163A) were engineered, allowing them to act on GlcNAc at r6. New analogs thereby made show marked antimicrobial activity against MRSA and VRSA by 2-3 orders of magnitude better than teicoplanin and vancomycin. The lipid side chain of the Tei-analogs armed with a terminal mono- or diguanidino group extends the antimicrobial specificity from Gram-(+) to Gram-negative (Gram-(-)), comparable to that of kanamycin. In addition to low cytotoxicity and high safety, the Tei analogs exhibit new modes of action as a result of resensitization of VRSA and Acinetobacter baumannii. The redirection of the biosynthetic pathway for the N-acyl-Glc pharmacophore from r4 to r6 bodes well for large-scale production of selected r6,Tei congeners in an environmentally friendly synthetic biology approach.

Evidence of Diradicals Involved in the Yeast Transketolase Catalyzed Keto-Transferring Reactions.

Transketolase (TK) catalyzes a reversible transfer of a two-carbon (C2 ) unit between phosphoketose donors and phosphoaldose acceptors, for which the group-transfer reaction that follows a one- or two-electron mechanism and the force that breaks the C2"-C3" bond of the ketose donors remain unresolved. Herein, we report ultrahigh-resolution crystal structures of a TK (TKps) from Pichia stipitis in previously undiscovered intermediate states and support a diradical mechanism for a reversible group-transfer reaction. In conjunction with MS, NMR spectroscopy, EPR and computational analyses, it is concluded that the enzyme-catalyzed non-Kekulé diradical cofactor brings about the C2"-C3" bond cleavage/formation for the C2 -unit transfer reaction, for which suppression of activation energy and activation and destabilization of enzymatic intermediates are facilitated.