Structural insights into a novel nonheme iron-dependent oxygenase in selenoneine biosynthesis.

Selenoneine (SEN) is a natural histidine derivative with radical-scavenging activity and shows higher antioxidant potential than its sulfur-containing isolog ergothioneine (EGT). Recently, the SEN biosynthetic pathway in Variovorax paradoxus was reported. Resembling EGT biosynthesis, the committed step of SEN synthesis is catalyzed by a nonheme Fe-dependent oxygenase termed SenA. This enzyme catalyzes oxidative carbon­selenium (C-Se) bond formation to conjugate N-α-trimethyl histidine (TMH) and selenosugar to yield selenoxide; the process parallels the EGT biosynthetic route, in which sulfoxide synthases known as EgtB members catalyze the conjugation of TMH and cysteine or γ-glutamylcysteine to afford sulfoxides. Here, we report the crystal structures of SenA and its complex with TMH and thioglucose (SGlc), an analog of selenoglucose (SeGlc) at high resolution. The overall structure of SenA adopts the archetypical fold of EgtB, which comprises a DinB-like domain and an FGE-like domain. While the TMH-binding site is highly conserved to that of EgtB, a various substrate-enzyme interaction network in the selenosugar-binding site of SenA features a number of water-mediated hydrogen bonds. The obtained structural information is beneficial for understanding the mechanism of SenA-mediated C-Se bond formation.

Remodeling the polymer-binding cavity to improve the efficacy of PBAT-degrading enzyme.

Poly(butylene adipate-co-terephthalate) (PBAT) is among the most widely applied synthetic polyesters that are utilized in the packaging and agricultural industries, but the accumulation of PBAT wastes has posed a great burden to ecosystems. Using renewable enzymes to decompose PBAT is an eco-friendly solution to tackle this problem. Recently, we demonstrated that cutinase is the most effective PBAT-degrading enzyme and that an engineered cutinase termed TfCut-DM could completely decompose PBAT film to terephthalate (TPA). Here, we report crystal structures of a variant of leaf compost cutinase in complex with soluble fragments of PBAT, including BTa and TaBTa. In the TaBTa complex, one TPA moiety was located at a polymer-binding site distal to the catalytic center that has never been experimentally validated. Intriguingly, the composition of the distal TPA-binding site shows higher diversity relative to the one proximal to the catalytic center in various cutinases. We thus modified the distal TPA-binding site of TfCut-DM and obtained variants that exhibit higher activity. Notably, the time needed to completely degrade the PBAT film to TPA was shortened to within 24 h by TfCut-DM Q132Y (5813 mol per mol protein). Taken together, the structural information regarding the substrate-binding behavior of PBAT-degrading enzymes could be useful guidance for direct enzyme engineering.

The structural and functional investigation into an unusual nitrile synthase.

The biosynthesis of neurotoxin aetokthonotoxin (AETX) that features a unique structure of pentabrominated biindole nitrile involves a first-of-its-kind nitrile synthase termed AetD, an enzyme that shares very low sequence identity to known structures and catalyzes an unprecedented mechanism. In this study, we resolve the crystal structure of AetD in complex with the substrate 5,7-di-Br-L-Trp. AetD adopts the heme oxygenase like fold and forms a hydrophobic cavity within a helical bundle to accommodate the indole moiety. A diiron cluster comprising two irons that serves as a catalytic center binds to the carboxyl O and the amino N of the substrate. Notably, we demonstrate that the AetD-catalyzed reaction is independent of the bromination of the substrate and also solved crystal structures of AetD in complex with 5-Br-L-Trp and L-Trp. Altogether, the present study reveals the substrate-binding pattern and validates the diiron cluster-comprising active center of AetD, which should provide important basis to support the mechanistic investigations into this class of nitrile synthase.

Phosphoantigens glue butyrophilin 3A1 and 2A1 to activate Vγ9Vδ2 T cells.

In both cancer and infections, diseased cells are presented to human Vγ9Vδ2 T cells through an 'inside out' signalling process whereby structurally diverse phosphoantigen (pAg) molecules are sensed by the intracellular domain of butyrophilin BTN3A11-4. Here we show how-in both humans and alpaca-multiple pAgs function as 'molecular glues' to promote heteromeric association between the intracellular domains of BTN3A1 and the structurally similar butyrophilin BTN2A1. X-ray crystallography studies visualized that engagement of BTN3A1 with pAgs forms a composite interface for direct binding to BTN2A1, with various pAg molecules each positioned at the centre of the interface and gluing the butyrophilins with distinct affinities. Our structural insights guided mutagenesis experiments that led to disruption of the intracellular BTN3A1-BTN2A1 association, abolishing pAg-mediated Vγ9Vδ2 T cell activation. Analyses using structure-based molecular-dynamics simulations, 19F-NMR investigations, chimeric receptor engineering and direct measurement of intercellular binding force revealed how pAg-mediated BTN2A1 association drives BTN3A1 intracellular fluctuations outwards in a thermodynamically favourable manner, thereby enabling BTN3A1 to push off from the BTN2A1 ectodomain to initiate T cell receptor-mediated γδ T cell activation. Practically, we harnessed the molecular-glue model for immunotherapeutics design, demonstrating chemical principles for developing both small-molecule activators and inhibitors of human γδ T cell function.

The impact of preoperative single low-dose dexamethasone on in-hospital prognosis in geriatric intertrochanteric fracture patients: Analysis of secondary outcomes in a randomized controlled trial.

BACKGROUND: Intertrochanteric fracture in the geriatric population is associated with poor prognosis, which may be attributed to consistent stress and the systemic inflammatory response. Dexamethasone is an exogenous glucocorticoid commonly used in clinical practice for broad anti-inflammatory action. The purpose is to investigate whether a single preoperative low-dose dexamethasone can improve the in-hospital prognosis in geriatric intertrochanteric fracture patients undergoing internal fixation surgery. METHODS: Between June 2020 and October 2022, 219 eligible patients with intertrochanteric fractures were in this study. After meeting the inclusion and exclusion criteria, 160 patients were randomly allocated to the dexamethasone or placebo groups (80 patients who are geriatric with an intertrochanteric fracture in each group). The patients in the dexamethasone group received 10 mg (2 mL) of dexamethasone intravenously, whereas the patients in the placebo group received 2 mL of saline intravenously within 30 minutes before being sent to the operating room. The efficacy-related outcomes (the first bed-chair transfer ability, in-hospital mortality, and length of stay) and safety-related outcomes (infection events and hyperglycemia) were collected for analysis. RESULTS: There were no significant differences in the baseline characteristics between the 2 groups. The dexamethasone group had a significantly higher rate of the first bed-chair transfer than the placebo group (65.0% [52/80] vs 48.8% [39/80], relative risk = 1.46, 95% confidence interval = 1.02 to 2.11; P = .038). One patient in the dexamethasone group and 7 patients in the placebo group died during hospitalization (1.3% [1/80] vs 8.8% [7/80], relative risk = 0.92, 95% confidence interval = 0.86 to 0.99; P = .07). No differences were found in the length of stay, infections, and hyperglycemia between the 2 groups. CONCLUSION: A single preoperative low-dose of dexamethasone can improve the in-hospital prognosis (increase the ability of the first bed-chair transfer and potentially decrease the in-hospital mortality) in geriatric intertrochanteric fracture patients after internal fixation surgery.

Cryo-EM structure and rational engineering of a superefficient ochratoxin A-detoxifying amidohydrolase.

Ochratoxin A (OTA) is among the most prevalent mycotoxins detected in agroproducts, posing serious threats to human and livestock health. Using enzymes to conduct OTA detoxification is an appealing potential strategy. The recently identified amidohydrolase from Stenotrophomonas acidaminiphila, termed ADH3, is the most efficient OTA-detoxifying enzyme reported thus far and can hydrolyze OTA to nontoxic ochratoxin α (OTα) and L-ß-phenylalanine (Phe). To elucidate the catalytic mechanism of ADH3, we solved the single-particle cryo-electron microscopy (cryo-EM) structures of apo-form, Phe- and OTA-bound ADH3 to an overall resolution of 2.5-2.7 Å. The role of OTA-binding residues was investigated by structural, mutagenesis and biochemical analyses. We also rationally engineered ADH3 and obtained variant S88E, whose catalytic activity was elevated by 3.7-fold. Structural analysis of variant S88E indicates that the E88 side chain provides additional hydrogen bond interactions to the OTα moiety. Furthermore, the OTA-hydrolytic activity of variant S88E expressed in Pichia pastoris is comparable to that of Escherichia coli-expressed enzyme, revealing the feasibility of employing the industrial yeast strain to produce ADH3 and its variants for further applications. These results unveil a wealth of information about the catalytic mechanism of ADH3-mediated OTA degradation and provide a blueprint for rational engineering of high-efficiency OTA-detoxifying machineries.

A single preoperative low-dose dexamethasone may reduce the incidence and severity of postoperative delirium in the geriatric intertrochanteric fracture patients with internal fixation surgery: an exploratory analysis of a randomized, placebo-controlled trial.

OBJECTIVE: Postoperative delirium (POD) is a common complication along with poor prognosis in geriatric intertrochanteric fracture (ITF) patients. However, the prevention and treatment of POD remain unclear. Previous studies have confirmed that POD is essentially a consequence of neuro-inflammatory responses. Dexamethasone is a glucocorticoid with comprehensive anti-inflammatory effects, while a high dose of dexamethasone correlates with many side effects or even adverse consequences. Thus, this prospective study aims to discuss whether a single preoperative low-dose dexamethasone can reduce the impact of POD on geriatric ITF patients with internal fixation surgery. METHODS: Between June 2020 and October 2022, there were 219 consecutive ITF patients assessed in our department. Of the 219 ITF patients, 160 cases who met the inclusion and exclusion criteria were finally enrolled and randomly allocated to the dexamethasone group and the placebo group (80 geriatric ITF patients in each group) in this prospective study. The patients in the dexamethasone group received intravenous 10 mg (2 ml) dexamethasone while the patients in the placebo group received intravenous 2 ml saline in 30 min before being sent to the operating room, respectively. The baseline characteristics, surgical information, incidence and severity of POD as the efficacy-related outcomes, and infection events and hyperglycemia as safety-related outcomes (adverse events), were collected and analyzed between the two groups. The severity of POD was evaluated by Memorial Delirium Assessment Scale (MDAS) score. RESULTS: There were no differences in baseline characteristics and surgical information between the dexamethasone group and the placebo group. The dexamethasone group had a lower incidence of POD than the placebo group within the first 5 days after surgery [(9/80, 11.3% vs. 21/80, 26.3%, RR = 0.83, 95% CI 0.71-0.97, P = 0.015]. The dexamethasone group had lower MDAS scores (Mean ± SD) than the placebo group [13.2 ± 1.0 (range 11 to 15) vs. 15.48 ± 2.9 (range 9 to 20), P = 0.011, effect size = 0.514]. There were no differences in infection events and hyperglycemia between the two groups. CONCLUSIONS: A single preoperative low-dose dexamethasone may reduce the incidence and severity of POD in geriatric ITF patients with internal fixation surgery. TRIAL REGISTRATION: ChiCTR2200055281.

Standardized Tip-Apex Distance (STAD): a modified individualized measurement of cephalic fixator position based on its own femoral head diameter in geriatric intertrochanteric fractures with internal fixation.

OBJECTIVE: To design a standardized Tip-Apex Distance (STAD) and analyze the clinical significance of STAD in predicting cut-out in geriatric intertrochanteric fractures with internal fixation. METHODS: Firstly, we designed STAD according to the rule of TAD. We measured the STAD individually based on its own femoral head diameter (iFHD) instead of the known diameter of the lag screw in calculating TAD, resulting in that the STAD is simply the relative quantitation relationship of iFHD (the times of iFHD). In this study, we assumed that all the iFHD was 6D (1iFHD = 6D, or 1D = 1/6 of iFHD) in order for complete match of the Cleveland zone system, easy comparison of the STAD, and convenient identification for artificial intelligence. Secondly, we calculated and recorded all the STAD of cephalic fixator in 123 eligible ITF patients. Thirdly, we grouped all the ITF patients into the Failure and Non-failure groups according to whether cut-out or not, and analyzed the correlation between the cut-out and the STAD. RESULTS: Cleveland zone, Parker's ratio (AP), TAD, and STAD were associated with the cut-out in univariate analysis. However, only STAD was the independent predictor of the cut-out by multivariate analysis. No cut-out was observed when STAD ≤ 2D (1/3 of iFHD). The Receiver Operating Characteristic (ROC) curve indicated that STAD was a reliable predictor of cut-out, and the best cut-off value of STAD was 2.92D. Cut-out rate increased dramatically when STAD increased, especially when STAD > 3D (1/2 of iFHD). CONCLUSION: Essentially, the STAD is a relative quantitation relationship of iFHD. The STAD is a reliable measurement of cephalic fixator position in predicting cut-out in geriatric ITF patients with single-screw cephalomedullary nail fixations. For avoiding cut-out, the STAD should be no more than a half of iFHD. LEVEL OF EVIDENCE: Level III, Prognostic Study.

Complete bio-degradation of poly(butylene adipate-co-terephthalate) via engineered cutinases.

Poly(butylene adipate-co-terephthalate) (PBAT), a polyester made of terephthalic acid (TPA), 1,4-butanediol, and adipic acid, is extensively utilized in plastic production and has accumulated globally as environmental waste. Biodegradation is an attractive strategy to manage PBAT, but an effective PBAT-degrading enzyme is required. Here, we demonstrate that cutinases are highly potent enzymes that can completely decompose PBAT films in 48 h. We further show that the engineered cutinases, by applying a double mutation strategy to render a more flexible substrate-binding pocket exhibit higher decomposition rates. Notably, these variants produce TPA as a major end-product, which is beneficial feature for the future recycling economy. The crystal structures of wild type and double mutation of a cutinase from Thermobifida fusca in complex with a substrate analogue are also solved, elucidating their substrate-binding modes. These structural and biochemical analyses enable us to propose the mechanism of cutinase-mediated PBAT degradation.

Functional tailoring of a PET hydrolytic enzyme expressed in Pichia pastoris.

Using enzymes to hydrolyze and recycle poly(ethylene terephthalate) (PET) is an attractive eco-friendly approach to manage the ever-increasing PET wastes, while one major challenge to realize the commercial application of enzyme-based PET degradation is to establish large-scale production methods to produce PET hydrolytic enzyme. To achieve this goal, we exploited the industrial strain Pichia pastoris to express a PET hydrolytic enzyme from Caldimonas taiwanensis termed CtPL-DM. In contrast to the protein expressed in Escherichia coli, CtPL-DM expressed in P. pastoris is inactive in PET degradation. Structural analysis indicates that a putative N-glycosylation site N181 could restrain the conformational change of a substrate-binding Trp and hamper the enzyme action. We thus constructed N181A to remove the N-glycosylation and found that the PET hydrolytic activity of this variant was restored. The performance of N181A was further enhanced via molecular engineering. These results are of valuable in terms of PET hydrolytic enzyme production in industrial strains in the future.

Eccentric distance zone analysis system: New regional evaluation of cephalic fixator tip location for predicting cut-out in geriatric intertrochanteric fractures with internal fixation.

Objective: The aim of this study was to investigate an eccentric distance (ED) zone analysis system for regional evaluation of the cephalic fixator tip based on the ED of the cephalic fixator tip referenced to the radius of its own femoral head to predict cut-out in intertrochanteric fractures (ITF) with internal fixation. Methods: First, we assumed all the femoral heads were regular spheres with the radius (R FD) of "3" for a complete match of the Cleveland zone system and calculated the ED of the cephalic fixator tip by measuring the distances from the cephalic fixator tip to the geometric central axis in the femoral neck and head on both anteroposterior (AP) view and lateral view radiographs. Second, we defined the maximum transverse section of the femoral head into three zones named ED Zone A with ED less than "1," Zone B with ED ranging in "1-2," and Zone C with ED ranging in "2-3" in turns by concentric circles (circles A, B, and C) with the radius of 1/3, 2/3, and 3/3 times of R FD, respectively. Third, we evaluated the ED zones according to the ED and location of the cephalic fixator tip in the eligible 123 ITF patients with single-screw cephalomedullary nail (SCMN) fixation and then analyzed the correlation between the cut-out rate and the ED zones. Results: The cut-out rates in ED Zones A, B, and C were 4.17%, 38.46%, and 100%, respectively. Multivariate logistic regression indicated that ED Zone A had at least a 14 times lower rate of cut-out compared with ED Zone B. The cephalic fixator tip located in ED Zone A has a lower cut-out rate than that in Cleveland Zone 5. The cut-out rate in ED Zone A is significantly lower than that in the region inside Cleveland Zone 5 but outside ED Zone A. Conclusion: ED zone analysis system is a reliable regional evaluation of the cephalic fixator tip position for predicting cut-out in geriatric ITF patients with SCMN fixations and potentially an artificial intelligence measurement during surgery. For decreasing the cut-out rate, the cephalic fixator tip should be located in ED Zone A.

Surgical Position of Lateral-Tilted Supine is Suitable for Proximal Humeral Fracture Operations in Geriatric Patients.

Objective: To report a new surgical position of lateral-tilted supine (LTS) for geriatric proximal humeral fracture operations. Methods: Between January 2016 and December 2020, we adopted the LTS position for operations in 65 geriatric patients with proximal humeral fractures. Results: Sixty-five patients including 25 males and 40 females aged 80.3 ± 8.5 years. The LTS position could be used for almost all proximal humeral fracture surgeries, such as ORIF with plate, suture anchor, and other fixation in 4 patients, open reduction and internal fixation (ORIF) with multiLoc nailing in 48, and shoulder hemiarthroplasty (SHA) in 13. Surgical position setting times were 11.47 ± 2.14 min. The systolic blood pressure changes before and after positioning were 15.07 ± 8.72 mmHg. All of the C-arm X-ray directions, including the cephalic side, contralateral side, and ipsilateral side, can be used in the LTS position surgeries. No surgical complications or no surgical position-related complications were found in these 65 cases. Conclusion: The surgical position of LTS is suitable for geriatric proximal humeral fracture operations.

Structure-based rational design of a short-chain dehydrogenase/reductase for improving activity toward mycotoxin patulin.

Patulin is a fatal mycotoxin that is widely detected in drinking water and fruit-derived products contaminated by diverse filamentous fungi. CgSDR from Candida guilliermondii represents the first NADPH-dependent short-chain dehydrogenase/reductase that catalyzes the reduction of patulin to the nontoxic E-ascladiol. To elucidate the catalytic mechanism of CgSDR, we solved its crystal structure in complex with cofactor and substrate. Structural analyses indicate that patulin is situated in a hydrophobic pocket adjacent to the cofactor, with the hemiacetal ring orienting toward the nicotinamide moiety of NADPH. In addition, we conducted structure-guided engineering to modify substrate-binding residue V187 and obtained variant V187F, V187K and V187W, whose catalytic activity was elevated by 3.9-, 2.2- and 1.7-fold, respectively. The crystal structures of CgSDR variants suggest that introducing additional aromatic stacking or hydrogen-bonding interactions to bind the lactone ring of patulin might account for the observed enhanced activity. These results illustrate the catalytic mechanism of SDR-mediated patulin detoxification for the first time and provide the upgraded variants that exhibit tremendous potentials in industrial applications.

Structural analysis and engineering of aldo-keto reductase from glyphosate-resistant Echinochloa colona.

Glyphosate is a dominant organophosphate herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) of the shikimate pathway. Glyphosate is extensively applied since manufactured, which has led to the emergence of various glyphosate-resistant crops and weeds. However, the molecular mechanism of many glyphosate-resistance machineries remains unclear. Recently, the upregulated expression of two homologous aldo-keto reductases (AKRs), designated as AKR4C16 and AKR4C17, were found to contribute to the glyphosate resistance in Echinochloa colona. This represents the first naturally evolved glyphosate-degrading machinery reported in plants. Here, we report the three-dimensional structure of these two AKR enzymes in complex with cofactor by performing X-ray crystallography. Furthermore, the binding-mode of glyphosate were elucidated in a ternary complex of AKR4C17. Based on the structural information and the previous study, we proposed a possible mechanism of action of AKR-mediated glyphosate degradation. In addition, a variant F291D of AKR4C17 that was constructed based on structure-based engineering showed a 70% increase in glyphosate degradation. In conclusion, these results demonstrate the structural features and glyphosate-binding mode of AKR4C17, which increases our understanding of the enzymatic mechanism of glyphosate bio-degradation and provides an important basis for the designation of AKR-based glyphosate-resistance for further applications.

Structural insights to a bi-functional isoprenyl diphosphate synthase that can catalyze head-to-tail and head-to-middle condensation.

Isoprenoids represent the largest group of natural products, whose basal skeletons are synthesized by various isoprenyl diphosphate synthases (IDSs). As majority of IDSs catalyze head-to-tail reaction to produce linear form isoprenoids, some catalyze head-to-middle reaction to produce branched form products. In a previous study, an IDS termed MA1831 from Methanosarcina acetivorans was found to be capable of catalyzing both types of reaction. In addition to the canonical linear product of C35 in length, MA1831 also catalyzes head-to-middle condensation of farnesyl diphosphate (FPP) and dimethylallyl diphosphate (DMAPP) to produce geranyllavandulyl diphosphate. In order to investigate the mechanism of action of MA1831, we determined its crystal structures in apo-form and in complex with substrates and analogues. The complex structures that contain isopentenyl S-thiolodiphosphate and DMAPP as homoallylic substrates were also reported, which should represent the reaction modes of MA1831-mediated head-to-tail and head-to-middle reaction, respectively. Based on the structural information, the mechanism of MA1831 catalyze head-to-tail and head-to-middle condensation reaction was proposed.

Bibliometric Analysis of γδ T Cells as Immune Regulators in Cancer Prognosis.

γδ T cells are one of only three immune cell types that express antigen receptors that undergo somatic recombination, and they contribute to immune responses to infection, cellular transformation, and tissue damage. As a "bridge" between the innate and adaptive immune systems, γδ T cells have been noted to be involved in various immune responses during cancer progression. The purpose of our study was to review current published information on γδ T cells and investigate their functions in different types of malignancy using bibliometric and bioinformatic methods. Our results indicated that studies on γδ T cells and cancer progression increased from 2014, and the number had peaked by 2021. We discovered that there is international cooperation in the performance of studies among 26 countries, where China was identified as the most productive with the highest citations. Using keyword co-occurrence analysis, we found that among all the cancer types investigated, gastric and breast cancers were most closely related to γδ T cells. Furthermore, interleukin (IL)-17 and IL-2 were the most common cytokines linked to γδ T cells and our investigation of their potential involvement in the prognosis of gastric and breast cancers, identified their different roles in various malignancies. Thus, we concluded that γδ T cells might influence the progression of different cancers in diverse ways.

The correlation between cutout and eccentric distance (ED) of the cephalic fixator tip in geriatric intertrochanteric fractures with internal fixation.

BACKGROUND: The location of cephalic fixator tip with different eccentric distance (ED) should have different risks of cutout. This study aims to evaluate the cephalic fixator tip position by measuring ED of the cephalic fixator tip in geriatric ITF patients with single-screw cephalomedullary nail (SCMN) fixation and analyze the correlation between the cutout and the ED. METHODS: Firstly, we assumed all the femoral head was a regular sphere and standardized the radius of the femoral head (RFD) as "3" no matter how big the RFD was for complete match of the Cleveland zone system and convenient identification of artificial intelligence. Secondly, we measured the ED of the cephalic fixator tip by calculating the distances from the cephalic fixator tip to the geometric central axis of the femoral neck and head on both AP view and lateral view radiographs. Thirdly, we evaluated all the ED of the cephalic fixator tip in the eligible 123 geriatric ITF patients and analyzed the correlation between the cutout and the ED. RESULTS: The ED in cutout group (1.25 ± 0.43) is much bigger than that in non-cutout group (0.64 ± 0.34) with significant difference (OR = 50.01, 95% CI 8.42-297.19, p < 0.001). The probability of cutout increased with ED increasing, especially when "ED ≥ 1." The best cutoff value of ED for predicting cutout was "1.022" ("1.022" was just a little bit more than 1/3 times of RFD because "RFD = 3," sensitivity = 73.3%, specificity = 86.1%, and AUC = 0.867, p < 0.001). CONCLUSION: ED is suitable for evaluation of the cephalic fixator tip position for predicting cutout in geriatric ITF patients with SCMN fixation, and ED can potentially be used as artificial intelligence application during surgery. The smaller the ED, the lower the cutout rate. For avoiding cutout, the ED of the cephalic fixator tip should be less than one-third times of the radius of the femoral head.

Structural insights into the cyclization of unusual brasilane-type sesquiterpenes.

The biosynthesis of brasilane-type sesquiterpenoids (BTSs) attracts much attention owing to their unique skeleton of 5/6 bicyclic structure that contains five Me groups. Here, the crystal structures of a BTS cyclase TaTC6 from Trichoderma atroviride FKI-3849 and its complexes with farnesyl pyrophosphate (FPP) and analogue were reported. These structural information reveal that TaTC6 exploits a hydrophobic pocket to constrain the hydrocarbon region of FPP in a "U-shape" to facilitate the initial C1-C11 bond formation after pyrophosphate ionization. Following, four carbocations of reaction intermediates were molecularly docked into the hydrophobic pocket to reveal critical residues involved in the cyclization cascade. Finally, an S239-stabilized water molecule that is 3.9 Å away from the C8 of the last allyl cation may conduct hydration to quench the reaction cascade. Mutating S239 to alanine led to ca. 40% reduction in activity compared with the wild-type enzyme. The conservation of the residues that constitute the hydrophobic pocket is also discussed. Overall, this study will give an insight into the mechanism of how the active site of STCs constrain the conformation of the flexible FPP and series allylic carbocations for the complicated-ring formation and unusual carbon rearrangement in the biosynthesis of BTSs.

Structural and Functional Insights into a Nonheme Iron- and α-Ketoglutarate-Dependent Halogenase That Catalyzes Chlorination of Nucleotide Substrates.

Nonheme iron- and α-ketoglutarate (αKG)-dependent halogenases (NHFeHals), which catalyze the regio- and stereoselective halogenation of the unactivated C(sp3)-H bonds, exhibit tremendous potential in the challenging asymmetric halogenation. AdeV from Actinomadura sp. ATCC 39365 is the first identified carrier protein-free NHFeHal that catalyzes the chlorination of nucleotide 2'-deoxyadenosine-5'-monophosphate (2'-dAMP) to afford 2'-chloro-2'-deoxyadenosine-5'-monophosphate. Here, we determined the complex crystal structures of AdeV/FeII/Cl and AdeV/FeII/Cl/αKG at resolutions of 1.76 and 1.74 Å, respectively. AdeV possesses a typical ß-sandwich topology with H194, H252, αKG, chloride, and one water molecule coordinating FeII in the active site. Molecular docking, mutagenesis, and biochemical analyses reveal that the hydrophobic interactions and hydrogen bond network between the substrate-binding pocket and the adenine, deoxyribose, and phosphate moieties of 2'-dAMP are essential for substrate recognition. Residues H111, R177, and H192 might play important roles in the second-sphere interactions that control reaction partitioning. This study provides valuable insights into the catalytic selectivity of AdeV and will facilitate the rational engineering of AdeV and other NHFeHals for synthesis of halogenated nucleotides. IMPORTANCE Halogenated nucleotides are a group of important antibiotics and are clinically used as antiviral and anticancer drugs. AdeV is the first carrier protein-independent nonheme iron- and α-ketoglutarate (αKG)-dependent halogenase (NHFeHal) that can selectively halogenate nucleotides and exhibits restricted substrate specificity toward several 2'-dAMP analogues. Here, we determined the complex crystal structures of AdeV/FeII/Cl and AdeV/FeII/Cl/αKG. Molecular docking, mutagenesis, and biochemical analyses provide important insights into the catalytic selectivity of AdeV. This study will facilitate the rational engineering of AdeV and other carrier protein-independent NHFeHals for synthesis of halogenated nucleotides.

Risk factors for cut-outs in geriatric intertrochanteric fractures with cephalomedullary nailing after obtaining acceptable reduction: a case-control study.

BACKGROUND: It is irresponsible if we disregard reduction quality to talk about cut-outs in intertrochanteric fractures (ITF) with internal fixation. The aim of this study is to analyze the risk-factors for cut-outs in geriatric ITF with cephalomedullary nailing after obtaining acceptable reduction. METHODS: In order to investigate the risk-factors for cut-outs in geriatric ITF after obtaining acceptable reduction, we retrospectively reviewed 367 patients who underwent cephalomedullary nail for ITF in our department between September 2016 and December 2021. Potential variables including demographic data and radiological parameters (namely the fracture type, Singh index, lateral wall fracture, cephalic nail position, Parker's ratio index, tip-apex-distance (TAD), and calcar-referenced TAD (CalTAD)) were collected. Logistic regression analysis was performed to identify the significant risk factors for cut-outs. RESULTS: One hundred twenty-one patients were suitable for this study. Of the 121 cases, nine cases (7.4%) were observed with cut-out or pending cut-out. We found that Age (adjusted odds ratio (OR) 1.158, 95% confidence interval (CI) 1.016 to 1.318, p = 0.028), lateral wall fracture (adjusted OR 11.07, 95%CI 1.790 to 68.380, p = 0.01), and CalTAD (adjusted OR 1.277, 95%CI 1.005 to 1.622, p = 0.045) were independent risk-factors for cut-outs. CONCLUSIONS: Age, lateral wall fracture and CalTAD are independent risk-factors for cut-outs in geriatric ITF with cephalomedullary nailing after obtaining acceptable reduction. In order to avoid cut-outs, an optimal CalTAD is necessary even obtaining acceptable reduction, especially in the over-aged patients with lateral wall fracture.