Development and Validation of a Clinical Prediction Model for Diagnosing Mycoplasma Infections in Gynecological Patients.

BACKGROUND: In adult females, mycoplasma infection is common and challenging to diagnose. This study aimed to use retrospective laboratory data to construct a nomogram for predicting the mycoplasma infection of individuals with probable urogenital tract mycoplasma infection. METHODS: A total of 2,859 patients with suspected urogenital tract mycoplasma infection were retrospectively enrolled in this study. Demographics and routine examinations of leucorrhea were used to develop a nomogram for predicting mycoplasma infection. The least absolute shrinkage and selection operator (LASSO) method was applied to filter variables and select predictors, and multivariable logistic regression was used to construct a nomogram. The discriminatory ability of the model was determined by calculating the area under the curve (AUC). The performance and clinical utility of the nomogram were generated by using Harrell's concordance index, calibration curve, and decision curve analysis (DCA). RESULTS: By using the LASSO regression method, seven variables (age, white blood cell, epithetical cell, cleanliness, candidiasis vaginalis, sialidases, and leukocyte esterase) were chosen, and a nomogram was constructed using these variables. The prediction nomogram (0.676, 95% CI: 0.611 - 0.744) demonstrated a satisfactory performance. The prediction model's AUC was 0.679 (95% CI: 0.660 - 0.691). Furthermore, the DCA showed a good clinical net benefit based on the mycoplasma infection nomogram. CONCLUSIONS: A nomogram was created in this study, which included seven demographic and clinical characteristics of female patients. The nomogram could be of great value for the diagnosis of mycoplasma infection.

Identification and biophysical characterization of potential phytochemical inhibitors of carboxyl/choline esterase from Helicoverpa armigera for advancing integrated pest management strategies.

In the realm of disease vectors and agricultural pest management, insecticides play a crucial role in preserving global health and ensuring food security. The pervasive use, particularly of organophosphates (OPs), has given rise to a substantial challenge in the form of insecticide resistance. Carboxylesterases emerge as key contributors to OP resistance, owing to their ability to sequester or hydrolyze these chemicals. Consequently, carboxylesterase enzymes become attractive targets for the development of novel insecticides. Inhibiting these enzymes holds the potential to restore the efficacy of OPs against which resistance has developed. This study aimed to screen the FooDB library to identify potent inhibitory compounds targeting carboxylesterase, Ha006a from the agricultural pest Helicoverpa armigera. The ultimate objective is to develop effective interventions for pest control. The compounds with the highest scores underwent evaluation through docking studies and pharmacophore analysis. Among them, four phytochemicals-donepezil, protopine, 3',4',5,7-tetramethoxyflavone, and piperine-demonstrated favorable binding affinity. The Ha006a-ligand complexes were subsequently validated through molecular dynamics simulations. Biochemical analysis, encompassing determination of IC50 values, complemented by analysis of thermostability through Differential Scanning Calorimetry and interaction kinetics through Isothermal Titration Calorimetry was conducted. This study comprehensively characterizes Ha006a-ligand complexes through bioinformatics, biochemical, and biophysical methods. This investigation highlights 3',4',5,7-tetramethoxyflavone as the most effective inhibitor, suggesting its potential for synergistic testing with OPs. Consequently, these inhibitors offer a promising solution to OP resistance and address environmental concerns associated with excessive insecticide usage, enabling a significant reduction in their overuse.

Increased serum carboxylesterase-1 levels are associated with metabolic dysfunction associated steatotic liver disease and metabolic syndrome in children with obesity.

BACKGROUND: Carboxylesterase 1(CES1) is expressed mainly in the liver and adipose tissue and is highly hypothesized to play an essential role in metabolism. Our study aimed to investigate the association between CES1 and metabolic syndrome (MetS) and metabolic dysfunction associated steatotic liver disease (MASLD) in children with obesity in China. METHODS: This study included 72 children with obesity aged 6-13years (including 25(35%) diagnosed as MetS and 36(50%) diagnosed as MASLD). All subjects were measured in anthropometry, serum level of biochemical parameters related to obesity, circumstance levels of insulin-like growth factor1, adipokines (adiponectin, leptin and growth differentiation factor 15) and CES1. RESULTS: Higher serum CES1 level were found in the MetS group (P = 0.004) and the MASLD group (P < 0.001) of children with obesity. Serum CES1 levels were positively correlated with alanine aminotransferase, aspartate aminotransferase, triglyceride, cholesterol, low-density lipoprotein cholesterol, GDF15, Leptin and negatively correlated with high-density lipoprotein cholesterol, adiponectin and IGF1. We also found a multivariable logistic regression analysis of MASLD and MetS predicted by CES1 significantly (MASLD P < 0.01, MetS P < 0.05). The combination of CES1, sex, age and BMI Z-score showed a sensitivity and specificity of 92.7% for the identification of MASLD and 78.6% for the identification of MetS. The cutoff for CES1 of MASLD is 56.30 ng/mL and of MetS is 97.79 ng/mL. CONCLUSIONS: CES1 is associated with an increasing risk of MetS and MASLD and can be established as a biomarker for metabolic syndrome and MASLD of children with obesity.

An Exploratory Study of the Enzymatic Hydroxycinnamoylation of Sucrose and Its Derivatives.

Phenylpropanoid sucrose esters are a large and important group of natural substances with significant therapeutic potential. This work describes a pilot study of the enzymatic hydroxycinnamoylation of sucrose and its derivatives which was carried out with the aim of obtaining precursors of natural phenylpropanoid sucrose esters, e.g., vanicoside B. In addition to sucrose, some chemically prepared sucrose acetonides and substituted 3'-O-cinnamates were subjected to enzymatic transesterification with vinyl esters of coumaric, ferulic and 3,4,5-trimethoxycinnamic acid. Commercial enzyme preparations of Lipozyme TL IM lipase and Pentopan 500 BG exhibiting feruloyl esterase activity were tested as biocatalysts in these reactions. The substrate specificity of the used biocatalysts for the donor and acceptor as well as the regioselectivity of the reactions were evaluated and discussed. Surprisingly, Lipozyme TL IM catalyzed the cinnamoylation of sucrose derivatives more to the 1'-OH and 4'-OH positions than to the 6'-OH when the 3'-OH was free and the 6-OH was blocked by isopropylidene. In this case, Pentopan reacted comparably to 1'-OH and 6'-OH positions. If sucrose 3'-O-coumarate was used as an acceptor, in the case of feruloylation with Lipozyme in CH3CN, 6-O-ferulate was the main product (63%). Pentopan feruloylated sucrose 3'-O-coumarate comparably well at the 6-OH and 6'-OH positions (77%). When a proton-donor solvent was used, migration of the 3'-O-cinnamoyl group from fructose to the 2-OH position of glucose was observed. The enzyme hydroxycinnamoylations studied can shorten the targeted syntheses of various phenylpropanoid sucrose esters.

GhPME36 aggravates susceptibility to Liriomyza sativae by affecting cell wall biosynthesis in cotton leaves.

BACKGROUND: Cotton is an important economic crop and a host of Liriomyza sativae. Pectin methylesterase (PME)-mediated pectin metabolism plays an indispensable role in multiple biological processes in planta. However, the pleiotropic functions of PME often lead to unpredictable effects on crop resistance to pests. Additionally, whether and how PME affects susceptibility to Liriomyza sativae remain unclear. RESULTS: Here, we isolated GhPME36, which is located in the cell wall, from upland cotton (Gossypium hirsutum L.). Interestingly, the overexpression of GhPME36 in cotton caused severe susceptibility to Liriomyza sativae but increased leaf biomass in Arabidopsis. Cytological observations revealed that the cell wall was thinner with more demethylesterified pectins in GhPME36-OE cotton leaves than in WT leaves, whereas the soluble sugar content of GhPME36-OE cotton leaf cell walls was accordingly higher; both factors attracted Liriomyza sativae to feed on GhPME36-OE cotton leaves. Metabolomic analysis demonstrated that glucose was significantly differentially accumulated. Transcriptomic analysis further revealed DEGs enriched in glucose metabolic pathways when GhPME36 was overexpressed, suggesting that GhPME36 aggravates susceptibility to Liriomyza sativae by affecting both the structure and components of cell wall biosynthesis. Moreover, GhPME36 interacts with another pectin-modifying enzyme, GhC/VIF1, to maintain the dynamic stability of pectin methyl esterification. CONCLUSIONS: Taken together, our results reveal the cytological and molecular mechanisms by which GhPME36 aggravates susceptibility to Liriomyza sativae. This study broadens the knowledge of PME function and provides new insights into plant resistance to pests and the safety of genetically modified plants.

Genome sequencing and CAZymes repertoire analysis of Diaporthe eres P3-1W causing postharvest fruit rot of 'Hongyang' kiwifruit in China.

Postharvest rot caused by various fungal pathogens is a damaging disease affecting kiwifruit production and quality, resulting in significant annual economic losses. This study focused on isolating the strain P3-1W, identified as Diaporthe eres, as the causal agent of 'Hongyang' postharvest rot disease in China. The investigation highlighted cell wall degrading enzymes (CWDEs) as crucial pathogenic factors. Specially, the enzymatic activities of cellulase, ß-galactosidase, polygalacturonase, and pectin methylesterases peaked significantly on the second day after infection of D. eres P3-1W. To gain a comprehensive understanding of these CWDEs, the genome of this strain was sequenced using PacBio and Illumina sequencing technologies. The analysis revealed that the genome of D. eres P3-1W spans 58,489,835 bp, with an N50 of 5,939,879 bp and a GC content of 50.7%. A total of 15,407 total protein-coding genes (PCGs) were predicted and functionally annotated. Notably, 857 carbohydrate-active enzymes (CAZymes) were identified in D. eres P3-1W, with 521 CWDEs consisting of 374 glycoside hydrolases (GHs), 108 carbohydrate esterase (CEs) and 91 polysaccharide lyases (PLs). Additionally, 221 auxiliary activities (AAs), 91 glycosyltransferases (GTs), and 108 carbohydrate binding modules (CBMs) were detected. These findings offer valuable insights into the CAZymes of D. eres P3-1W.

Leukaemia-related protein 16 is highly expressed in oestrogen-dependent endometrial carcinoma and potentially promotes Ishikawa human endometrial cancer cells growth - a histopathological study.

Leukaemia-related protein 16 (LRP16) has been found to be highly expressed in various tumours and to be related to poor prognosis. However, the role of LRP16 in endometrial carcinoma remains to be explored. We aimed to investigate the prognosis and role of LRP16 in endometrial carcinoma. Overall, 160 endometrial carcinoma (EC) tissues and 60 benign samples were collected. The expression of LRP16 protein in EC tissues was significantly increased compared with that in normal endometrial tissues, and high LRP16 expression was related to poor patient prognosis. Reduced LRP16 expression markedly inhibited cancer cell growth. The proliferation rates in the prophylactic bilateral salpingectomy (PBS) group and the shNon group were 0.727 ±0.015 and 0.743 ±0.009, respectively, while the proliferation rate in the shLRP16 group was only 0.373 ±0.012. The migration experiment showed that the number of cells passing through the basement membrane in the shLRP16 group was 34.2 ±5.1, which was significantly different to the shNon (161.6 ±7.8) and PBS groups (138.0 ±7.2). The results of the invasion experiment showed that the number of cells was 39.2 ±6.2 in the shLRP16 group, 146.7 ±8.2 in the shNon group, and 141.2 ±8.1 in the PBS group ( p < 0.05). Leukaemia-related protein 16 is highly expressed in oestrogen-dependent EC and may promote cancer cell growth.

DepoScope: Accurate phage depolymerase annotation and domain delineation using large language models.

Bacteriophages (phages) are viruses that infect bacteria. Many of them produce specific enzymes called depolymerases to break down external polysaccharide structures. Accurate annotation and domain identification of these depolymerases are challenging due to their inherent sequence diversity. Hence, we present DepoScope, a machine learning tool that combines a fine-tuned ESM-2 model with a convolutional neural network to identify depolymerase sequences and their enzymatic domains precisely. To accomplish this, we curated a dataset from the INPHARED phage genome database, created a polysaccharide-degrading domain database, and applied sequential filters to construct a high-quality dataset, which is subsequently used to train DepoScope. Our work is the first approach that combines sequence-level predictions with amino-acid-level predictions for accurate depolymerase detection and functional domain identification. In that way, we believe that DepoScope can greatly enhance our understanding of phage-host interactions at the level of depolymerases.

An Innovative Aggregation-Induced Emission-Based NIR Fluorescent Probe for Visualizing Carboxylesterases in Living Cells, Zebrafish, and Tumor-Bearing Mice.

In the human body, carboxylesterases (CEs) play crucial roles in xenobiotic metabolism and lipid homeostasis. But abnormal expression of CEs is highly associated with some diseases, such as hyperlipidemia, diabetes, and liver cancer. Therefore, it is of great importance to develop an efficient tool for the accurate detection of CEs in living organisms. Herein, an innovative near-infrared (NIR) fluorescent probe, TTAP-AB, was designed for CE detection based on the aggregation-induced emission (AIE) mechanism. This probe exhibits rapid response (2 min), excellent sensitivity (limit of detection = 8.14 × 10-6 U/mL), and high selectivity to CEs. Additionally, owing to its good biocompatibility, the TTAP-AB probe enables the monitoring of dynamic changes in CE levels under drug-induced modulation in living cells and zebrafish. More importantly, the TTAP-AB probe was successfully employed to image liver tumors and assist in tumor resection through the real-time monitoring of CEs, indicating that TTAP-AB is promising to guide liver cancer surgery. Therefore, the TTAP-AB probe can not only enrich the strategies for CE detection in biological systems but also has great potential for some clinical imaging applications, including medical diagnosis, preclinical research, and imaging-guided surgery.

Identification and Analysis of the Mechanism of Stem Mechanical Strength Enhancement for Maize Inbred Lines QY1.

Enhancing stalk strength is a crucial strategy to reduce lodging. We identified a maize inbred line, QY1, with superior stalk mechanical strength. Comprehensive analyses of the microstructure, cell wall composition, and transcriptome of QY1 were performed to elucidate the underlying factors contributing to its increased strength. Notably, both the vascular bundle area and the thickness of the sclerenchyma cell walls in QY1 were significantly increased. Furthermore, analyses of cell wall components revealed a significant increase in cellulose content and a notable reduction in lignin content. RNA sequencing (RNA-seq) revealed changes in the expression of numerous genes involved in cell wall synthesis and modification, especially those encoding pectin methylesterase (PME). Variations in PME activity and the degree of methylesterification were noted. Additionally, glycolytic efficiency in QY1 was significantly enhanced. These findings indicate that QY1 could be a valuable resource for the development of maize varieties with enhanced stalk mechanical strength and for biofuel production.

Determining the accuracy of the leukocyte esterase reagent strip test in the rapid diagnosis of adult septic arthritis.

BACKGROUNDS: Septic arthritis is a dangerous disease that occurs when microorganisms enter synovial fluid. It needs fast and accurate management; otherwise, it can harm the patient's life. Currently, the tests measure WBC and PMN in SF, so we hypothesized to use a proxy that is easier and faster to measure. Leukocyte esterase is an enzyme secreted by neutrophils that can be found in the synovial fluid of SA patients. In this study, we tried to investigate the sensitivity and specificity of leukocyte esterase in diagnosing septic arthritis. METHODS: We obtained synovial fluid samples from forty-six patients suspected of having septic arthritis and fifty-eight healthy individuals and measured the WBCs, ESR, CRP, PMN, glucose, and protein of SF in 2021. We also used the leukocyte esterase dipstick test to investigate the level of LE in synovial fluid for one minute. RESULTS: Based on clinical and paraclinical criteria, sixteen out of the forty-six patients were diagnosed with SA. When (++) was considered positive, the sensitivity and specificity of the LE dipstick test for the diagnosis of SA were 93.7% (95% CI: 81.8-100%) and 60% (95% CI: 42.4-77.5%, P = 0.000), respectively. When both (+) and (++) were considered positive, they were 100% and 43.3% (95% CI: 25.6-61.0% P = 0.000), respectively. All the patients in the control group had negative cultures and LE test readings (specificity = 100%). CONCLUSION: The LE dipstick test can be a valuable diagnostic tool in the initial diagnosis of SA since it is affordable, fast, and reliable.

A 3-component module maintains sepal flatness in Arabidopsis.

As in origami, morphogenesis in living systems heavily relies on tissue curving and folding through the interplay between biochemical and biomechanical cues. By contrast, certain organs maintain their flat posture over several days. Here, we identified a pathway that is required for the maintenance of organ flatness, taking the sepal, the outermost floral organ, in Arabidopsis as a model system. Through genetic, cellular, and mechanical approaches, our results demonstrate that the global gene expression regulator VERNALIZATION INDEPENDENCE 4 (VIP4) fine-tunes the mechanical properties of sepal cell walls and maintains balanced growth on both sides of the sepals, mainly by orchestrating the distribution pattern of AUXIN RESPONSE FACTOR 3 (ARF3). vip4 mutation results in softer cell walls and faster cell growth on the adaxial sepal side, which eventually cause sepals to bend outward. Downstream of VIP4, ARF3 works through modulating auxin to downregulate pectin methylesterase VANGUARD1, resulting in decreased cell wall stiffness. Thus, our work unravels a 3-component module that relates hormonal patterns to organ curvature and actively maintains sepal flatness during its growth.

A comparative assessment of treatment methods to release ferulic and p-cumaric acids from Brewer's Spent Grains.

Brewers' spent grain (BSG) is the main byproduct from the brewing industry, which accounts for 85 % of the total waste generated during beer production. This lignocellulosic material is traditionally used as livestock feed and sold at a low price. However, BSG can be used as a low-cost feedstock for the production of bioactive molecules and chemicals precursors, upgrading the value of this byproduct. In this context, BSG is a promising feedstock for the extraction of antioxidants like ferulic acid (FA) and p-coumaric acid (p-Cu). The effectiveness of three hydrolysis treatments were evaluated for the extraction of FA and p-Cu from BSG, namely enzymatic (based on the synergistic cooperation between a feruloyl esterase and an endo-1,4-ß-xylanase), alkaline and hydrothermal. The hydrothermal treatment produced the highest extraction yields (7.2 g/kgBSG and 1.4 g/kgBSG for FA and p-Cu, respectively) in a short extraction time (an hour). On the other hand, enzymatic hydrolysis extracted 4.3 g/kgBSG for FA and negligible yields for p-Cu in 4 h of incubation at 25 °C. Yields of 5.5 g/kgBSG for FA and 0.6 g/kgBSG for p-Cu were obtained in more than 5 h of alkaline treatment at 120 °C. The mass and energy balances revealed the high dependence of the operating costs on the concentration of BSG used during the extraction process, with costs of 34.5 €, 6607 € and 205.5 € per kg of FA for the chemical, enzymatic and hydrothermal extraction methods at 100 kg BSG/m3.

Assay Analysis of Tannase from Lactobacillus plantarum.

Tannin, which is an astringent taste in the mouth, is a polyphenol compound contained in some plants. Tannin causes denaturation of proteins of the tongue or oral mucosa. Tannase, a hydrolase that cleaves carboxylic ester bonds specifically, is used in many industrial fields. Some tannase (tannin acyl hydrolase, EC3.1.1.20) is used widely to prevent or reduce creaming of some foods and beverages. Because some tannins are formed of insoluble salts combined with protein, they reduce creaming such as the white hazing of iced tea. Moreover, they can clarify beverages such as fruit juices during wine and beer production. Tannase is produced by microorganisms under conditions with tannic acid present, mainly from plants. Tannase characteristics differ according to its microorganism of origin. Therefore, it is important to study the microbes used as lactic acid bacteria (LAB), evaluate new methods of tannase assay, and apply them in food or other industries. In this chapter, assay of tannase in LAB is demonstrated using methyl gallate as substrate, with color development by rhodanine and potassium hydroxide solution, using a spectrophotometer. Actual data of high tannase-producing LAB, Lactobacillus plantarum, and enzyme characteristics in optimum conditions are presented in this chapter.

The evolution of cutinase Est1 based on the clustering strategy and its application for commercial PET bottles degradation.

The rapid increase in global plastic consumption, especially the worldwide use of polyethylene terephthalate (PET), has caused serious pollution problems. Due to the low recycling rate of PET, a substantial amount of waste accumulates in the environment, which prompts a growing focus on enzymatic degradation for its efficiency and environmentally friendliness. This study systematically designed and modified a cutinase, Est1 from Thermobifida alba AHK119, known for its potential of plastic-degradation at high temperatures. Additionally, the introduction of clustering algorithms provided the ability to understand and modify biomolecules, to accelerate the process of finding the optimal mutations. K-means was further proceeded based on the positive mutations. After comprehensive screening for thermostability and activity mutation sites, the dominant mutation Est1_5M (Est1 with the mutations of N213M, T215P, S115P, Q93A, and L91W) exhibited satisfying degradation ability for commercial PET bottles. The results showed that Est1_5M achieved a degradation rate of 90.84% in 72 h, 65-fold higher than the wild type. This study offers reliable theoretical and practical support for the development of efficient PET-degrading enzymes, providing a reference for plastic pollution management.

Monitoring Fruit Growth and Development in Apricot (Prunus armeniaca L.) through Gene Expression Analysis.

The main objective of this study was to monitor apricot development and ripening through gene expression analysis of key candidate genes using the RT-qPCR technique. Eight apricot cultivars were selected to analyze phenological and genetic patterns from pre-ripening stages through to postharvest. In addition, 19 selected genes were analyzed in the contrasting cultivars 'Cebas Red' and 'Rojo Pasión' in different stages (two preharvest stages S1 and S2, one harvest stage S3, and two postharvest stages S4 and S5). This pool of genes included genes related to fruit growth and ripening, genes associated with fruit color, and genes linked to the fruit's nutraceutical aspects. Among the studied genes, Polygalacturonase (PG), Pectin methylesterase (PME), Aminocyclopropane-1-carboxylate synthase (ACS), and Myo-inositol-1-phosphate synthase (INO1) were directly related to fruit maturation and quality. Significant differential expression was observed between the cultivars, which correlated with variations in firmness, shelf life, and sensory characteristics of the apricots. 'Rojo Pasión' displayed high levels of PG, associated with rapid maturation and shorter postharvest shelf life, whereas 'Cebas Red' exhibited lower levels of this gene, resulting in greater firmness and extended shelf life. Genes CCD4, CRTZ, and ZDS, related to carotenoids, showed varied expression patterns during growth and postharvest stages, with higher levels in 'Rojo Pasión'. On the other hand, Sucrose synthase (SUSY) and Lipoxygenase (LOX2) were prominent during the postharvest and growth stages, respectively. Additionally, GDP-L-galactose phosphorylase (VTC2_5) was linked to better postharvest performance. This research provides valuable insights for future breeding initiatives aimed at enhancing the quality and sustainability of apricot cultivation.

High expression of oleoyl-ACP hydrolase underpins life-threatening respiratory viral diseases.

Respiratory infections cause significant morbidity and mortality, yet it is unclear why some individuals succumb to severe disease. In patients hospitalized with avian A(H7N9) influenza, we investigated early drivers underpinning fatal disease. Transcriptomics strongly linked oleoyl-acyl-carrier-protein (ACP) hydrolase (OLAH), an enzyme mediating fatty acid production, with fatal A(H7N9) early after hospital admission, persisting until death. Recovered patients had low OLAH expression throughout hospitalization. High OLAH levels were also detected in patients hospitalized with life-threatening seasonal influenza, COVID-19, respiratory syncytial virus (RSV), and multisystem inflammatory syndrome in children (MIS-C) but not during mild disease. In olah-/- mice, lethal influenza infection led to survival and mild disease as well as reduced lung viral loads, tissue damage, infection-driven pulmonary cell infiltration, and inflammation. This was underpinned by differential lipid droplet dynamics as well as reduced viral replication and virus-induced inflammation in macrophages. Supplementation of oleic acid, the main product of OLAH, increased influenza replication in macrophages and their inflammatory potential. Our findings define how the expression of OLAH drives life-threatening viral disease.

Substrate specificity modification of paraben hydrolase and tannase from Aspergillus oryzae.

Paraben hydrolase and tannase catalyze the hydrolysis of parabens (4-hydroxybenzoic acid esters) and gallic acid (3,4,5-trihydroxybenzoic acid) esters, respectively. Paraben hydrolase (AoPrbA) and tannase (AoTanB) from Aspergillus oryzae belong to the tannase family in the ESTHER database. However, the substrate specificities of AoPrbA and AoTanB are narrow. Based on structural information of Aspergillus niger tannase (PDB code 7k4o), we constructed five single variants of AoPrbA (Thr200Glu, Phe231Gln, Leu232Gln, Ile361Tyr, and Leu428Ser) and four of AoTanB (Glu203Asp, Glu203Thr, His237Ala, and Ser440Leu) to investigate substrate discrimination between AoPrbA and AoTanB. Each variant was expressed in Pichia pastoris and were purified from the culture supernatant. Five purified variants of AoPrbA and four variants of AoTanB showed reduced paraben hydrolase and tannase activities compared with AoPrbA and AoTanB wild types, respectively. Interestingly, the AoPrbA wild type did not hydrolyze gallic acid methyl ester, whereas the Thr200Glu, Leu232Gln, and Leu428Ser variants did, indicating that these three variants acquired tannase activity. In particular, the Leu428Ser variant exhibited considerably greater hydrolysis of gallic acid and protocatechuic acid methyl esters. Meanwhile, the AoTanB wild type, and Glu203Asp, His237Ala and Ser440Leu variants hydrolyzed the protocatechuate methyl and 4-hydroxybenzoate ethyl esters; however, the Glu203Thr variant did not hydrolyze above-mentioned substrates. Additionally, the ratio of paraben hydrolase activity to tannase activity in Ser440Leu was markedly elevated.

Unveiling the crystal structure of thermostable dienelactone hydrolase exhibiting activity on terephthalate esters.

Dienelactone hydrolase (DLH) is one of numerous hydrolytic enzymes with an α/ß-hydrolase fold, which catalyze the hydrolysis of dienelactone to maleylacetate. The DLHs share remarkably similar tertiary structures and a conserved arrangement of catalytic residues. This study presents the crystal structure and comprehensive functional characterization of a novel thermostable DLH from the bacterium Hydrogenobacter thermophilus (HtDLH). The crystal structure of the HtDLH, solved at a resolution of about 1.67 Å, exhibits a canonical α/ß-hydrolase fold formed by eight ß-sheet strands in the core, with one buried α-helix and six others exposed to the solvent. The structure also confirmed the conserved catalytic triad of DHLs formed by Cys121, Asp170, and His202 residues. The HtDLH forms stable homodimers in solution. Functional studies showed that HtDLH has the expected esterase activity over esters with short carbon chains, such as p-nitrophenyl acetate, reaching optimal activity at pH 7.5 and 70 °C. Furthermore, HtDLH maintains more than 50 % of its activity even after incubation at 90 °C for 16 h. Interestingly, HtDLH exhibits catalytic activity towards polyethylene terephthalate (PET) monomers, including bis-1,2-hydroxyethyl terephthalate (BHET) and 1-(2-hydroxyethyl) 4-methyl terephthalate, as well as other aliphatic and aromatic esters. These findings associated with the lack of activity on amorphous PET indicate that HtDLH has characteristic of a BHET-degrading enzyme. This work expands our understanding of enzyme families involved in PET degradation, providing novel insights for plastic biorecycling through protein engineering, which could lead to eco-friendly solutions to reduce the accumulation of plastic in landfills and natural environments.

Structural insights into strigolactone catabolism by carboxylesterases reveal a conserved conformational regulation.

Phytohormone levels are regulated through specialized enzymes, participating not only in their biosynthesis but also in post-signaling processes for signal inactivation and cue depletion. Arabidopsis thaliana (At) carboxylesterase 15 (CXE15) and carboxylesterase 20 (CXE20) have been shown to deplete strigolactones (SLs) that coordinate various growth and developmental processes and function as signaling molecules in the rhizosphere. Here, we elucidate the X-ray crystal structures of AtCXE15 (both apo and SL intermediate bound) and AtCXE20, revealing insights into the mechanisms of SL binding and catabolism. The N-terminal regions of CXE15 and CXE20 exhibit distinct secondary structures, with CXE15 characterized by an alpha helix and CXE20 by an alpha/beta fold. These structural differences play pivotal roles in regulating variable SL hydrolysis rates. Our findings, both in vitro and in planta, indicate that a transition of the N-terminal helix domain of CXE15 between open and closed forms facilitates robust SL hydrolysis. The results not only illuminate the distinctive process of phytohormone breakdown but also uncover a molecular architecture and mode of plasticity within a specific class of carboxylesterases.