Biochemically Characterized Research Articles

Biochemical characterization and structure prediction of the Cerrado soil CRB2(1) metagenomic dioxygenase

Dioxygenases are enzymes involved in the conversion of polyconic aromatic hydroxycarbons (PAHs), attracting significant biotechnological interest for the conversion of recalcitrant organic compounds. Furthermore, few studies show that dioxygenases can take on the function of resistance genes in clones. This enzymatic versatility opens up new opportunities for elucidating the mechanisms of microbial resistance, as well as its biotechnological application. In this work, a Cerrado soil dioxygenase named CRB2(1) was biochemically characterized. The enzyme was shown to have optimal activity at pH 7; a temperature of 30 °C; and using iron ions as a cofactor for substrate cleavage. The kinetic catalytic parameters of CRB2(1) were Vmax = 0.02281 µM/min and KM = 97.6. Its predicted three-dimensional structure obtained using the Modeller software v9.22 based on the crystal structure of gentisate 1,2-dioxygenase from Silicibacter pomeroyi (GDOsp) (PDB ID 3BU7, resolution 2.80 Å, residues 17–374) revealed substrate binding to the cupin domain, where the active site is located. The analyzed substrates interact directly with the iron ion, coordinated by three histidine residues. Changing the iron ion charge modifies the binding between the active site and the substrates. Currently, there is a demand for enzymes that have biotechnological activities of interest. Metagenomics allows analyzing the biotechnological potential of several organisms at the same time, based on sequence and functional activity analyses.

In Silico Analysis and Biochemical Characterization of Streptomyces PET Hydrolase with Bis(2-Hydroxyethyl) Terephthalate Biodegradation Activity.

Polyethylene terephthalate (PET), one of the most widely used plastics in the world, causes serious environmental problems. Recently, scientists have been focused on the enzymatic degradation of PET, an environmentally friendly method that offers an attractive approach to the degradation and recycling of PET. In this work, PET hydrolase from Streptomyces sp. W2061 was biochemically characterized, and the biodegradation of PET was performed using the PET model substrate bis (2- hydroxyethyl terephthalate) (BHET). PET hydrolase has an isoelectric point of 5.84, and a molecular mass of about 50.31 kDa. The optimum pH and temperature were 7.0 and 40°C, respectively. LC-MS analysis of the enzymatic products showed that the PET hydrolase successfully degraded a single ester bond of BHET, leading to the formation of MHET. Furthermore, in silico characterization of the PET hydrolase protein sequence and its predicted three-dimensional structure was designed and compared with the well-characterized IsPETase from Ideonella sakaiensis. The structural analysis showed that the (Gly-x1-Ser-x2-Gly) serine hydrolase motif and the catalytic triad (Ser, Asp, and His) were conserved in all sequences. In addition, we integrated molecular dynamics (MD) simulations to analyze the variation in the structural stability of the PET hydrolase in the absence and presence of BHET. These simulations showed the formation of a stable complex between the PET hydrolase and BHET. To the best of our knowledge, this is the first study on Streptomyces sp. W2061 to investigate the BHET degradation activity of PET hydrolase, which has potential application in the biodegradation of plastics in the environment.

The biochemical characterization of a TatD nuclease from Thermus thermophilus

Nucleases play pivotal roles in DNA repair and apoptosis. Moreover, they have various applications in biotechnology and industry. Among nucleases, TatD has been characterized as an exonuclease with various biological functions in different organisms. Here, we biochemically characterized the potential TatD nuclease from Thermus thermophilus. The tatD gene from T. thermophilus was cloned, then the recombinant TatD nuclease was expressed and purified. Our results revealed that the TthTatD nuclease could degrade both single-stranded and double-stranded DNA, and its activity is dependent on the divalent metal ions Mg2+ and Mn2+. Remarkably, the activity of TthTatD nuclease is highest at 37 °C and decreases with increasing temperature. TthTatD is not a thermostable enzyme, even though it is from a thermophilic bacterium. Based on the sequence similarity and molecular docking of the DNA substrate into the modeled TthTatD structure, several key conserved residues were identified and their roles were confirmed by analyzing the enzymatic activities of the site-directed mutants. The residues E86 and H149 play key roles in binding metal ions, residues R124/K126 and K211/R212 had a critical role in binding DNA substrate. Our results confirm the enzymatic properties of TthTatD and provide a primary basis for its possible application in biotechnology.

Biochemical characterization of a novel C-terminally truncated β-galactosidase from Paenibacillus antarcticus with high transglycosylation activity

The transgalactosylase activity of β-galactosidases offers a convenient and promising strategy for conversion of lactose into high-value oligosaccharides, such as galacto-oligosaccharides (GOS) and human milk oligosaccharides (HMOs). In this study, we cloned and biochemically characterized a novel C-terminally truncated β-galactosidase (PaBgal2A-D) from Paenibacillus antarcticus with high transglycosylation activity. PaBgal2A-D is a member of glycoside hydrolase (GH) family 2. The optimal pH and temperature of PaBgal2A-D were determined to be pH 6.5 and 50°C, respectively. It was relatively stable within pH 5.0-8.0 and up to 50°C. PaBgal2A-D showed high transglycosylation activity for GOS synthesis, and the maximum yield of 50.8% (wt/wt) was obtained in 2 h. Moreover, PaBgal2A-D could synthesize lacto-N-neotetraose (LNnT) using lactose and lacto-N-triose II (LNT2), with a conversion rate of 16.4%. This study demonstrated that PaBgal2A-D could be a promising tool to prepare GOS and LNnT.

Purification and biochemical characterization of a novel thermostable endo-polygalacturonase from Aspergillus niger strain HO32 and its suitability for clarification of orange juice

A novel endo-polygalacturonase (PGC-AN64) from Aspergillus niger HO32 was produced (88.22 U/mL) on orange peel by-product (OPP), purified, biochemically characterized, and its utility in clarification of orange juice was elucidated. The enzyme was purified by ammonium sulfate fractionation (80 %) and gel filtration chromatography on Sephacryl® S-200 HR column. Its pH and temperature optima were 6.5 and 70 °C, respectively, and was stable over a pH range of 5–7 and temperature 60–80 °C. The 26-residue NH2-terminal sequence of PGC-AN64 showed high homology with those of Aspergillus pectinases. Metal ions (Na+, Ca2+, Mg2+, Mn2+, and Zn2+) positively affect PGC-AN64 activity, while Ba2+, Cd2+, and Cr3+ negatively affect its original activity. Interestingly, PGC-AN64 exhibited a considerable substrate specificity and catalytic efficiency compared to the commercial enzymes PECLYVE V (P1) and PECLYVE CP (P2). More interestingly and compared to P1 and P2, PGC-AN64 showed that the clarification process had a significant effect on the transmittance percentages with 84.68 ± 2.08, 88.05 ± 1.79, and 93.13 ± 3.58 % for PGC-AN64, P1, and P2, respectively. Furthermore, color parameters after the clarification process increase in the L* and b* values, and led to a decrease in the a* value. The obtained results suggest that PGC-AN64 revealed its potential in orange juice clarification.

Exploring class III cellobiose dehydrogenase: sequence analysis and optimized recombinant expression

BackgroundCellobiose dehydrogenase (CDH) is an extracellular fungal oxidoreductase with multiple functions in plant biomass degradation. Its primary function as an auxiliary enzyme of lytic polysaccharide monooxygenase (LPMO) facilitates the efficient depolymerization of cellulose, hemicelluloses and other carbohydrate-based polymers. The synergistic action of CDH and LPMO that supports biomass-degrading hydrolases holds significant promise to harness renewable resources for the production of biofuels, chemicals, and modified materials in an environmentally sustainable manner. While previous phylogenetic analyses have identified four distinct classes of CDHs, only class I and II have been biochemically characterized so far.ResultsFollowing a comprehensive database search aimed at identifying CDH sequences belonging to the so far uncharacterized class III for subsequent expression and biochemical characterization, we have curated an extensive compilation of putative CDH amino acid sequences. A sequence similarity network analysis was used to cluster them into the four distinct CDH classes. A total of 1237 sequences encoding putative class III CDHs were extracted from the network and used for phylogenetic analyses. The obtained phylogenetic tree was used to guide the selection of 11 cdhIII genes for recombinant expression in Komagataella phaffii. A small-scale expression screening procedure identified a promising cdhIII gene originating from the plant pathogen Fusarium solani (FsCDH), which was selected for expression optimization by signal peptide shuffling and subsequent production in a 5-L bioreactor. The purified FsCDH exhibits a UV-Vis spectrum and enzymatic activity similar to other characterized CDH classes.ConclusionThe successful production and functional characterization of FsCDH proved that class III CDHs are catalytical active enzymes resembling the key properties of class I and class II CDHs. A detailed biochemical characterization based on the established expression and purification strategy can provide new insights into the evolutionary process shaping CDHs and leading to their differentiation into the four distinct classes. The findings have the potential to broaden our understanding of the biocatalytic application of CDH and LPMO for the oxidative depolymerization of polysaccharides.

Structural and biochemical characterization of a nucleotide hydrolase from Streptococcus pneumonia

In this report, we structurally and biochemically characterized the unknown gene product SP1746 from Streptococcus pneumoniae serotype 4. Various crystal structures of SP1746 in the apo form and in complex with different nucleotides were determined. SP1746 is a globular protein, which belongs to the histidine-aspartate (HD) domain superfamily with two Fe3+ ions in the active site that are coordinated by key active site residues and water molecules. All nucleotides bind in a similar orientation in the active site with their phosphate groups anchored to the diiron cluster. Biochemically, SP1746 hydrolyzes different nucleotide substrates. SP1746 most effectively hydrolyzes diadenosine tetraphosphate (Ap4A) to two ADPs. Based on the aforementioned data, we annotated SP1746 as an Ap4A hydrolase, belonging to the YqeK family. Our invitro data indicate a potential role for SP1746 in regulating Ap4A homeostasis, which requires validation with invivo experiments in bacteria in the future.

Substrate specificity mapping of fungal CAZy AA3_2 oxidoreductases

BackgroundOxidative enzymes targeting lignocellulosic substrates are presently classified into various auxiliary activity (AA) families within the carbohydrate-active enzyme (CAZy) database. Among these, the fungal AA3 glucose–methanol–choline (GMC) oxidoreductases with varying auxiliary activities are attractive sustainable biocatalysts and important for biological function. CAZy AA3 enzymes are further subdivided into four subfamilies, with the large AA3_2 subfamily displaying diverse substrate specificities. However, limited numbers of enzymes in the AA3_2 subfamily are currently biochemically characterized, which limits the homology-based mining of new AA3_2 oxidoreductases. Importantly, novel enzyme activities may be discovered from the uncharacterized parts of this large subfamily.ResultsIn this study, phylogenetic analyses employing a sequence similarity network (SSN) and maximum likelihood trees were used to cluster AA3_2 sequences. A total of 27 AA3_2 proteins representing different clusters were selected for recombinant production. Among them, seven new AA3_2 oxidoreductases were successfully produced, purified, and characterized. These enzymes included two glucose dehydrogenases (TaGdhA and McGdhA), one glucose oxidase (ApGoxA), one aryl alcohol oxidase (PsAaoA), two aryl alcohol dehydrogenases (AsAadhA and AsAadhB), and one novel oligosaccharide (gentiobiose) dehydrogenase (KiOdhA). Notably, two dehydrogenases (TaGdhA and KiOdhA) were found with the ability to utilize phenoxy radicals as an electron acceptor. Interestingly, phenoxy radicals were found to compete with molecular oxygen in aerobic environments when serving as an electron acceptor for two oxidases (ApGoxA and PsAaoA), which sheds light on their versatility. Furthermore, the molecular determinants governing their diverse enzymatic functions were discussed based on the homology model generated by AlphaFold.ConclusionsThe phylogenetic analyses and biochemical characterization of AA3_2s provide valuable guidance for future investigation of AA3_2 sequences and proteins. A clear correlation between enzymatic function and SSN clustering was observed. The discovery and biochemical characterization of these new AA3_2 oxidoreductases brings exciting prospects for biotechnological applications and broadens our understanding of their biological functions.

A novel endo-1,4-β-xylanase from Alicyclobacillus mali FL18: Biochemical characterization and its synergistic action with β-xylosidase in hemicellulose deconstruction

A novel endo-1,4-β-xylanase-encoding gene was identified in Alicyclobacillus mali FL18 and the recombinant protein, named AmXyn, was purified and biochemically characterized. The monomeric enzyme worked optimally at pH 6.6 and 80 °C on beechwood xylan with a specific activity of 440.00 ± 0.02 U/mg and a good catalytic efficiency (kcat/KM = 91.89 s−1mLmg−1). In addition, the enzyme did not display any activity on cellulose, suggesting a possible application in paper biobleaching processes. To develop an enzymatic mixture for xylan degradation, the association between AmXyn and the previously characterized β-xylosidase AmβXyl, deriving from the same microorganism, was assessed. The two enzymes had similar temperature and pH optima and showed the highest degree of synergy when AmXyn and AmβXyl were added sequentially to beechwood xylan, making this mixture cost-competitive and suitable for industrial use. Therefore, this enzymatic cocktail was also employed for the hydrolysis of wheat bran residue. TLC and HPAEC-PAD analyses revealed a high conversion rate to xylose (91.56 %), placing AmXyn and AmβXyl among the most promising biocatalysts for the saccharification of agricultural waste.

Biochemical characterisation of a PL24 ulvan lyase from seaweed-associated Vibrio sp. FNV38

Ulvan is a green macroalgal cell wall polysaccharide that has tremendous potential for valorisation due to its unique composition of sulphated rhamnose, glucuronic acid, iduronic acid and xylose. Several potential applications such as production of biofuels, bioplastics and other value-added products necessitate the breakdown of the polysaccharide to oligomers or monomers. Research on ulvan saccharifying enzymes has been continually increasing over the last decade, with the increasing focus on valorisation of seaweed biomass for a biobased economy. Lyases are the first of several enzymes that are involved in saccharifying the polysaccharide and several ulvan lyases have been structurally and biochemically characterised to enable their effective use in the valorisation processes. This study investigates the whole genome of Vibrio sp. FNV38, an ulvan metabolising organism and biochemical characteristics of a PL24 ulvan lyase that it possesses. The genome of Vibrio sp. FNV38 has a diverse CAZy profile with several genes involved in the metabolism of ulvan, cellulose, agar, and alginate. The enzyme exhibits optimal activity at pH 8.5 in 100 mM Tris–HCl buffer and 30 °C. However, its thermal stability is poor with significant loss of activity after 2 h of incubation at temperatures above 25 °C. Breakdown product analysis reveals that the enzyme depolymerised the polysaccharide predominantly to disaccharides and tetrasaccharides.

Biochemical Characterization of a Novel Alkaline-Tolerant Xaa-Pro Dipeptidase from Aspergillus phoenicis

Xaa-Pro dipeptidase (XPD, EC 3.4.13.9; also known as prolidase) catalyzes the hydrolysis of the iminopeptide bond in the trans-Xaa-Pro dipeptides (Xaa represents any amino acid except proline), which makes it find wide applications in food, medical and environmental protection fields. In the present study, a novel Xaa-Pro dipeptidase from Aspergillus phoenicis ATCC 14332 (ApXPD) was heterologously expressed and biochemically characterized. Reclassification based on phylogenetic analysis and the version 12.5 MEROPS database showed that this enzyme was the only fungal XPD in the unassigned subfamily that shared the highest sequence identity with Xanthomonas campestris prolidase but not with that from the more related fungal species A. niudulans. As compared with other prolidases, ApXPD also contained a long N-terminal tail (residues 1–63) and an additional region (PAPARLREKL) and used a different arginine residue for dipeptide selectivity. After heterologous expression and partial purification, recombinant ApXPD was highly active and stable over the alkaline range from 8.5 to 10.0, with maximum activity at pH 9.0 and more than 80% activity retained after 1 h incubation at pHs of 8.5–10.0 (55 °C). It also had an apparent optimum temperature of 55 °C and remained stable at 20–30 °C. Moreover, this enzyme was a cobalt-dependent prolidase that only cleaved dipeptides Lys-Pro, Gly-Pro, and Ala-Pro rather than other dipeptides, tripeptides, and tetrapeptides. All these distinct features make A. phoenicis ATCC 14332 XPD unique among currently known prolidases, thus defining a novel Xaa-Pro dipeptidase subfamily.

Unraveling the terpene synthase family and characterization of BsTPS2 contributing to (S)-( +)-linalool biosynthesis in Boswellia.

Boswellia tree bark exudes oleo-gum resin in response to wounding, which is rich in terpene volatiles. But, the molecular and biochemical basis of wound-induced formation of resin volatiles remains poorly understood. Here, we combined RNA-sequencing (RNA-seq) and metabolite analysis to unravel the terpene synthase (TPS) family contributing to wound-induced biosynthesis of resin volatiles in B. serrata, an economically-important Boswellia species. The analysis of large-scale RNA-seq data of bark and leaf samples representing more than 600 million sequencing reads led to the identification of 32 TPSs, which were classified based on phylogenetic relationship into various TPSs families found in angiosperm species such as TPS-a, b, c, e/f, and g. Moreover, RNA-seq analysis of bark samples collected at 0-24h post-wounding shortlisted 14 BsTPSs that showed wound-induced transcriptional upregulation in bark, suggesting their important role in wound-induced biosynthesis of resin volatiles. Biochemical characterization of a bark preferentially-expressed and wound-inducible TPS (BsTPS2) in vitro and in planta assays revealed its involvement in resin terpene biosynthesis. Bacterially-expressed recombinant BsTPS2 catalyzed the conversion of GPP and FPP into (S)-( +)-linalool and (E)-(-)-nerolidol, respectively, in vitro assays. However, BsTPS2 expression in Nicotiana benthamiana found that BsTPS2 is a plastidial linalool synthase. In contrast, cytosolic expression of BsTPS2 did not form any product. Overall, the present work unraveled a suite of TPSs that potentially contributed to the biosynthesis of resin volatiles in Boswellia and biochemically characterized BsTPS2, which is involved in wound-induced biosynthesis of (S)-( +)-linalool, a monoterpene resin volatile with a known role in plant defense.

Expression and Characterization of a β-Galactosidase from the Pacific Oyster, Crassostrea gigas, and Evaluation of Strategies for Testing Substrate Specificity.

β-Galactosidases (EC 3.2.1.23) are exoglycosidases that catalyze the cleavage of glycoconjugates with terminal β-D-galactose residues in β1,3-, β1,4- or β1,6-linkage. Although this family of exoglycosidases has been extensively studied in vertebrates, plants, yeast, and bacteria, little information is available for mollusks. Mollusks are a diverse and highly successful group of animals that play many different roles in their ecosystems, including filter feeders and detritivores. Here, the first β-galactosidase from the Pacific oyster, Crassostrea gigas was discovered, biochemically characterized, and compared to our previously characterized slug enzyme from Arion vulgaris (UniProt Ref. Nr.: A0A0B7AQJ9). Overall, the mussel enzyme showed similar biochemical parameters to the snail enzyme. The enzyme from C. gigas was most active in an acidic environment (pH 3.5) and at a reaction temperature of 50 °C. Optimal storage conditions were up to 37 °C. In contrast to the enzyme from A. vulgaris, the supplementation of cations (Ni2+, Co2+, Mn2+, Mg2+, Ca2+, Cu2+, Ba2+) increased the activity of the enzyme from C. gigas. Substrate specificity studies of the β-galactosidases from the mussel, C. gigas, and the slug, A. vulgaris, revealed activity towards terminal β1,3- and β1,4-linked galactose residues for both enzymes. Using the same substrates in labeled and unlabeled form, we were able to detect the effect of labeling on the β-galactosidase activity using MALDI-TOF MS, HPTLC, and HPLC. While lactose was cleaved by the enzymes in an unlabeled or labeled state, galacto-N-biose was not cleaved as soon as a 2-amino benzoic acid label was added. In this study we present the biochemical characterization of the first recombinantly expressed β-galactosidase from the Pacific oyster, C. gigas, and we compare different analytical methods for the determination of β-galactosidase activity using the enzyme from C. gigas and A. vulgaris.

Cloning, expression, and biochemical characterization of β-etherase LigF from Altererythrobacter sp. B11

Lignin, a complex heteropolymer present in plant cell walls, is now recognized as a valuable renewable resource with potential applications in various industries. The lignin biorefinery concept, which aims to convert lignin into value-added products, has gained significant attention in recent years. β-etherases, enzymes that selectively cleave β-O-4 aryl ether bonds in lignin, have shown promise in lignin depolymerization. In this study, the β-etherase LigF from Altererythrobacter sp. B11 was cloned, expressed, purified, and biochemically characterized. The LigF-AB11 enzyme exhibited optimal activity at 32 °C and pH 8.5 when catalyzing the substrate PNP-AV. The enzyme displayed mesophilic behavior and demonstrated higher activity at moderate temperatures. Stability analysis revealed that LigF-AB11 was not thermostable, with a complete loss of activity at 60 °C within an hour. Moreover, LigF-AB11 exhibited excellent pH stability, retaining over 50 % of its activity after 1 h under pH conditions ranging from 3.0 to 11.0. Metal ions and surface impregnation agents were found to affect the enzyme's activity, highlighting the importance of considering these factors in enzymatic processes for lignin depolymerization. This study provides valuable insights into the biochemical properties of LigF-AB11 and contributes to the development of efficient enzymatic processes for lignin biorefineries. Further optimization and understanding of β-etherases will facilitate their practical application in the valorization of lignin.

Biochemical characterization of a novel bifunctional alginate lyase from Microbulbifer arenaceous

Bio‒production of alginate oligosaccharides (AOSs), a type of functional food additive, is a promising way for green utilization of algae, in which alginate hydrolyzing enzymes play a key role. A novel alginate lyase gene (MiAly17A) from a marine bacterium Microbulbifer arenaceous was heterologously expressed in E. coli. The coding sequence of the gene shared the highest identity of 86% with a polysaccharide lyase (PL) family 17 alginate lyase (AlgL17) from Microbulbifer sp. ALW1. The recombinant enzyme (MiAly17A) was purified and biochemically characterized. MiAly17A showed maximal enzyme activity at 40 °C and pH 7.5, respectively. It was stable at the temperatures below 35 °C and within pH 5.0–8.0. The enzyme activities were increased by 5.3 and 5.6 folds in the presence of 100 mM of K+ and Na+, respectively. MiAly17A was bifunctional and could hydrolyze sodium alginate to release unsaturated monosaccharides and oligosaccharides with degrees of polymerization (DP) 2–7. The enzyme catalyzed the cleavage of glycosidic bonds from the non-reducing ends and the backbone of the tested oligosaccharides (DP ≥ 4), exhibiting both exolytic and endo-lytic activities. Moreover, MiAly17A was used for the production of alginate oligosaccharides from sodium alginate, and the highest conversion ratio of 68% was obtained. The unique properties may possess the enzyme great potential for preparation of alginate oligosaccharides.

Developing Connections Between LINC00298 RNA and Alzheimer's Disease Through Mapping Its Interactome and Through Biochemical Characterization.

Long non-coding RNAs are ubiquitous throughout the human system, yet many of their biological functions remain unknown. LINC00298 RNA, a long intergenic non-coding RNA, has been shown to have preferential expression in the central nervous system where it contributes to neuronal differentiation and development. Furthermore, previous research has indicated that LINC00298 RNA is known to be a genetic risk factor for the development of Alzheimer's disease. To biochemically characterize LINC00298 RNA and to elucidate its biological function within hippocampal neuronal cells, thereby providing a greater understanding of its role in Alzheimer's disease pathogenesis. LINC00298 RNA was in vitro transcribed and then subjected to structural analysis using circular dichroism, and UV-Vis spectroscopy. Additionally, affinity column chromatography was used to capture LINC00298 RNA's protein binding partners from hippocampal neuronal cells, which were then identified using liquid chromatography and mass spectrometry (LC/MS). LINC00298 RNA is comprised of stem-loop secondary structural elements, with a cylindrical tertiary structure that has highly dynamic regions, which result in high positional entropy. LC/MS identified 24 proteins within the interactome of LINC00298 RNA. Through analysis of LINC00298 RNA's 24 protein binding partners, it was determined that LINC00298 RNA may play significant roles in neuronal development, proliferation, and cellular organization. Furthermore, analysis of LINC00298 RNA's interactome indicated that LINC00298 RNA is capable of intracellular motility with dual localization in the nucleus and the cytosol. This biochemical characterization of LINC00298 RNA has shed light on its role in Alzheimer's disease pathogenesis.

Biochemical and cellular studies of three human 3-phosphoglycerate dehydrogenase variants responsible for pathological reduced L-serine levels.

In the brain, the non-essential amino acid L-serine is produced through the phosphorylated pathway (PP) starting from the glycolytic intermediate 3-phosphoglycerate: among the different roles played by this amino acid, it can be converted into D-serine and glycine, the two main co-agonists of NMDA receptors. In humans, the enzymes of the PP, namely phosphoglycerate dehydrogenase (hPHGDH, which catalyzes the first and rate-limiting step of this pathway), 3-phosphoserine aminotransferase, and 3-phosphoserine phosphatase are likely organized in the cytosol as a metabolic assembly (a "serinosome"). The hPHGDH deficiency is a pathological condition biochemically characterized by reduced levels of L-serine in plasma and cerebrospinal fluid and clinically identified by severe neurological impairment. Here, three single-point variants responsible for hPHGDH deficiency and Neu-Laxova syndrome have been studied. Their biochemical characterization shows that V261M, V425M, and V490M substitutions alter either the kinetic (both maximal activity and Km for 3-phosphoglycerate in the physiological direction) and the structural properties (secondary, tertiary, and quaternary structure, favoring aggregation) of hPHGDH. All the three variants have been successfully ectopically expressed in U251 cells, thus the pathological effect is not due to hindered expression level. At the cellular level, mistargeting and aggregation phenomena have been observed in cells transiently expressing the pathological protein variants, as well as a reduced L-serine cellular level. Previous studies demonstrated that the pharmacological supplementation of L-serine in hPHGDH deficiencies could ameliorate some of the related symptoms: our results now suggest the use of additional and alternative therapeutic approaches.

Characterization of drought stress-mitigating Rhizobium from faba bean (Vicia faba L.) in the Chinese Qinghai-Tibet Plateau.

Rhizobium-driven symbiotic nitrogen-fixation in legumes not only benefits the growth but also enhances the stress tolerance of plants. Isolating and characterizing efficient, drought-tolerant rhizobia is a central goal for improving crop yields in arid regions. Here, we phylogenetically and biochemically characterized a novel strain of Rhizobium ('QHCD11') sampled from the root nodules of faba beans growing in an arid agricultural area in Qinghai-Tibet. We further tested the drought tolerance of the strain as well as of 'Qingcan 14' faba bean seedlings inoculated with it. Biochemical characterization involved bromothymol blue (BTB) tests, carbon metabolic profiling (Biolog GENIII), DNA-DNA hybridization (dDDH) assays, average nucleotide identity (ANI) analyses, and 16S rRNA sequencing. The result indicated that strain 'QHCD11' likely belongs to the Rhizobium indicum species. Drought stress tolerance was assessed by exposure to polyethylene glycol (PEG-6000) at concentrations of 0, 10, 15, and 20%. Increasing concentrations of PEG-6000 tended to result in decreased growth of 'QHCD11', although the strain performed better at 20% PEG 6000 than at 15%. Inoculation of drought-stressed faba bean seedlings with strain 'QHCD11' improved root vitality, chlorophyll content, antioxidant enzyme activities, and plant height. We suggest that inoculation of faba beans with 'QHCD11' is an environmentally sound strategy for mitigating crop drought stress in arid and semi-arid regions. In addition, the results presents here will benefit future studies into faba bean-rhizobia symbioses under drought stress.

Isolation, biochemical characterization, and genome sequencing of two high-quality genomes of a novel chitinolytic Jeongeupia species.

Chitin is the second most abundant polysaccharide worldwide as part of arthropods' exoskeletons and fungal cell walls. Low concentrations in soils and sediments indicate rapid decomposition through chitinolytic organisms in terrestrial and aquatic ecosystems. The enacting enzymes, so-called chitinases, and their products, chitooligosaccharides, exhibit promising characteristics with applications ranging from crop protection to cosmetics, medical, textile, and wastewater industries. Exploring novel chitinolytic organisms is crucial to expand the enzymatical toolkit for biotechnological chitin utilization and to deepen our understanding of diverse catalytic mechanisms. In this study, we present two long-read sequencing-based genomes of highly similar Jeongeupia species, which have been screened, isolated, and biochemically characterized from chitin-amended soil samples. Through metabolic characterization, whole-genome alignments, and phylogenetic analysis, we could demonstrate how the investigated strains differ from the taxonomically closest strain Jeongeupia naejangsanensis BIO-TAS4-2T (DSM 24253). In silico analysis and sequence alignment revealed a multitude of highly conserved chitinolytic enzymes in the investigated Jeongeupia genomes. Based on these results, we suggest that the two strains represent a novel species within the genus of Jeongeupia, which may be useful for environmentally friendly N-acetylglucosamine production from crustacean shell or fungal biomass waste or as a crop protection agent.

Structural and Biochemical Insights into Bis(2-hydroxyethyl) Terephthalate Degrading Carboxylesterase Isolated from Psychrotrophic Bacterium Exiguobacterium antarcticum.

This study aimed to elucidate the crystal structure and biochemically characterize the carboxylesterase EaEst2, a thermotolerant biocatalyst derived from Exiguobacterium antarcticum, a psychrotrophic bacterium. Sequence and phylogenetic analyses showed that EaEst2 belongs to the Family XIII group of carboxylesterases. EaEst2 has a broad range of substrate specificities for short-chain p-nitrophenyl (pNP) esters, 1-naphthyl acetate (1-NA), and 1-naphthyl butyrate (1-NB). Its optimal pH is 7.0, losing its enzymatic activity at temperatures above 50 °C. EaEst2 showed degradation activity toward bis(2-hydroxyethyl) terephthalate (BHET), a polyethylene terephthalate degradation intermediate. We determined the crystal structure of EaEst2 at a 1.74 Å resolution in the ligand-free form to investigate BHET degradation at a molecular level. Finally, the biochemical stability and immobilization of a crosslinked enzyme aggregate (CLEA) were assessed to examine its potential for industrial application. Overall, the structural and biochemical characterization of EaEst2 demonstrates its industrial potency as a biocatalyst.