Search Results (486)

Rapeseed meal is severely restricted in its utilization as unconventional animal feed due to anti-nutritive compounds, such as glucosinolate, that are degraded to toxic nitriles such as 3-butenenitrile and 4-pentenenitrile in animals. Few studies on nitrilases that can degrade glucosinolate-derived nitriles have been reported thus far. In the present study, a nitrilase gene GiNIT from Gibberella intermedia was over-expressed in Escherichia coli and the purified recombinant nitrilase rGiNIT showed specific activities of 134.48 U/mg and 122.16 U/mg when using 3-butenenitrile and 4-pentenenitrile as substrates at the optimal pH, 7.5, and temperature, 45 °C, which is the highest reported in the literature. The conversion of 3-butenenitrile and 4-pentenenitrile by rGiNIT reached 81.89% and 80.23% after hydrolysis for 15 min and 300 min, respectively. Site-directed mutagenesis and molecular docking analysis revealed that the catalytic ability of rGiNIT depended on the substrate binding pocket comprising 13 key amino acid residues. These results provide a potential enzyme resource for rapeseed meal detoxification and theoretical guidance for protein engineering. Full article

In the present review, we summarize genome mining of genomic data obtained from the psychrophilic Antarctic marine ciliate Euplotes focardii and its evolutionary-close mesophilic cosmopolitan counterpart E. crassus. This analysis highlights adaptation strategies that are unique to the Antarctic ciliate, including antioxidant gene duplication and distinctive substitutions that may play roles in increased drug binding affinity and enzyme reaction rate in cold environments. Enzymes from psychrophiles are usually characterized by high activities and reaction rates at low temperatures compared with their counterparts from mesophiles and thermophiles. As a rule, catalyst cold activity derives from an increased structural flexibility that may lead to protein denaturation in response to temperature fluctuation. Molecular thermolability has been a major drawback of using macromolecules from psychrophiles in industrial applications. Here, we report a case study in which the role of peculiar amino acid substitution in cold adaptation is demonstrated by site-directed mutagenesis. Combined with a rational design approach, these substitutions can be used for site-directed mutagenesis to obtain cold-active catalysts that are structurally stable. Furthermore, molecular docking analysis of β-tubulin isotypes extrapolated from E. focardii and E. crassus genomes allowed us to obtain additional insight on the taxol binding site and drug affinity. E. focardii genome mining and the comparison with the mesophilic sibling counterpart can be used as an inspiration for molecular engineering for medical and industrial applications. Full article

Epoxide hydrolases (EHs) catalyze the conversion of epoxides into vicinal diols. The epoxide hydrolase gene from P. chrysosporium was previously cloned and subjected to site-directed mutation to study its enzyme activity, but the results were unsatisfactory. This study used error prone PCR and DNA shuffling to construct a PchEHA mutation library. We performed mutation-site combinations on PchEHA based on enzyme activity measurement results combined with directed evolution technology. More than 15,000 mutants were randomly selected for the preliminary screening of PchEHA enzyme activity alongside 38 mutant strains with increased enzyme activity or enantioselectivity. Protein expression and purification were conducted to determine the hydrolytic activity of PchEHA, and three mutants increased their activity by more than 95% compared with that of the wt. After multiple rounds of screening and site-specific mutagenesis, we found that F3 offers the best enzyme activity and enantioselectivity; furthermore, the molecular docking results confirmed this result. Overall, this study uncovered novel mutants with potential value as industrial biocatalysts. Full article

Lipoxygenases (LOXs) from lower organisms have substrate flexibility and function versatility in fatty acid oxidation, but it is not clear how these LOXs acquired the ability to execute multiple functions within only one catalytic domain. This work studied a multifunctional LOX from red alga Pyropia haitanensis (PhLOX) which combined hydroperoxidelyase (HPL) and allene oxide synthase (AOS) activity in its active pocket. Molecular docking and site-directed mutagenesis revealed that Phe642 and Phe826 jointly regulated the double peroxidation of fatty acid, Gln777 and Asn575 were essential to the AOS function, and the HPL activity was improved when Asn575, Gln777, or Phe826 was replaced by leucine. Phylogenetic analysis indicated that Asn575 and Phe826 were unique amino acid sites in the separated clades clustered with PhLOX, whereas Phe642 and Gln777 were conserved in plant or animal LOXs. The N-terminal START/RHO_alpha_C/PITP/Bet_v1/CoxG/CalC (SRPBCC) domain of PhLOX was another key variable, as the absence of this domain disrupted the versatility of PhLOX. Moreover, the functions of two homologous LOXs from marine bacterium Shewanella violacea and red alga Chondrus crispus were examined. The HPL activity of PhLOX appeared to be inherited from a common ancestor, and the AOS function was likely acquired through mutations in some key residues in the active pocket. Taken together, our results suggested that some LOXs from red algae attained their versatility by amalgamating functional domains of ancestral origin and unique amino acid mutations. Full article

Analysis of the structure of two-domain laccase ScaSL from Streptomyces carpinensis VKM Ac-1300 (with a middle-redox potential) revealed determinants that could affect the increased potential of ScaSL. Site-directed mutagenesis of the ScaSL laccase was carried out, and mutants H286A, H286T, H286W, and F232Y/F233Y were obtained. Replacement of His 286 with Ala led to a decrease in redox potential (0.45 V) and an increase in stability at pH 9 and 11; replacement with Thr led to an increase in redox potential (0.51 V) but to a decrease in the thermal stability of the protein; replacement with Trp did not affect the enzyme properties. Replacement of Phe residues 232 and 233 with Tyr led to a shift in enzyme activity to the acidic pH range without changing the redox potential and a decrease in the thermostability and pH stability of the enzyme. All mutants more efficiently oxidized phenolic substrate 2,6-DMP and were able to participate in direct electron transfer (DET) with MWCNT-modified electrodes. The F232Y/F233/Y mutant was unable to degrade triphenylmethane dyes without a mediator but showed a greater degree of decolorization of azo dyes in the presence of the mediator. The crystal structure of laccase with the highest potential was determined with high resolution. Full article

Here, we report on a bifunctional alginate lyase (Vnalg7) expressed in Pichia pastoris, which can degrade natural Undaria pinnatifida into unsaturated guluronic acid di- and trisaccharide without pretreatment. The enzyme activity of Vnalg7 (3620.00 U/mL-culture) was 15.81-fold higher than that of the original alg (228.90 U/mL-culture), following engineering modification. The degradation rate reached 52.75%, and reducing sugar reached 30.30 mg/mL after combining Vnalg7 (200.00 U/mL-culture) and 14% (w/v) U. pinnatifida for 6 h. Analysis of the action mode indicated that Vnalg7 could degrade many substrates to produce a variety of unsaturated alginate oligosaccharides (AOSs), and the minimal substrate was tetrasaccharide. Site-directed mutagenesis showed that Glu²³⁸, Glu²⁴¹, Glu³¹², Arg²³⁶, His³⁰⁷, Lys⁴¹⁴, and Tyr⁴¹⁸ are essential catalytic sites, while Glu³³⁴, Glu³⁴⁴, and Asp³¹¹ play auxiliary roles. Mechanism analysis revealed the enzymatic degradation pattern of Vnalg7, which mainly recognizes and attacks the third glycosidic linkage from the reducing end of oligosaccharide substrate. Our findings provide a novel alginate lyase tool and a sustainable and commercial production strategy for value-added biomolecules using seaweeds. Full article

Parasporin PS2Aa1, recently renamed Mpp46Aa1, is an anti-cancer protein known for its selectivity against various human cancer cell lines. We genetically modified native PS2Aa1 to create a library of approximately 100 mutants. From this library, we selected promising mutants based on their half-maximal inhibitory concentration (IC₅₀) and sequence variations. In this study, Variant 3–35, with the G257V substitution, demonstrated increased cytotoxicity and selectivity against the colon cancer cell line SW480. Conversely, Variant N65, featuring substitutions N92D, K175R, and S218G, yielded the most favorable results against the cancer cell lines SW-620, MOLT-4, and Jurkat. The caspase 3/7 and 9, Annexin V-Cy3 and 6-GFDA activities, and, most notably, mitochondrial membrane permeabilization assays confirmed the apoptotic marker elevation. These findings indicate that residues 92, 175, 218, and 257 may play a critical role in the cytotoxic activity and selectivity. We successfully obtained genetically improved variants with substitutions at these key amino acid positions. Additionally, we conducted molecular dynamic simulations to explore the potential interactions between PS2Aa1 and the CD59 GPI-anchored protein. The simulation results revealed that residues 57, 92, and 101 were consistently present, suggesting their possible significance in the interactions between parasporin and the CD59 protein. Full article

System x_c⁻, the cystine/glutamate exchanger, is a membrane transporter that plays a critical role in the antioxidant response of cells. Recent work has shown that System x_c⁻ localizes to the plasma membrane during oxidative stress, allowing for increased activity to support the production of glutathione. In this study, we used site-directed mutagenesis to examine the role of C-terminal lysine residues (K422, K472, and K473) of xCT (SLC7A11) in regulating System x_c⁻. We observed that K473R exhibits loss of transporter activity and membrane localization and is 7.5 kD lower in molecular weight, suggesting that K473 regulates System x_c⁻ trafficking and is modified under basal conditions. After ruling out ubiquitination and neddylation, we demonstrated that unlike WT xCT, K473R lacks N- and O-glycosylation and is sequestered in the endoplasmic reticulum. Next, we demonstrated that K473Q, a constitutively acetylated lysine mimic, also exhibits loss of transporter activity, decreased membrane expression, and a 4 kD decrease in molecular weight; however, it is N- and O-glycosylated and localized to the endoplasmic reticulum and Golgi. These results suggest that acetylation and deacetylation of K473 in the endoplasmic reticulum and Golgi, respectively, serve to regulate the progression of the transporter through the biosynthetic pathway. Full article

This study identified a salt-tolerant GH11 xylanase, Xyn^st, which was isolated from a soil bacterium Bacillus sp. SC1 and can resist as high as 4 M NaCl. After rational design and high-throughput screening of site-directed mutant libraries, a double mutant W6F/Q7H with a 244% increase in catalytic activity and a 10 °C increment in optimal temperature was obtained. Both Xyn^st and W6F/Q7H xylanases were stimulated by high concentrations of salts. In particular, the activity of W6F/Q7H was more than eight times that of Xyn^st in the presence of 2 M NaCl at 65 °C. Kinetic parameters indicated they have the highest affinity for beechwood xylan (K_m = 0.30 mg mL⁻¹ for Xyn^st and 0.18 mg mL⁻¹ for W6F/Q7H), and W6F/Q7H has very high catalytic efficiency (K_cat/K_m = 15483.33 mL mg⁻¹ s⁻¹). Molecular dynamic simulation suggested that W6F/Q7H has a more compact overall structure, improved rigidity of the active pocket edge, and a flexible upper-end alpha helix. Hydrolysis of different xylans by W6F/Q7H released more xylooligosaccharides and yielded higher proportions of xylobiose and xylotriose than Xyn^st did. The conversion efficiencies of Xyn^st and W6F/Q7H on all tested xylans exceeded 20%, suggesting potential applications in the agricultural and food industries. Full article

The human cytomegalovirus (HCMV) glycoprotein B (gB) is the viral fusogen required for entry into cells and for direct cell-to-cell spread of the virus. We have previously demonstrated that the exchange of the carboxy-terminal domain (CTD) of gB for the CTD of the structurally related fusion protein G of the vesicular stomatitis virus (VSV-G) resulted in an intrinsically fusion-active gB variant (gB/VSV-G). In this present study, we employed a dual split protein (DSP)-based cell fusion assay to further characterize the determinants of fusion activity in the CTD of gB. We generated a comprehensive library of gB CTD truncation mutants and identified two mutants, gB-787 and gB-807, which were fusion-competent and induced the formation of multinucleated cell syncytia in the absence of other HCMV proteins. Structural modeling coupled with site-directed mutagenesis revealed that gB fusion activity is primarily mediated by the CTD helix 2, and secondarily by the recruitment of cellular SH2/WW-domain-containing proteins. The fusion activity of gB-807 was inhibited by gB-specific monoclonal antibodies (MAbs) targeting the antigenic domains AD-1 to AD-5 within the ectodomain and not restricted to MAbs directed against AD-4 and AD-5 as observed for gB/VSV-G. This finding suggested a differential regulation of the fusion-active conformational state of both gB variants. Collectively, our findings underscore a pivotal role of the CTD in regulating the fusogenicity of HCMV gB, with important implications for understanding the conformations of gB that facilitate membrane fusion, including antigenic structures that could be targeted by antibodies to block this essential step in HCMV infection. Full article

The carnitine/acylcarnitine carrier (CAC) is a crucial protein for cellular energy metabolism, facilitating the exchange of acylcarnitines and free carnitine across the mitochondrial membrane, thereby enabling fatty acid β-oxidation and oxidative phosphorylation (OXPHOS). Although CAC has not been crystallised, structural insights are derived from the mitochondrial ADP/ATP carrier (AAC) structures in both cytosolic and matrix conformations. These structures underpin a single binding centre-gated pore mechanism, a common feature among mitochondrial carrier (MC) family members. The functional implications of this mechanism are well-supported, yet the structural organization of the CAC, particularly the formation of dimeric or oligomeric assemblies, remains contentious. Recent investigations employing biochemical techniques on purified and reconstituted CAC, alongside molecular modelling based on crystallographic AAC dimeric structures, suggest that CAC can indeed form dimers. Importantly, this dimerization does not alter the transport mechanism, a phenomenon observed in various other membrane transporters across different protein families. This observation aligns with the ping–pong kinetic model, where the dimeric form potentially facilitates efficient substrate translocation without necessitating mechanistic alterations. The presented findings thus contribute to a deeper understanding of CAC’s functional dynamics and its structural parallels with other MC family members. Full article

Acetylation modification has become one of the most popular topics in protein post-translational modification (PTM) research and plays an important role in bacterial virulence. A previous study indicated that the virulence-associated caseinolytic protease proteolytic subunit (ClpP) is acetylated at the K165 site in Vibrio alginolyticus strain HY9901, but its regulation regarding the virulence of V. alginolyticus is still unknown. We further confirmed that ClpP undergoes lysine acetylation (Kace) modification by immunoprecipitation and Western blot analysis and constructed the complementation strain (C-clpP) and site-directed mutagenesis strains including K165Q and K165R. The K165R strain significantly increased biofilm formation at 36 h of incubation, and K165Q significantly decreased biofilm formation at 24 h of incubation. However, the acetylation modification of ClpP did not affect the extracellular protease (ECPase) activity. In addition, we found that the virulence of K165Q was significantly reduced in zebrafish by in vivo injection. To further study the effect of lysine acetylation on the pathogenicity of V. alginolyticus, GS cells were infected with four strains, namely HY9901, C-clpP, K165Q and K165R. This indicated that the effect of the K165Q strain on cytotoxicity was significantly reduced compared with the wild-type strain, while K165R showed similar levels to the wild-type strain. In summary, the results of this study indicate that the Kace of ClpP is involved in the regulation of the virulence of V. alginolyticus. Full article

Acid-sensing ion channels (ASICs), which act as proton-gating sodium channels, have garnered attention as pharmacological targets. ASIC1a isoform, notably prevalent in the central nervous system, plays an important role in synaptic plasticity, anxiety, neurodegeneration, etc. In the peripheral nervous system, ASIC1a shares prominence with ASIC3, the latter well established for its involvement in pain signaling, mechanical sensitivity, and inflammatory hyperalgesia. However, the precise contributions of ASIC1a in peripheral functions necessitate thorough investigation. To dissect the specific roles of ASICs, peptide ligands capable of modulating these channels serve as indispensable tools. Employing molecular modeling, we designed the peptide targeting ASIC1a channel from the sea anemone peptide Ugr9-1, originally targeting ASIC3. This peptide (A23K) retained an inhibitory effect on ASIC3 (IC₅₀ 9.39 µM) and exhibited an additional inhibitory effect on ASIC1a (IC₅₀ 6.72 µM) in electrophysiological experiments. A crucial interaction between the Lys23 residue of the A23K peptide and the Asp355 residue in the thumb domain of the ASIC1a channel predicted by molecular modeling was confirmed by site-directed mutagenesis of the channel. However, A23K peptide revealed a significant decrease in or loss of analgesic properties when compared to the wild-type Ugr9-1. In summary, using A23K, we show that negative modulation of the ASIC1a channel in the peripheral nervous system can compromise the efficacy of an analgesic drug. These results provide a compelling illustration of the complex balance required when developing peripheral pain treatments targeting ASICs. Full article

The Lactobacillales (LB) stand apart among bacterial orders, using manganese (Mn) instead of iron to support their growth and swiftly ferment complex foods while acidifying their environment. The present work investigates whether a shift in the use of Mn could mark the origin of LB. Transmembrane carriers of the ubiquitous Slc11 family play key roles in LB physiology by catalyzing proton-dependent Mn import. In prior studies, the Slc11 clade found in LB (MntH Cb, MCb) showed both remarkable structural plasticity and highly efficient Mn uptake, and another Slc11 clade, MCg1, demonstrated divergent evolution coinciding with emergence of bacterial genera (e.g., Bordetella, Achromobacter). Herein, the Slc11 clade MCb is subdivided in sister groups: MCb_ie and MCb_gut. MCb_ie derives directly from the Slc11 clade MCa, pointing an intermediate stage in the evolution of MCb_gut. MCb_ie predominates in marine Bacillaceae, is more conserved than MCb_gut, lacks the structural plasticity that typify MCb_gut carriers, and responds differently to identical mutagenesis. Exchanging MCb_ie/MCb_gut amino acid residues at sites that distinguish these clades showed conformation-dependent effects with both MCb_ie and MCb_gut templates, and the 3D location of the targeted sites in the carrier structure together suggests that the mechanism to open the inner gate, and release Mn into the cytoplasm, differs between MCb_ie and MCb_gut. Building on the established phylogeny for Enterococcus revealed that a pair of genes encoding MCb_gut was present in the common ancestor of LB, as MCb_gu1 and MCb_gu2 templates exhibited distinct structural dynamics properties. These data are discussed when examining whether MCb_gut⁺ LB could emerge in the upper gut of early vertebrates (ca. 540 mya), through genome contraction and evolution toward Mn-centrism, as they specialized as gastric aids favoring stomach establishment in jawed vertebrates through bi-directional communication with host nervous, endocrine and immune systems. Full article

An imbalance in estrogen signaling is a critical event in breast tumorigenesis. The majority of breast cancers (BCs) are hormone-sensitive; they majorly express the estrogen receptor (ER+) and are activated by 17β-estradiol (E2). The steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step in steroid biosynthesis. The dysregulation of the epigenetic machinery, modulating E2 levels, is a primary occurrence for promoting breast tumorigenesis. StAR expression, concomitant with E2 synthesis, was reported to be aberrantly high in human and mouse hormone-dependent BC cells compared with their non-cancerous counterparts. However, the mechanism of action of StAR remains poorly understood. We discovered StAR as an acetylated protein and have identified a number of lysine (K) residues that are putatively acetylated in malignant and non-malignant breast cells, using LC-MS/MS (liquid chromatography–tandem mass spectrometry), suggesting they differently influence E2 synthesis in mammary tissue. The treatment of hormone-sensitive MCF7 cells with a variety of histone deacetylase inhibitors (HDACIs), at therapeutically and clinically relevant doses, identified a few additional StAR acetylated lysine residues. Among a total of fourteen StAR acetylomes undergoing acetylation and deacetylation, K111 and K253 were frequently recognized either endogenously or in response to HDACIs. Site-directed mutagenesis studies of these two StAR acetylomes, pertaining to K111Q and K253Q acetylation mimetic states, resulted in increases in E2 levels in ER+ MCF7 and triple negative MB-231 BC cells, compared with their values seen with human StAR. Conversely, these cells carrying K111R and K253R deacetylation mutants diminished E2 biosynthesis. These findings provide novel and mechanistic insights into intra-tumoral E2 regulation by elucidating the functional importance of this uncovered StAR post-translational modification (PTM), involving acetylation and deacetylation events, underscoring the potential of StAR as a therapeutic target for hormone-sensitive BC. Full article