Double-site Mutants Research Articles

N-glycosylation of Wnt3 regulates the progression of hepatocellular carcinoma by affecting Wnt/β-catenin signal pathway.

Wnt/FZD-mediated signaling pathways are activated in more than 90% of hepatocellular carcinoma (HCC) cell lines. As a well-known secretory glycoprotein, Wnt3 can interact with FZD receptors on the cell surface, thereby activating the Wnt/β-catenin signaling pathway. However, the N-glycosylation modification site of Wnt3 and the effect of this modification on the biological function of the protein are still unclear. To investigate the effect of Wnt3 N-glycosylation on the biological function of HCC cells. Site-directed mutagenesis was used to verify the Wnt3 N-glycosylation sites, actinomycin D treatment was used to detect the stability of Wnt3 after site-directed mutation, the binding of the N-glycosylation site-directed mutant Wnt3 to FZD7 was observed by laser confocal microscopy, and the effects of the N-glycosylation site-directed mutation of Wnt3 on the Wnt/β-catenin signaling pathway and the progression of HCC cells were detected by western blot and cell function experiments. Wnt3 has two N-glycosylation-modified sites (Asn90 and Asn301); when a single site at amino acid 301 is mutated, the stability of Wnt3 is weakened; the binding ability of Wnt3 to FZD7 decreases when both sites are mutated simultaneously; and the level of proteins related to the Wnt/β-catenin signaling pathway is downregulated. Cell proliferation, migration and invasion are also weakened in the case of single 301 site and double-site mutations. These results indicate that by inhibiting the N-glycosylation of Wnt3, the proliferation, migration, invasion and colony formation abilities of liver cancer cells can be weakened, which might provide new therapeutic strategies for clinical liver cancer in the future.

Enhancing the Thermal Stability and Enzyme Activity of Ketopantoate Hydroxymethyltransferase through Interface Modification Engineering.

Ketopantoate hydroxymethyltransferase (KPHMT) plays a pivotal role in d-pantothenic acid biosynthesis. Most KPHMTs are homodecamers with low thermal stability, posing challenges for protein engineering and limiting output enhancement. Previously, a high-enzyme activity KPHMT mutant (K25A/E189S) from Corynebacterium glutamicum was screened as mother strain (M0). Building upon this strain, our study focused on interface engineering modifications, employing a multifaceted approach including integrating folding-free energy calculation, B-factor analysis, and conserved site analysis. Preliminary screening led to the selection of five mutants in the interface─E106S, E98T, E98N, S247I, and S247D─showing improved thermal stability, culminating in the double-site mutant M8 (M0-E98N/S247D). M8 exhibited a T1/2 value of 288.79 min at 50 °C, showing a 3.29-fold increase compared to M0. Meanwhile, the Tm value of M8 was elevated from 53.2 to 59.6 °C. Investigations of structural and molecular dynamics simulations revealed alterations in surface electrostatic charge distribution and the formation of increased hydrogen bonds between subunits, contributing to enhanced thermal stability. This investigation corroborates the efficacy of interface engineering modifications in bolstering KPHMT stability while showing its potential for positively impacting industrial d-pantothenic acid synthesis.

Simultaneous Enhancement of the Thermostability and Catalytic Activity of D-Allulose 3-Epimerase from Clostridium bolteae ATTC BAA-613 Based on the "Back to Consensus Mutations" Hypothesis.

D-Allulose is a high value rare sugar with multiple physiological functions and commercial potential that can be enzymatically synthesized from D-fructose by D-allulose 3-epimerase (DAEase). Poor catalytic activity and thermostability of DAEase prevent the industrial production of D-allulose. In this work, rational design was applied to a previously identified DAEase from Clostridium bolteae ATCC BAA-613 based on the "back to consensus mutations" hypothesis, and the catalytic activity of the Cb-I265 V variant was enhanced 2.5-fold. Furthermore, the Cb-I265 V/E268D double-site variant displayed 2.0-fold higher specific catalytic activity and 1.4-fold higher thermostability than the wild-type enzyme. Molecular docking and kinetic simulation results indicated increased hydrogen bonds between the active pocket and substrate, possibly contributing to the improved thermal stability and catalytic activity of the double-site mutant. The findings outlined a feasible approach for the rational design of multiple preset functions of target enzymes simultaneously.

Production of 14α-Hydroxy Progesterone Using a Steroidal Hydroxylase from Cochliobolus lunatus Expressed in Escherichia coli

Steroids with hydroxylation at C14 are drawing increased attention because of their diverse biological activities and applications. P-450lun from Cochliobolus lunatus is the first fungal cytochrome P450 reported to have 14α-hydroxylase activity. Studies have shown that P-450lun catalyzes the hydroxylation of progesterone (PROG) at C14α with low regiospecificity and activity. To improve its regiospecificity and activity for PROG, truncated forms of P-450lun and its cognate redox partner CPRlun were functionally co-expressed in Escherichia coli. Then, a semi-rational protein engineering approach was applied to P-450lun, resulting in a double-site mutant E109A/F297W with enhanced 14α-position selectivity for PROG compared with the wild-type P-450lun (97% vs. 28%). Protein structure analysis revealed that the F297W substitution can hinder the binding pose for 11β-hydroxylation product formation. Finally, whole-cell catalysis was optimized, and the final titer of 14α-OH-PROG reached 16.0 mg/L. This is the first report where a fungal 14α-hydroxylase was functionally expressed in Escherichia coli. The steroid hydroxylation system obtained in this study can serve as a basis for the synthesis of 14α-hydroxylated PROG and the rapid evolution of eukaryotic cytochrome P-450lun.

Semirational Design Based on Consensus Sequences to Balance the Enzyme Activity-Stability Trade-Off.

In this study, the phenomenon of the stability-activity trade-off, which is increasingly recognized in enzyme engineering, was explored. Typically, enhanced stability in enzymes correlates with diminished activity. Utilizing Rosa roxburghii copper-zinc superoxide dismutase (RrCuZnSOD) as a model, single-site mutations were introduced based on a semirational design derived from consensus sequences. The initial set of mutants was selected based on activity, followed by combinatorial mutation. This approach yielded two double-site mutants, D25/A115T (18,688 ± 206 U/mg) and A115T/S135P (18,095 ± 1556 U/mg), exhibiting superior enzymatic properties due to additive and synergistic effects. These mutants demonstrated increased half-lives (T1/2) at 80 °C by 1.2- and 1.6-fold, respectively, and their melting temperatures (Tm) rose by 3.4 and 2.5 °C, respectively, without any loss in activity relative to the wild type. Via an integration of structural analysis and molecular dynamics simulations, we elucidated the underlying mechanism facilitating the concurrent enhancement of both thermostability and enzymatic activity.

Specificity Enhancement of Glutenase Bga1903 toward Celiac Disease-Eliciting Pro-Immunogenic Peptides via Active-Site Modification.

Celiac disease is an autoimmune disease triggered by oral ingestion of gluten, with certain gluten residues resistant to digestive tract enzymes. Within the duodenum, the remaining peptides incite immunogenic responses, including the generation of autoantibodies and inflammation, leading to irreversible damage. Our previous exploration unveiled a glutenase called Bga1903 derived from the Gram-negative bacterium Burkholderia gladioli. The cleavage pattern of Bga1903 indicates its moderate ability to mitigate the toxicity of pro-immunogenic peptides. The crystal structure of Bga1903, along with the identification of subsites within its active site, was determined. To improve its substrate specificity toward prevalent motifs like QPQ within gluten peptides, the active site of Bga1903 underwent site-directed mutagenesis according to structural insights and enzymatic kinetics. Among the double-site mutants, E380Q/S387L exhibits an approximately 34-fold increase in its specificity constant toward the QPQ sequence, favoring glutamines at the P1 and P3 positions compared to the wild type. The increased specificity of E380Q/S387L not only enhances its ability to break down pro-immunogenic peptides but also positions this enzyme variant as a promising candidate for oral therapy for celiac disease.

Engineering a BsBDHA substrate-binding pocket entrance for the improvement in catalytic performance toward (R)-phenyl-1,2-ethanediol based on the computer-aided design

Oxidation reactions of vicinal diols using (2,3)-butanediol dehydrogenases hold promise for producing α-hydroxy ketones or in cascade biocatalytic processes for chiral amino alcohols. However, their application is restricted by low specific activities and catalytic efficiencies. In this study, to enhance the catalytic activity toward (R)-phenyl-1,2-ethanediol (PED), a Bacillus subtilis (2,3)-butanediol dehydrogenase (BsBDHA) was engineered by reshaping its substrate-binding pocket (SBP) entrance based on computer-aided design. Firstly, based on molecular docking simulation, seven residues of BsBDHA were identified and subjected to leucine scanning mutagenesis. Two E. coli transformants, E. coli/bsbdhaI49L and /bsbdhaV266L, were confirmed with oxidation activities of 0.85 and 0.87 U/g wet cell, respectively, being 1.20- and 1.23-fold that of E. coli/bsbdha. Secondly, both Ile49 and Val266 were subjected to partial site-saturation and combinatorial mutagenesis. One double-site mutant, BsBDHAI49L/V266L, was obtained and its specific activity and kcat/Km toward (R)-PED increased to 1.79 U/mg and 0.39 mM−1 s−1, respectively. These values were 3.3- and 4.9-fold those of BsBDHA. Finally, the analysis of molecular mechanism for BsBDHAI49L/V266L with enhanced catalytic efficiency was performed by molecular dynamics simulation. This study is the first report on the improvement of catalytic performance of BsBDHA toward (R)-PED and provides useful reference data for the further studies.

Improved Bioproduction of the Nylon 12 Monomer by Combining the Directed Evolution of P450 and Enhancing Heme Synthesis.

The nylon 12 (PA12) monomer ω-aminododecanoic acid (ω-AmDDA) could be synthesized from lauric acid (DDA) through multi-enzyme cascade transformation using engineered E. coli, with the P450 catalyzing terminal hydroxylation of DDA as a rate-limiting enzyme. Its activity is jointly determined by the heme domain and the reductase domain. To obtain a P450 mutant with higher activity, directed evolution was conducted using a colorimetric high-throughput screening (HTS) system with DDA as the real substrate. After two rounds of directed evolution, a positive double-site mutant (R14R/D629G) with 90.3% higher activity was obtained. Molecular docking analysis, kinetic parameter determination and protein electrophoresis suggested the improved soluble expression of P450 resulting from the synonymous mutation near the N-terminus and the shortened distance of the electron transfer between FMN and FAD caused by D629G mutation as the major reasons for activity improvement. The significantly increased kcat and unchanged Km provided further evidence for the increase in electron transfer efficiency. Considering the important role of heme in P450, its supply was strengthened by the metabolic engineering of the heme synthesis pathway. By combining P450-directed evolution and enhancing heme synthesis, 2.02 ± 0.03 g/L of ω-AmDDA was produced from 10 mM DDA, with a yield of 93.6%.

Mutation of two residues converts the ligand-binding domain of RXRα into a uniform monomer without impairing the binding of retinoic acid and cofactors

Retinoid X receptor (RXRα) is a nuclear receptor (NR) for retinoic acid (RA) and regulates various NR signaling pathways. Ligand-binding domain (LBD) of RXRα can bind with its ligand 9-cis-RA and cofactors, and mediate the forming of homodimer and homotetramer of RXRα and its heterodimer with other NRs, conferring RXRα the ability to play complicated roles in development and diseases. Due to the coexistence of monomer, dimer and tetramer, there are difficulties to study the structure and interaction of RXRα-LBD with its ligands and cofactors in solution and to distinguish the roles of different forms of RXRα in cell. Here, through analyzing available structures of RXRα-LBD, we selected two residues, D379 and L420, in the homodimer interface to design three mutants of RXRα-LBD. Recombinant proteins of the three mutants showed decreased proportions of dimer and tetramer but unchanged overall structure and binding affinities to 9-cis-RA, corepressor SMRT, and coactivator SRC2. Especially, the double-site mutant RXRα-LBDD379A−L420G existed as a uniform monomer. Furthermore, L420 was found to play a similar role in forming RXRα-LBD homodimer and its heterodimer with various NRs, while the role of D379 varies a lot, as it shows almost no interaction with RARα/β, LXRα/β, and THRα/β. This study provides a new insight into the mechanism for forming RXRα-LBD homodimer and its heterodimer with other NRs, and will facilitate the studies on the structure and interaction of RXRα-LBD with ligands, cofactors and drugs in solution, and the broad physiological functions of RXRα cooperating with various NRs in cell.

Tuning the catalytic performances of a sucrose isomerase for production of isomaltulose with high concentration.

Obtaining a sucrose isomerase (SIase) with high catalytic performance is of great importance in industrial production of isomaltulose (a reducing sugar). In order to obtain such SIase mutant, a high-throughput screening system in microtiter plate format was developed based on a widely used 2,4-dinitrosalicylic acid (DNS) method for determination of reducing sugar. An SIase from Erwiniasp. Ejp617 (ErSIase) was selected to improve its catalytic efficiency. After screening of ~ 8000 mutants from a random mutagenesis library, Q209 and R456 were identified as beneficial positions. Saturation mutagenesis of the two positions resulted in a double-site mutant ErSIase_Q209S-R456H that showed the highest catalytic efficiency, and its specific activity reached 684 U/mg that is 17.5-fold higher than that of the wild-type ErSIase. By employing the lyophilized Escherichia coli (E. coli) cells harboring ErSIase_Q209S-R456H, a high space-time yield (STY = 3.9kg/(L·d)) was achieved toward 600g/L sucrose. Furthermore, the in silico analysis suggested that the hydrogen bond network was improved and steric hindrance was reduced due to the beneficial substitutions.Key points• A sucrose isomerase mutant with high catalytic efficiency was obtained.• The highest space-time yield was achieved toward high-concentration sucrose.• The optimized H-bond network contributed to the enhanced catalytic efficiency.

Using multiple-fragment amplification combined with Gibson assembly to clone genes with site-directed mutations

In order to develop a simple and efficient site-directed mutagenesis solution, the Gibson assembly technique was used to clone the cyclin dependent kinase 4 gene with single or double site mutations, with the aim to simplify the overlap extension PCR. The gene fragments containing site mutations were amplified using a strategy similar to overlap extension PCR. Meanwhile, an empty plasmid was digested by double restriction endonucleases to generate a linearized vector with a short adaptor overlapping with the targeted gene fragments. The gene fragments were directly spliced with the linearized vector by Gibson assembly in an isothermal, single-reaction, creating a recombinant plasmid. After the recombinant plasmids were transformed into competent Escherichia coli DH5α, several clones were screened from each group. Through restriction analysis and DNA sequencing, it was found that the randomly selected clones were 100% target mutants. Since there was neither tedious multiple-round PCR amplification nor frequent DNA extraction operation, and there was no need to digest the original plasmid, this protocol circumvents many factors that may interfere with the conventional site-directed mutagenesis. Hence, genes with single or multiple mutations could be cloned easily and efficiently. In summary, the major defects associated with overlap extension PCR and rolling circle amplification were circumvented in this protocol, making it a good solution for site-directed mutagenesis.

A multifunctional enolase mediates cytoadhesion and interaction with host plasminogen and fibronectin in Mycoplasma hyorhinis

Mycoplasma hyorhinis may cause systemic inflammation of pigs, typically polyserositis and arthritis, and is also associated with several types of human cancer. However, the pathogenesis of M. hyorhinis colonizing and breaching the respiratory barrier to establish systemic infection is poorly understood. Glycolytic enzymes are important moonlighting proteins and virulence-related factors in various bacteria. In this study, we investigated the functions of a glycolytic critical enzyme, enolase in the infection and systemic spread of M. hyorhinis. Bacterial surface localization of enolase was confirmed by flow cytometry and colony hybridization assay. Recombinant M. hyorhinis enolase (rEno) was found to adhere to pig kidney (PK-15) cells, and anti-rEno serum significantly decreased adherence. The enzyme was also found to bind host plasminogen and fibronectin, and interactions were specific and strong, with dissociation constant (KD) values of 1.4 nM and 14.3 nM, respectively, from surface plasmon resonance analysis. Activation of rEno-bound plasminogen was confirmed by its ability to hydrolyze plasmin-specific substrates and to degrade a reconstituted extracellular matrix. To explore key sites during these interactions, C-terminal lysine residues of enolase were replaced with leucine, and the resulting single-site and double-site mutants show significantly reduced interaction with plasminogen in far-Western blotting and surface plasmon resonance tests. The binding affinities of all mutants to fibronectin were reduced as well. Collectively, these results imply that enolase moonlights as an important adhesin of M. hyorhinis, and interacts with plasminogen and fibronectin. The two lysine residues in the C-terminus are important binding sites for its multiple binding activities.

Structure Based Affinity Maturation and Characterizing of SARS-CoV Antibody CR3022 against SARS-CoV-2 by Computational and Experimental Approaches.

The COVID-19 epidemic is raging around the world. Neutralizing antibodies are powerful tools for the prevention and treatment of SARS-CoV-2 infection. Antibody CR3022, a SARS-CoV neutralizing antibody, was found to cross-react with SARS-CoV-2, but its affinity was lower than that of its binding with SARS-CoV, which greatly limited the further development of CR3022 against SARS-CoV-2. Therefore, it is necessary to improve its affinity to SARS-CoV-2 in vitro. In this study, the structure-based molecular simulations were utilized to virtually mutate the possible key residues in the complementarity-determining regions (CDRs) of the CR3022 antibody. According to the criteria of mutation energy, the mutation sites that have the potential to impact the antibody affinity were then selected. Then optimized CR3022 mutants with the enhanced affinity were further identified and verified by enzyme-linked immunosorbent assay (ELISA), surface plasma resonance (SPR) and autoimmune reactivity experiments. Finally, molecular dynamics (MD) simulation and binding free energy calculation (MM/PBSA) were performed on the wild-type CR3022 and its two double-site mutants to understand in more detail the contribution of these sites to the higher affinity. It was found that the binding affinity of the CR3022 antibody could be significantly enhanced more than ten times after the introduction of the S103F/Y mutation in HCDR–3 and the S33R mutation in LCDR–1. The additional hydrogen-bonding, hydrophobic interactions, as well as salt-bridges formed between the modified double-site mutated antibody and SARS-CoV-2 RBD were identified. The computational and experimental results clearly demonstrated that the affinity of the modified antibody has been greatly enhanced. This study indicates that CR3022 as a neutralizing antibody recognizing the conserved region of RBD against SARS-CoV with cross-reactivity with SARS-CoV-2, a different member in a large family of coronaviruses, could be improved by the computational and experimental approaches which provided insights for developing antibody drugs against SARS-CoV-2.

Molecular genotyping of G6PD mutations for neonates in Ningbo area.

The aim of this study is to determine the cut‐off value of glucose‐6‐phosphate dehydrogenase (G6PD) activity and the mutation spectrum of G6PD gene in neonates with G6PD deficiency at Ningbo. Around 82233 neonatal blood samples were measured to determine G6PD activity. The positive samples were further detected with gene analysis. A total of 445 neonates were confirmed as G6PD deficiency, and the incidence in Ningbo was 1/185. 17 types of G6PD gene mutations were found, including 11 single‐site mutations and 6 double‐site mutations. Considering the significant differences in G6PD activity, the cut‐off value was detected to be 2.35 and 3.65 U/gHb for males and females, respectively. Significant differences in G6PD activities were noted and found to be varied from 4.61 to 6.02 U/gHb in different seasons (p < 0.0001). G6PD deficiency screening is a significant detection test for neonatal G6PD deficiency prevention. Our study highlights that the screening should be done using different cut‐off values according to the sexes in different seasons.

Remarkable improvement in the regiocomplementarity of a Glycine max epoxide hydrolase by reshaping its substrate-binding pocket for the enantioconvergent preparation of (R)-hexane-1,2-diol

• A superior double-site mutant of Gm EH3, Gm EH3 W102V/P187F , was obtained by semi-rational design. • The α S of Gm EH3 W102V/P187F for ( S )- 1a was obviously increased to 89.7% from 72.4% of Gm EH3. • Enantioconvergent hydrolysis of rac - 1a by E. coli / gmeh3 W102V/P187F gave ( R )- 1b with 83.1% ee p . E. coli / gmeh3 , an E. coli transformant expressing Gm EH3, had the best regiocomplementarity (α S = 72.4% and β R = 97.6%) for ( S )- and ( R )-1,2-epoxyhexanes among five tested aliphatic chain rac -1,2-epoxides ( 1a – 5a ). To prepare ( R )-hexane-1,2-diol ( 1b ) with high ee p via enantioconvergent hydrolysis of rac - 1a , the regioselectivity coefficient α S of Gm EH3 for ( S )- 1a was improved by reshaping its substrate-binding pocket (SBP). Based on the semi-rational design, Trp 102 , Ile 105 , Ile 178 , Pro 187 and Leu 189 lining Gm EH3’s SBP were identified, each of which was substituted with four residues, respectively. From 17 transformants harboring single-site variants of gmeh3, E. coli / gmeh3 W102L , / gmeh3 W102I , / gmeh3 W102V and / gmeh3 P187F were selected, catalyzing the conversion of rac - 1a into ( R )- 1b with obviously enhanced ee p values of 59.3–78.3%. Then, three transformants containing double-site variants were constructed by combinatorial mutagenesis of gmeh3 P187F separately with gmeh3 W102L , gmeh3 W102I and gmeh3 W102V . Among the three transformants, E. coli / gmeh3 W102V/P187F displayed the largest α S of 89.7% with β R of 96.2%. The enantioconvergent hydrolysis of 500 mM rac -1a was conducted using 200 mg/mL wet cells of E. coli / gmeh3 W102V/P187F at 25°C for 12 h, affording ( R )- 1b with 83.1% ee p and 91.5% yield. The molecular docking simulation analysis demonstrated that Gm EH3 W102V/P187F more regiopreferentially attacks C α in the oxirane ring of ( S )- 1a than Gm EH3.

Discovering and Characterizing of Survivin Dominant Negative Mutants With Stronger Pro-apoptotic Activity on Cancer Cells and CSCs.

Survivin as a member of the inhibitor of apoptosis proteins (IAPs) family is undetectable in normal cells, but highly expressed in cancer cells and cancer stem cells (CSCs) which makes it an attractive target in cancer therapy. Survivin dominant negative mutants have been reported as competitive inhibitors of endogenous survivin protein in cancer cells. However, there is a lack of systematic comparative studies on which mutants have stronger effect on promoting apoptosis in cancer cells, which will hinder the development of novel anti-cancer drugs. Here, based on the previous study of survivin and its analysis of the relationship between structure and function, we designed and constructed a series of different amino acid mutants from survivin (TmSm34, TmSm48, TmSm84, TmSm34/48, TmSm34/84, and TmSm34/48/84) fused cell-permeable peptide TATm at the N-terminus, and a dominant negative mutant TmSm34/84 with stronger pro-apoptotic activity was selected and evaluated systematically in vitro. The double-site mutant of survivin (TmSm34/84) showed more robust pro-apoptotic activity against A549 cells than others, and could reverse the resistance of A549 CSCs to adriamycin (ADM) (reversal index up to 7.01) by decreasing the expression levels of survivin, P-gp, and Bcl-2 while increasing cleaved caspase-3 in CSCs. This study indicated the selected survivin dominant negative mutant TmSm34/84 is promising to be an excellent candidate for recombinant anti-cancer protein by promoting apoptosis of cancer cells and their stem cells and sensitizing chemotherapeutic drugs.

Structure-guided improvement in the enantioselectivity of an Aspergillus usamii epoxide hydrolase for the gram-scale kinetic resolution of ortho-trifluoromethyl styrene oxide

Microtuning the substrate-binding pocket (SBP) of EHs has emerged as an effective approach to manipulate their enantio- or regio-selectivities and activities towards target substrates. Here, the enantioselectivity (enantiomeric ratio, E) of AuEH2 towards a racemic (rac-) ortho-trifluoromethyl styrene oxide (o-TFMSO) was improved via microtuning its SBP. Based on the analysis on the crystal structure of AuEH2, its specific residues I192, Y216, R322 and L344 lining the SBP in close to the catalytic triad were identified for site-saturation mutagenesis. After screening, five single-site mutants were selected with E values elevated from 8 to 12―25 towards rac-o-TFMSO. To further improve E, four double-site mutants were constructed by combinatorial mutagenesis of AuEH2R322V separately with AuEH2I192V, AuEH2Y216F, AuEH2L344A and AuEH2L344C. Among all the mutants, AuEH2R322V/L344C possessed the largest E of 83 with activity of 67 U/g wet cell. The kinetic resolution of 200 mM rac-o-TFMSO was conducted at 0 °C for 5.5 h using 80 mg/mL wet cells of E. coli/Aueh2R322V/L344C, a transformant expressing AuEH2R322V/L344C, retaining (S)-o-TFMSO with 98.4 % ees and 49.3 % yields. Furthermore, the molecular docking simulation analysis indicated that AuEH2R322V/L344C more enantiopreferentially attacks the terminal carbon (Cβ) in the oxirane ring of (R)-o-TFMSO than AuEH2.

Glycosylation of β1 subunit plays a pivotal role in the toxin sensitivity and activation of BK channels

Background:The accessory β1 subunits, regulating the pharmacological and biophysical properties of BK channels, always undergo post-translational modifications, especially glycosylation. To date, it remains elusive whether the glycosylation contributes to the regulation of BK channels by β1 subunits. Methods:Herein, we combined the electrophysiological approach with molecular mutations and biochemical manipulation to investigate the function roles of N-glycosylation in β1 subunits.Results: The results show that deglycosylation of β1 subunits through double-site mutations (β1 N80A/N142A or β1 N80Q/N142Q) could significantly increase the inhibitory potency of iberiotoxin, a specific BK channel blocker. The deglycosylated channels also have a different sensitivity to martentoxin, another BK channel modulator with some remarkable effects as reported before. On the contrary to enhancing effects of martentoxin on glycosylated BK channels under the presence of cytoplasmic Ca2+, deglycosylated channels were not affected by the toxin. However, the deglycosylated channels were surprisingly inhibited by martentoxin under the absence of cytoplasmic Ca2+, while the glycosylated channels were not inhibited under this same condition. In addition, wild type BK (α+β1) channels treated with PNGase F also showed the same trend of pharmacological results to the mutants. Similar to this modulation of glycosylation on BK channel pharmacology, the deglycosylated forms of the channels were activated at a faster speed than the glycosylated ones. However, the V1/2 and slope were not changed by the glycosylation.Conclusion: The present study reveals that glycosylation is an indispensable determinant of the modulation of β1-subunit on BK channel pharmacology and its activation. The loss of glycosylation of β1 subunits could lead to the dysfunction of BK channel, resulting in a pathological state.

Effect of mutations in capsid shell protein on the assembly of BmCPV virus-like particles.

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a typical single-layer capsid dsRNA virus belonging to the genus Cypovirus in the family Reoviridae. The results of cryo-electron microscopy showed that the BmCPV capsid consists of 60 asymmetric units, and each asymmetric unit contains one turret protein (TP), two large protrusion proteins (LPP) and two capsid shell proteins (CSP). CSP has the ability to self-assemble into virus-like particles (VLPs), and the small protrusion domain (SPD) in CSP may play an essential role in the assembly of viral capsids. In this study, three critical amino acid sites, D828, S829 and V945, in the SPD were efficiently mutated (point mutation) based on the principle of PCR circular mutagenesis. Moreover, a multi-gene expression system, Ac-MultiBac baculovirus, was used to produce eight different recombinant VLPs in vitro. Transmission electron microscopy showed that the single site and double site mutations had little effect on the efficiency and morphology of the assembly of VLPs. Still, the simultaneous mutation of the three sites had a significant impact. The experimental results demonstrate that the SPD of CSP plays an essential role in assembly of the viral capsid, which lays the foundation for further analysis of the molecular and structural mechanism of BmCPV capsid assembly.

Enhancing the decolorization activity of Bacillus pumilus W3 CotA-laccase to Reactive Black 5 by site-saturation mutagenesis.

Reactive Black 5 (RB5) is a typical refractory azo dye. Widespread utilization of RB5 has caused a variety of environmental and health problems. The enzymatic degradation of RB5 can be a promising solution due to its superiority as an eco-friendly and cost-competitive process. Bacterial CotA-laccase shows great application prospect to eliminate hazardous dyes from wastewater. However, efficient decolorization of RB5 CotA-laccase generally requires the participation of costly, toxic mediators. In the present study, we modified the amino acids Thr415 and Thr418 near the type 1 copper site and the amino acid Gln442 at the entrance of the substrate-binding pocket of Bacillus pumilus W3 CotA-laccase to boost its RB5 decolorization activity based on molecular docking analysis and site-saturation mutagenesis. Through the strategies, two double site mutants T415D/Q442A and T418K/Q442A obtained demonstrated 43.94 and 52.64% RB5 decolorization rates in the absence of a mediator at pH10.0, respectively, which were about 3.70- and 4.43-fold higher compared with the wild-type CotA-laccase. Unexpectedly, the catalytic efficiency of the T418K/Q442A to ABTS was enhanced by 5.33-fold compared with the wild-type CotA-laccase. The mechanisms of conferring enhanced activity to the mutants were proposed by structural analysis. In summary, the mutants T415D/Q442A and T418K/Q442A have good application potentials for the biodegradation of RB5. KEY POINTS: • Three amino acids of CotA-laccase were manipulated by site-saturation mutagenesis. • Decolorization rate of two mutants to RB5 was enhanced 3.70- and 4.43-fold, respectively. • The mechanisms of awarding enhanced activity to the mutants were supposed.