Unveiling novel insights into human IL-6 - IL-6R interaction sites through 3D computer-guided docking and systematic site mutagenesis.

The cytokine interleukin-6 (IL-6) plays a crucial role in autoimmune and inflammatory diseases. Understanding the precise mechanism of IL-6 interaction at the amino acid level is essential to develop IL-6-inhibiting compounds. In this study, we employed computer-guided drug design tools to predict the key residues that are involved in the interaction between IL-6 and its receptor IL-6R. Subsequently, we generated IL-6 mutants and evaluated their binding affinity to IL-6R and the IL-6R - gp130 complex, as well as monitoring their biological activities. Our findings revealed that the R167A mutant exhibited increased affinity for IL-6R, leading to enhanced binding to IL-6R - gp130 complex and subsequently elevated intracellular phosphorylation of STAT3 in effector cells. On the other hand, although E171A reduced its affinity for IL-6R, it displayed stronger binding to the IL-6R - gp130 complex, thereby enhancing its biological activity. Furthermore, we identified the importance of R178 and R181 for the precise recognition of IL-6 by IL-6R. Mutants R181A/V failed to bind to IL-6R, while maintaining an affinity for the IL-6 - gp130 complex. Additionally, deletion of the D helix resulted in complete loss of IL-6 binding affinity for IL-6R. Overall, this study provides valuable insights into the binding mechanism of IL-6 and establishes a solid foundation for future design of novel IL-6 inhibitors.

Gut microbe-generated phenylacetylglutamine is an endogenous allosteric modulator of ß2-adrenergic receptors.

Allosteric modulation is a central mechanism for metabolic regulation but has yet to be described for a gut microbiota-host interaction. Phenylacetylglutamine (PAGln), a gut microbiota-derived metabolite, has previously been clinically associated with and mechanistically linked to cardiovascular disease (CVD) and heart failure (HF). Here, using cells expressing ß1- versus ß2-adrenergic receptors (ß1AR and ß2AR), PAGln is shown to act as a negative allosteric modulator (NAM) of ß2AR, but not ß1AR. In functional studies, PAGln is further shown to promote NAM effects in both isolated male mouse cardiomyocytes and failing human heart left ventricle muscle (contracting trabeculae). Finally, using in silico docking studies coupled with site-directed mutagenesis and functional analyses, we identified sites on ß2AR (residues E122 and V206) that when mutated still confer responsiveness to canonical ß2AR agonists but no longer show PAGln-elicited NAM activity. The present studies reveal the gut microbiota-obligate metabolite PAGln as an endogenous NAM of a host GPCR.

Comprehensive review on Haloalkane dehalogenase (LinB): a ß-hexachlorocyclohexane (HCH) degrading enzyme.

Haloalkane dehalogenase, LinB, is a member of the α/ß hydrolase family of enzymes. It has a wide range of halogenated substrates, but, has been mostly studied in context of degradation of hexachlorocyclohexane (HCH) isomers, especially ß-HCH (5-12% of total HCH isomers), which is the most recalcitrant and persistent among all the HCH isomers. LinB was identified to directly act on ß-HCH in a one or two step transformation which decreases its toxicity manifold. Thereafter, many studies focused on LinB including its structure determination using X-ray crystallographic studies, structure comparison with other haloalkane dehalogenases, substrate specificity and kinetic studies, protein engineering and site-directed mutagenesis studies in search of better catalytic activity of the enzyme. LinB was mainly identified and characterized in bacteria belonging to sphingomonads. Detailed sequence comparison of LinB from different sphingomonads further revealed the residues critical for its activity and ability to catalyze either one or two step transformation of ß-HCH. Association of LinB with IS6100 elements is also being discussed in detail in sphingomonads. In this review, we summarized vigorous efforts done by different research groups on LinB for developing better bioremediation strategies against HCH contamination. Also, kinetic studies, protein engineering and site directed mutagenesis studies discussed here forms the basis of further exploration of LinB's role as an efficient enzyme in bioremediation projects.

Multistrategy Engineering of an Inulosucrase to Enhance the Activity and Thermostability for Efficient Production of Microbial Inulin.

Inulin has found commercial applications in the pharmaceutical, nutraceutical, and food industries due to its beneficial health effects. The enzymatic biosynthesis of microbial inulin has garnered increasing attention. In this study, molecular modification was applied to Lactobacillus mulieris UMB7800 inulosucrase, an enzyme that specifically produces high-molecular weight inulin, to enhance its catalytic activity and thermostability. Among the 18 variable regions, R5 was identified as a crucial region significantly impacting enzymatic activity by replacing it with more conserved sequences. Site-directed mutagenesis combined with saturated mutagenesis revealed that the mutant A250 V increased activity by 68%. Additionally, after screening candidate mutants by rational design, four single-point mutants, S344D, H434P, E526D, and G531P, were shown to enhance thermostability. The final combinational mutant, M5, exhibited a 66% increase in activity and a 5-fold enhancement in half-life at 55 °C. These findings are significant for understanding the catalytic activity and thermostability of inulosucrase and are promising for the development of microbial inulin biosynthesis platforms.

Rationally Engineered Novel Glycosyltransferase UGT74DD1 from Siraitia grosvenorii Catalyzes the Generation of the Sweetener Mogroside III.

Mogrosides are natural compounds highly valued in the food sector for their exceptional sweetness. Here, we report a novel O-glycosyltransferase (UGT74DD1) from Siraitia grosvenorii that catalyzes the conversion of mogrol to mogroside IIE. Site-directed mutagenesis yielded the UGT74DD1-W351A mutant, which exhibited the new capability to transform mogroside IIE into the valuable sweetener mogroside III, but with low catalytic activity. Subsequently, using structure-guided directed evolution with combinatorial active-site saturation testing, the superior mutant M6 (W351A/Q373 K/E49H/Q335W/S278C/D17F) were obtained, which showed a 46.1-fold increase in catalytic activity compared to UGT74DD1-W351A. Molecular dynamics simulations suggested that the enhanced activity and extended substrate profiles of M6 are due to its enlarged substrate-binding pocket and strengthened enzyme-substrate hydrogen bonding interactions. Overall, we redesigned UGT74DD1, yielding mutants that catalyze the conversion of mogrol into mogroside III. This study thus broadens the toolbox of UGTs capable of catalyzing the formation of valuable polyglycoside compounds.

Characterization of the Structural Requirements for the NADase Activity of Bacterial Toll/IL-1R domains in a Course-based Undergraduate Research Experience.

TLRs initiate innate immune signaling pathways via Toll/IL-1R (TIR) domains on their cytoplasmic tails. Various bacterial species also express TIR domain-containing proteins that contribute to bacterial evasion of the innate immune system. Bacterial TIR domains, along with the mammalian sterile α and TIR motif-containing protein 1 and TIRs from plants, also have been found to exhibit NADase activity. Initial X-ray crystallographic studies of the bacterial TIR from Acinetobacter baumannii provided insight into bacterial TIR structure but were unsuccessful in cocrystallization with the NAD+ ligand, leading to further questions about the TIR NAD binding site. In this study, we designed a Course-Based Undergraduate Research Experience (CURE) involving 16-20 students per year to identify amino acids crucial for NADase activity of A. baumannii TIR domain protein and the TIR from Escherichia coli (TIR domain-containing protein C). Students used structural data to identify amino acids that they hypothesized would play a role in TIR NADase activity, and created plasmids to express mutated TIRs through site-directed mutagenesis. Mutant TIRs were expressed, purified, and tested for NADase activity. The results from these studies provide evidence for a conformational change upon NAD binding, as was predicted by recent cryogenic electron microscopy and hydrogen-deuterium exchange mass spectrometry studies. Along with corroborating recent characterization of TIR NADases that could contribute to drug development for diseases associated with dysregulated TIR activity, this work also highlights the value of CURE-based projects for inclusion of a diverse group of students in authentic research experiences.

Structural and functional analysis of plant ELO-like elongase for fatty acid elongation.

ELO-like elongase is a condensing enzyme elongating long chain fatty acids in eukaryotes. Eranthis hyemalis ELO-like elongase (EhELO1) is the first higher plant ELO-type elongase that is highly active in elongating a wide range of polyunsaturated fatty acids (PUFAs) and some monounsaturated fatty acids (MUFAs). This study attempted using domain swapping and site-directed mutagenesis of EhELO1 and EhELO2, a close homologue of EhELO1 but with no apparent elongase activity, to elucidate the structural determinants critical for catalytic activity and substrate specificity. Domain swapping analysis of the two showed that subdomain B in the C-terminal half of EhELO1 is essential for MUFA elongation while subdomain C in the C-terminal half of EhELO1 is essential for both PUFA and MUFA elongations, implying these regions are critical in defining the architecture of the substrate tunnel for substrate specificity. Site-directed mutagenesis showed that the glycine at position 220 in the subdomain C plays a key role in differentiating the function of the two elongases. In addition, valine at 161 and cysteine at 165 in subdomain A also play critical roles in defining the architecture of the deep substrate tunnel, thereby contributing significantly to the acceptance of, and interaction with primer substrates.

The effect of phosphorylation efficiency on the oncogenic properties of the protein E7 from high-risk HPV.

The Human papillomavirus (HPV) causes tumors in part by hijacking the host cell cycle and forcing uncontrolled cellular division. While there are >200 genotypes of HPV, 15 are classified as high-risk and have been shown to transform infected cells and contribute to tumor formation. The remaining low-risk genotypes are not considered oncogenic and result in benign skin lesions. In high-risk HPV, the oncoprotein E7 contributes to the dysregulation of cell cycle regulatory mechanisms. High-risk E7 is phosphorylated in cells at two conserved serine residues by Casein Kinase 2 (CK2) and this phosphorylation event increases binding affinity for cellular proteins such as the tumor suppressor retinoblastoma (pRb). While low-risk E7 possesses similar serine residues, it is phosphorylated to a lesser degree in cells and has decreased binding capabilities. When E7 binding affinity is decreased, it is less able to facilitate complex interactions between proteins and therefore has less capability to dysregulate the cell cycle. By comparing E7 protein sequences from both low- and high-risk HPV variants and using site-directed mutagenesis combined with NMR spectroscopy and cell-based assays, we demonstrate that the presence of two key nonpolar valine residues within the CK2 recognition sequence, present in low-risk E7, reduces serine phosphorylation efficiency relative to high-risk E7. This results in significant loss of the ability of E7 to degrade the retinoblastoma tumor suppressor protein, thus also reducing the ability of E7 to increase cellular proliferation and reduce senescence. This provides additional insight into the differential E7-mediated outcomes when cells are infected with high-risk verses low-risk HPV. Understanding these oncogenic differences may be important to developing targeted treatment options for HPV-induced cancers.

Structure-Assisted Design of Chitosanase Product Specificity for the Production of High-Degree Polymerization Chitooligosaccharides.

Chitosanases are valuable enzymatic tools in the food industry for converting chitosan into functional chitooligosaccharides (COSs). However, most of the chitosanases extensively characterized produced a low degree of polymerization (DP) COSs (DP = 1-3, LdpCOSs), indicating an imperative for enhancements in the product specificity for the high DP COS (DP >3, HdpCOSs) production. In this study, a chitosanase from Methanosarcina sp. 1.H.T.1A.1 (OUC-CsnA4) was cloned and expressed. Analysis of the enzyme-substrate interactions and the subsite architecture of the OUC-CsnA4 indicated that a Ser49 mutation could modify its interaction pattern with the substrate, potentially enhancing product specificity for producing HdpCOSs. Site-directed mutagenesis provided evidence that the S49I and S49P mutations in OUC-CsnA4 enabled the production of up to 24 and 26% of (GlcN)5 from chitosan, respectively─the wild-type enzyme was unable to produce detectable levels of (GlcN)5. These mutations also altered substrate binding preferences, favoring the binding of longer-chain COSs (DP >5) and enhancing (GlcN)5 production. Furthermore, molecular dynamics simulations and molecular docking studies underscored the significance of +2 subsite interactions in determining the (GlcN)4 and (GlcN)5 product specificity. These findings revealed that the positioning and interactions of the reducing end of the substrate within the catalytic cleft are crucial factors influencing the product specificity of chitosanase.

Controlling DNA Fragments Translocation across Nanopores with the Synergic Use of Site-Directed Mutagenesis, pH-Dependent Charge Tuning, and Electroosmotic Flow.

Biological and solid-state nanopores are at the core of transformative techniques and nanodevices, democratizing the examination of matter and biochemical reactions at the single-molecule level, with low cost, portability, and simplicity in operation. One of the crucial hurdles in such endeavors is the fast analyte translocation, which limits characterization, and a rich number of strategies have been explored over the years to overcome this. Here, by site-directed mutagenesis on the α-hemolysin protein nanopore (α-HL), sought to replace selected amino acids with glycine, electrostatic binding sites were induced on the nanopore's vestibule and constriction region and achieved in the most favorable case a 20-fold increase in the translocation time of short single-stranded DNA (ssDNA) at neutral pH, with respect to the wild-type (WT) nanopore. We demonstrated an efficient tool of controlling the ssDNA translocation time, via the interplay between the nanopore-ssDNA surface electrostatic interactions and electroosmotic flow, all mediated by the pH-dependent ionization of amino acids lining the nanopore's translocation pathway. Our data also reveal the nonmonotonic, pH-induced alteration of ssDNA average translocation time. Unlike mildly acidic conditions (pH ∼ 4.7), at a pH ∼ 2.8 maintained symmetrically or asymmetrically across the WT α-HL, we evidenced the manifestation of a dominant electroosmotic flow, determining the speeding up of the ssDNA translocation across the nanopore by counteracting the ssDNA-nanopore attractive electrostatic interactions. We envision potential applications of the presented approach by enabling easy-to-use, real-time detection of short ssDNA sequences, without the need for complex biochemical modifications to the nanopore to mitigate the fast translocation of such sequences.

Identification of heparin-binding amino acid residues in antibody HS4C3 with the potential to design antibodies against heparan sulfate domains.

Heparan sulfate (HS) is a linear polysaccharide with high structural and functional diversity. Detection and localization of HS in tissues can be performed using single chain variable fragment (scFv) antibodies. Although several anti-HS antibodies recognizing different sulfation motifs have been identified, little is known about their interaction with HS. In this study the interaction between the scFv antibody HS4C3 and heparin was investigated. Heparin-binding lysine and arginine residues were identified using a protect and label methodology. Site-directed mutagenesis was applied to further identify critical heparin-binding lysine/arginine residues using immunohistochemical and biochemical assays. In addition, computational docking of a heparin tetrasaccharide towards a 3-D homology model of HS4C3 was applied to identify potential heparin-binding sites. Of the 12 lysine and 15 arginine residues within the HS4C3 antibody, 6 and 9, respectively, were identified as heparin-binding. Most of these residues are located within one of the complementarity determining regions (CDR) or in their proximity. All basic amino acid residues in the CDR3 region of the heavy chain were involved in binding. Computational docking showed a heparin tetrasaccharide close to these regions. Mutagenesis of heparin-binding residues reduced or altered reactivity towards HS and heparin. Identification of heparin-binding arginine and lysine residues in HS4C3 allows for better understanding of the interaction with HS and creates a framework to rationally design antibodies targeting specific HS motifs.

Using site-directed mutagenesis to further the understanding of insulin receptor-insulin like growth factor-1 receptor heterodimer structure.

Type 2 diabetes is characterised by the disruption of insulin and insulin-like growth factor (IGF) signalling. The key hubs of these signalling cascades - the Insulin receptor (IR) and Insulin-like growth factor 1 receptor (IGF1R) - are known to form functional IR-IGF1R hybrid receptors which are insulin resistant. However, the mechanisms underpinning IR-IGF1R hybrid formation are not fully understood, hindering the ability to modulate this for future therapies targeting this receptor. To pinpoint suitable sites for intervention, computational hotspot prediction was utilised to identify promising epitopes for targeting with point mutagenesis. Specific IGF1R point mutations F450A, R391A and D555A show reduced affinity of the hybrid receptor in a BRET based donor-saturation assay, confirming hybrid formation could be modulated at this interface. These data provide the basis for rational design of more effective hybrid receptor modulators, supporting the prospect of identifying a small molecule that specifically interacts with this target.

Conformational dynamics underlying atypical chemokine receptor 3 activation.

Atypical Chemokine Receptor 3 (ACKR3) belongs to the G protein-coupled receptor family but it does not signal through G proteins. The structural properties that govern the functional selectivity and the conformational dynamics of ACKR3 activation are poorly understood. Here, we combined hydrogen/deuterium exchange mass spectrometry, site-directed mutagenesis, and molecular dynamics simulations to examine the binding mode and mechanism of action of ACKR3 ligands of different efficacies. Our results show that activation or inhibition of ACKR3 is governed by intracellular conformational changes of helix 6, intracellular loop 2, and helix 7, while the DRY motif becomes protected during both processes. Moreover, we identified the binding sites and the allosteric modulation of ACKR3 upon ß-arrestin 1 binding. In summary, this study highlights the structure-function relationship of small ligands, the binding mode of ß-arrestin 1, the activation dynamics, and the atypical dynamic features in ACKR3 that may contribute to its inability to activate G proteins.

Through virtual saturation mutagenesis and rational design for superior substrate conversion in engineered d-amino acid oxidase.

The d-amino acid oxidase (DAAO) is pivotal in obtaining optically pure l-glufosinate (l-PPT) by converting d-glufosinate (d-PPT) to its deamination product. We screened and designed a Rasamsonia emersonii DAAO (ReDAAO), making it more suitable for oxidizing d-PPT. Using Caver 3.0, we delineated three substrate binding pockets and, via alanine scanning, identified nearby key residues. Pinpointing key residues influencing activity, we applied virtual saturation mutagenesis (VSM), and experimentally validated mutants which reduced substrate binding energy. Analysis of positive mutants revealed elongated side-chain prevalence in substrate binding pocket periphery. Although computer-aided approaches can rapidly identify advantageous mutants and guide further design, the mutations obtained in the first round may not be suitable for combination with other advantageous mutations. Therefore, each round of combination requires reasonable iteration. Employing VSM-assisted screening multiple times and after four rounds of combining mutations, we ultimately obtained a mutant, N53V/F57Q/V94R/V242R, resulting in a mutant with a 5097% increase in enzyme activity compared to the wild type. It provides valuable insights into the structural determinants of enzyme activity and introduces a novel rational design procedure.

Single-round QuikChange PCR for engineering multiple site-directed mutations in plasmid DNA.

Mutational study is a cornerstone methodology in biochemistry and genetics, and many mutagenesis strategies have been invented to promote the efficiency of gene engineering. In this study, we developed a simple and timesaving approach to integrate simultaneous mutagenesis at discrete sites. By using plasmid as a template and compatible oligonucleotide primers per the QuikChange strategy, our method was able to introduce multiple nucleotide insertions, deletions and replacements in one round of polymerase chain reaction. The longest insertion and deletion were achieved with 28 bp and 16 bp mismatch respectively. For minor nucleotide replacements (mismatch no more than 4 bp), mutations were achieved at up to 4 discrete locations. Usually, a successful clone with all desired mutations was found by screening 5 colonies. Clones with a subset of mutations may be stocked into the library of mutants or used as templates in the next rounds of mutagenic PCR to accomplish the entire construction project. This method can be applied to build up a combinatory library of mutants through saturation mutagenesis at multiple sites. It is promising to facilitate the research of protein biochemistry, forward genetics and synthetic biology.

A flexible linker of 8-amino acids between the membrane binding segment and the FMN domain of cytochrome P450 reductase is necessary for optimal activity.

The diflavin NADPH-cytochrome P450 reductase (CYPOR) plays a critical role in human cytochrome P450 (CYP) activity by sequentially delivering two electrons from NADPH to CYP enzymes during catalysis. Although electron transfer to forty-eight human CYP enzymes by the FMN hydroquinone of CYPOR is well-known, the role of the linker between the NH2-terminus membrane-binding domain (MBD) and FMN domain in supporting the activity of P450 enzymes remains poorly understood. Here we demonstrate that a linker with at least eight residues is required to form a functional CYPOR-CYP2B4 complex. The linker has been shortened in two amino-acid increments from Phe44 to Ile57 using site directed mutagenesis. The ability of the deletion mutants to support cytochrome P450 2B4 (CYP2B4) catalysis and reduce ferric CYP2B4 was determined using an in vitro assay and stopped-flow spectrophotometry. Steady-state enzyme kinetics showed that shortening the linker by 8-14 amino acids inhibited (63-99%) the ability of CYPOR to support CYP2B4 activity and significantly increased the Km of CYPOR for CYP2B4. In addition, the reductase mutants decreased the rate of reduction of ferric CYP2B4 (46-95%) compared to wildtype when the linker was shortened by 8-14 residues. These results indicate that a linker with a minimum length of eight residues is necessary to enable the FMN domain of reductase to interact with CYP2B4 to form a catalytically competent complex. Our study provides evidence that the length of the MBD-FMN domain linker is a major determinant of the ability of CYPOR to support CYP catalysis and drug metabolism by P450 enzymes. PREAMBLE: This manuscript is dedicated in memory of Dr. James R. Kincaid who was the doctoral advisor to Dr. Freeborn Rwere and a longtime collaborator and friend of Dr. Lucy Waskell. Dr. James R. Kincaid was a distinguished professor of chemistry specializing in resonance Raman (rR) studies of heme proteins. He inspired Dr. Rwere (a Zimbabwean native) and three other Zimbabweans (Dr. Remigio Usai, Dr. Daniel Kaluka and Ms. Munyaradzi E. Manyumwa) to use lasers to document subtle changes occurring at heme active site of globin proteins (myoglobin and hemoglobin) and cytochrome P450 enzymes. Dr. Rwere appreciate his contributions to the development of talented Black scientists from Africa.

Enhancing the amination activity of meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum by modifying the crucial residue His154 for deamination.

During the deamination and amination processes of meso-diaminopimelate dehydrogenase (meso-DAPDH) from Symbiobacterium thermophilum (StDAPDH), residue R71 was observed to display distinct functions. H154 has been proposed as a basic residue that facilitates water molecules to attack the D-chiral carbon of meso-DAP during deamination. Inspired by the phenomenon of R71, the effects of H154 during deamination and amination were investigated in this study with the goal of enhancing the amination activities of StDAPDH. Single site saturation mutagenesis indicated that almost all of the H154 mutants completely lost their deamination activity towards meso-DAP. However, some H154 variants showed enhanced kcat/Km values towards pyruvic acid and other bulky 2-keto acids, such as 2-oxovaleric acid, 4-methyl-2-oxopentanoic acid, 2-ketobutyric acid, and 3-methyl-2-oxobutanoic acid. When combined with the previously reported W121L/H227I mutant, triple mutants with significantly improved kcat/Km values (2.4-, 2.5-, 2.5-, and 4.0-fold) towards these 2-keto acids were obtained. Despite previous attempts, mutations at the H154 site did not yield the desired results. Moreover, this study not only recognizes the distinctive impact of H154 on both the deamination and amination reactions, but also provides guidance for further high-throughput screening in protein engineering and understanding the catalytic mechanism of StDAPDH.

Combining binding pocket mutagenesis and substrate tunnel engineering to improve an (R)-selective transaminase for the efficient synthesis of (R)-3-aminobutanol.

(R)-selective transaminases have the potential to act as efficient biocatalysts for the synthesis of important pharmaceutical intermediates. However, their low catalytic efficiency and unfavorable equilibrium limit their industrial application. Seven (R)-selective transaminases were identified using homologous sequence mining. Beginning with the optimal candidate from Mycolicibacterium hippocampi, virtual mutagenesis and substrate tunnel engineering were performed to improve catalytic efficiency. The obtained variant, T282S/Q137E, exhibited 3.68-fold greater catalytic efficiency (kcat/Km) than the wild-type enzyme. Using substrate fed-batch and air sweeping processes, effective conversion of 100 mM 4-hydroxy-2-butanone was achieved with a conversion rate of 93 % and an ee value > 99.9 %. This study provides a basis for mutation of (R)-selective transaminases and offers an efficient biocatalytic process for the asymmetric synthesis of (R)-3-aminobutanol.

Engineering of the Phytase YiAPPA to Improve Thermostability and Activity and Its Application Potential in Dephytinization of Food Ingredients.

The aim of this study was to modify phytase YiAPPA via protein surficial residue mutation to obtain phytase mutants with improved thermostability and activity, enhancing its application potential in the food industry. First, homology modeling of YiAPPA was performed. By adopting the strategy of protein surficial residue mutation, the lysine (Lys) and glycine (Gly) residues on the protein surface were selected for site-directed mutagenesis to construct single-site mutants. Thermostability screening was performed to obtain mutants (K189R and K216R) with significantly elevated thermostability. The combined mutant K189R/K216R was constructed via beneficial mutation site stacking and characterized. Compared with those of YiAPPA, the half-life of K189R/K216R at 80°C was extended from 14.81 min to 23.35 min, half-inactivation temperature (T50 30) was increased from 55.12°C to 62.44°C, and Tm value was increased from 48.36°C to 53.18°C. Meanwhile, the specific activity of K189R/K216R at 37°C and pH 4.5 increased from 3960.81 to 4469.13 U/mg. Molecular structure modeling analysis and molecular dynamics simulation showed that new hydrogen bonds were introduced into K189R/K216R, improving the stability of certain structural units of the phytase and its thermostability. The enhanced activity was primarily attributed to reduced enzyme-substrate binding energy and shorter nucleophilic attack distance between the catalytic residue His28 and the phytate substrate. Additionally, the K189R/K216R mutant increased the hydrolysis efficiency of phytate in food ingredients by 1.73-2.36 times. This study established an effective method for the molecular modification of phytase thermostability and activity, providing the food industry with an efficient phytase for hydrolyzing phytate in food ingredients.

Cysteine Oxidation in Human Galectin-1 Occurs Sequentially via a Folded Intermediate to a Fully Oxidized Unfolded Form.

Galectins are multifunctional effectors in cellular homeostasis and dysregulation. Oxidation of human galectin-1 (Gal-1) with its six sulfhydryls produces a disulfide-bridged oxidized form that lacks normal lectin activity yet gains new glycan-independent functionality. Nevertheless, the mechanistic details as to how Gal-1 oxidation occurs remain unclear. Here, we used 15N and 13C HSQC NMR spectroscopy to gain structural insight into the CuSO4-mediated path of Gal-1 oxidation and identified a minimum two-stage conversion process. During the first phase, disulfide bridges form slowly between C16-C88 and/or C42-C66 to produce a partially oxidized, conformationally flexible intermediate that retains the ability to bind lactose. Site-directed mutagenesis of C16 to S16 impedes the onset of this overall slow process. During the second phase, increased motional dynamics of the intermediate enable the relatively distant C2 and C130 residues to form the third and final disulfide bond, leading to an unfolded state and consequent dimer dissociation. This fully oxidized end state loses the ability to bind lactose, as shown by the hemagglutination assay. Consistent with this model, we observed that the Gal-1 C2S mutant maintains intermediate-state structural features with a free sulfhydryl group at C130. Incubation with dithiothreitol reduces all disulfide bonds and allows the lectin to revert to its native state. Thus, the sequential, non-random formation of three disulfide bridges in Gal-1 in an oxidative environment acts as a molecular switch for fundamental changes to its functionality. These data inspire detailed bioactivity analysis of the structurally defined oxidized intermediate in, e.g., acute and chronic inflammation.