Active Site Architecture Research Articles

Two highly selected mutations in the tandemly duplicated CYP6P4a and CYP6P4b genes drive pyrethroid resistance in Anopheles funestus in West Africa

BackgroundGaining a comprehensive understanding of the genetic mechanisms underlying insecticide resistance in malaria vectors is crucial for optimising the effectiveness of insecticide-based vector control methods and developing diagnostic tools for resistance management. Considering the heterogeneity of metabolic resistance in major malaria vectors, the implementation of tailored resistance management strategies is essential for successful vector control. Here, we provide evidence demonstrating that two highly selected mutations in CYP6P4a and CYP6P4b are driving pyrethroid insecticide resistance in the major malaria vector Anopheles funestus, in West Africa.ResultsContinent-wide polymorphism survey revealed escalated signatures of directional selection of both genes between 2014 and 2021. In vitro insecticide metabolism assays with recombinant enzymes from both genes showed that mutant alleles under selection exhibit higher metabolic efficiency than their wild-type counterparts. Using the GAL4-UAS expression system, transgenic Drosophila flies overexpressing mutant alleles exhibited increased resistance to pyrethroids. These findings were consistent with in silico predictions which highlighted changes in enzyme active site architecture that enhance the affinity of mutant alleles for type I and II pyrethroids. Furthermore, we designed two DNA-based assays for the detection of CYP6P4a-M220I and CYP6P4b-D284E mutations, showing their current confinement to West Africa. Genotype/phenotype correlation analyses revealed that these markers are strongly associated with resistance to types I and II pyrethroids and combine to drastically reduce killing effects of pyrethroid bed nets.ConclusionsOverall, this study demonstrated that CYP6P4a and CYP6P4b contribute to pyrethroid resistance in An. funestus and provided two additional insecticide resistance molecular diagnostic tools that would contribute to monitoring and better management of resistance.

Structures of vertebrate R2 retrotransposon complexes during target-primed reverse transcription and after second strand nicking.

R2 retrotransposons are model site-specific eukaryotic non-LTR retrotransposons that copy-and-paste into gene loci encoding ribosomal RNAs. Recently we demonstrated that avian A-clade R2 proteins achieve efficient and precise insertion of transgenes into their native safe-harbor loci in human cells. The features of A-clade R2 proteins that support gene insertion are not characterized. Here, we report high resolution cryo-electron microscopy structures of two vertebrate A-clade R2 proteins, avian and testudine, at the initiation of target-primed reverse transcription and one structure after cDNA synthesis and second strand nicking. Using biochemical and cellular assays we discover the basis for high selectivity of template use and unique roles for each of the expanded A-clade zinc-finger domains in nucleic acid recognition. Reverse transcriptase active site architecture is reinforced by an unanticipated insertion motif in vertebrate A-clade R2 proteins. Our work brings first insights to A-clade R2 protein structure during gene insertion and enables further improvement and adaptation of R2-based systems for precise transgene insertion.

Structural insights into the recognition of tetrapyrrole substrates by ancestral class II chelatase CfbA.

Nickel-chelatase CfbA, unlike descendant chelatases, is an ancestral class II chelatase with a symmetric active site architecture. CfbA utilizes sirohydrochlorin (SHC) as a physiological substrate in the biosynthesis of coenzyme F430. CbiXS, a structural analog of CfbA, can use uroporphyrin III (UPIII) and uroporphyrin I (UPI) as non-physiological substrates. Owing to the broad tetrapyrrole specificity of the unique active site of ancestral class II chelatases, the substrate recognition mechanism of CfbA has garnered interest. Herein, we conducted an X-ray crystallographic analysis of CfbA in complex with UPIII and UPI. Interestingly, the binding sites for UPIII and UPI were distinct. UPI was bound at the entrance of the active site, whereas UPIII was bound deep inside the active site cavity in a manner similar to SHC. Despite the difference in the binding positions of UPIII and UPI, Ser11 at the active site provided critical polar interactions for recognizing UPIII and UPI. Several CfbA variants with a Ser11 mutation were studied to confirm the significance of Ser11's position in the context of tetrapyrrole recognition. The CfbA S11T variant showed Ni2+-chelatase activity against coproporphyrin I (CPI), which is a more hydrophobic tetrapyrrole than UPIII and UPI. Using a CPI-docked model of the S11T variant, we proposed that balancing the hydrophobic/polar interactions at residue 11 could alter substrate selectivity. The structural and mutational analyses reported here highlight the importance of polar and hydrophobic interactions at the entry region of the active site for substrate tetrapyrrole recognition by ancestral and descendant class II chelatases.

Activity and cryo-EM structure of the polymerase domain of the human norovirus ProPol precursor.

Human norovirus (HuNV) is a leading cause of acute gastroenteritis worldwide with most infections caused by genogroup I and genogroup II (GII) viruses. Replication of HuNV generates both precursor and mature proteins during processing of the viral polyprotein that are essential to the viral lifecycle. One such precursor is protease-polymerase (ProPol), a multi-functional enzyme comprised of the norovirus protease and polymerase proteins. This work investigated HuNV ProPol by determining the de novo polymerase activity, protein structure, and antiviral inhibition profile. The GII ProPol de novo enzymatic efficiencies (kcat/Km) for RNA templates and ribonucleotides were equal or superior to those of mature GII Pol on all templates measured. Furthermore, GII ProPol was the only enzyme form active on a poly(A) template. The first structure of the polymerase domain of HuNV ProPol in the unliganded state was determined by cryo-electron microscopy at a resolution of 2.6 Å. The active site and overall architecture of ProPol are similar to those of mature Pol. In addition, both galidesivir triphosphate and PPNDS inhibited polymerase activity of GII ProPol, with respective half-maximal inhibitory concentration (IC50) values of 247.5 µM and 3.8 µM. In both instances, the IC50 obtained with ProPol was greater than that of mature Pol, indicating that ProPol can exhibit different responses to antivirals. This study provides evidence that HuNV ProPol possesses overlapping and unique enzyme properties compared with mature Pol and will aid our understanding of the replication cycle of the virus.IMPORTANCEDespite human norovirus (HuNV) being a leading cause of acute gastroenteritis, the molecular mechanisms surrounding replication are not well understood. Reports have shown that HuNV replication generates precursor proteins from the viral polyprotein, one of which is the protease-polymerase (ProPol). This precursor is important for viral replication; however, the polymerase activity and structural differences between the precursor and mature forms of the polymerase remain to be determined. We show that substrate specificity and polymerase activity of ProPol overlap with, but is distinct from, the mature polymerase. We employ cryo-electron microscopy to resolve the first structure of the polymerase domain of ProPol. This shows a polymerase architecture similar to mature Pol, indicating that the interaction of the precursor with substrates likely defines its activity. We also show that ProPol responds differently to antivirals than mature polymerase. Altogether, these findings enhance our understanding of the function of the important norovirus ProPol precursor.

Mechanistic Perspective on Oxygen Activation Chemistry by Flavoenzymes.

Flavin-dependent enzymes catalyze a panoply of chemical transformations essential for living organisms. Through oxygen activation, flavoenzymes could generate diverse flavin-oxygen species that mediate numerous redox and non-redox transformations. In this review, we highlight the extensive oxygen activation chemistry at two sites of the flavin cofactor: C4a and N5 sites. Oxygen activation at the C4a site generates flavin-C4aOO(H) species for various monooxygenation reactions, while activation at the N5 site produces negatively charged flavin-N5OOH species, which act as highly reactive nucleophiles or bases. The selective oxygen activation at either the C4a or N5 site depends on the nature of substrates and is controlled by the active site architecture. These insights have expanded our understanding of oxygen activation chemistry in flavoenzymes and will serve as a foundation for future efforts in enzyme engineering and redesign.

Biochemical and structural insights into a 5’ to 3’ RNA ligase reveal a potential role in tRNA ligation

ATP-grasp superfamily enzymes contain a hand-like ATP-binding fold and catalyze a variety of reactions using a similar catalytic mechanism. More than 30 protein families are categorized in this superfamily, and they are involved in a plethora of cellular processes and human diseases. Here, we identify C12orf29 (RLIG1) as an atypical ATP-grasp enzyme that ligates RNA. Human RLIG1 and its homologs autoadenylate on an active site Lys residue as part of a reaction intermediate that specifically ligates RNA halves containing a 5'-phosphate and a 3'-hydroxyl. RLIG1 binds tRNA in cells and can ligate tRNA within the anticodon loop in vitro. Transcriptomic analyses of Rlig1 knockout mice revealed significant alterations in global tRNA levels in the brains of female mice, but not in those of male mice. Furthermore, crystal structures of a RLIG1 homolog from Yasminevirus bound to nucleotides revealed a minimal and atypical RNA ligase fold with a conserved active site architecture that participates in catalysis. Collectively, our results identify RLIG1 as an RNA ligase and suggest its involvement in tRNA biology.

Enzymatic Degradation toward Herbicides: The Case of the Sulfonylureas.

Commercial herbicides, particularly sulfonylureas, are used worldwide and pose a significant challenge to environmental sustainability. The efficient degradation of sulfonylurea herbicides is critical. SulE, an esterase isolated from the bacterial strain Hansschlegelia zhihuaiae S113, shows degradation activity toward sulfonylurea herbicides. However, the detailed catalytic mechanism remains vague to a large extent. Herein, we decipher the SulEP44R-catalyzed degradation mechanism of sulfonylurea herbicides using hybrid quantum mechanics and molecular mechanics approaches. Our results show that the degradation of sulfonylureas catalyzed by SulEP44R involves four concerted elementary steps. The rate-determining step has an energy barrier range of 19.7-21.4 kcal·mol-1, consistent with the experimentally determined range of 16.0-18.0 kcal·mol-1. Distortion/interaction analysis demonstrates that active-site amino acids play a vital role in the enzymatic catalytic efficacy. The unique architecture of SulEP44R's active site can serve as an excellent template for designing artificial catalysts. Key structural and charge parameters affecting catalytic activity were systematically screened and identified. Based on the elucidated degradation mechanism, several new herbicides with both high herbicidal activity and biodegradability were developed with the aid of a high-throughput strategy. Our findings may advance the application of sulfonylurea herbicides within the framework of environmental sustainability.

Current Status of Amine Dehydrogenases: From Active Site Architecture to Diverse Applications Across a Broad Substrate Spectrum

AbstractAmine dehydrogenases (AmDHs) are NAD(P)H‐dependent oxidoreductases that catalyze the reductive amination between carbonyl compounds and ammonia as the amine donor yielding valuable amines, typically with excellent enantioselectivity. While nature has provided enzymes with inherent AmDH activities, protein engineering techniques allowed researchers to expand the toolbox of available AmDHs, extend their substrate scope, improve their catalytic activities and stability under synthetically relevant conditions and even enable new reactivity concepts. The biocatalytic synthesis of amines using AmDHs has matured to a point where hundreds of aldehydes or ketones, of varying steric demands and bearing diverse functional groups, can be efficiently transformed. This review offers an overview of the available AmDHs and their substrate spectrum, covering from structural and evolutionary analyses to diverse methods employing these enzymes. Depending on the catalytic activities of other enzymes as reaction partners, AmDHs were applied in kinetic resolution (KR) and deracemization processes, cascade reactions for the amination of alcohols and alkenes or for the synthesis of amines and amino alcohols featuring multiple stereogenic centers. Moreover, the synthetic potential of AmDHs in novel pathways, such as the synthesis of secondary amines or alcohols, presents exciting opportunities for expanding their catalytic repertoire.

Bimodal substrate binding in the active site of the glycosidase BcX.

Bacillus circulans xylanase (BcX) from the glycoside hydrolase family 11 degrades xylan through a retaining, double-displacement mechanism. The enzyme is thought to hydrolyze glycosidic bonds in a processive manner and has a large, active site cleft, with six subsites allowing the binding of six xylose units. Such an active site architecture suggests that oligomeric xylose substrates can bind in multiple ways. In the crystal structure of the catalytically inactive variant BcX E78Q, the substrate xylotriose is observed in the active site, as well as bound to the known secondary binding site and a third site on the protein surface. Nuclear magnetic resonance (NMR) titrations with xylose oligomers of different lengths yield nonlinear chemical shift trajectories for active site nuclei resonances, indicative of multiple binding orientations for these substrates for which binding and dissociation are in fast exchange on the NMR timescale, exchanging on the micro- to millisecond timescale. Active site binding can be modeled with a 2 : 1 model with dissociation constants in the low and high millimolar range. Extensive mutagenesis of active site residues indicates that tight binding occurs in the glycon binding site and is stabilized by Trp9 and the thumb region. Mutations F125A and W71A lead to large structural rearrangements. Binding at the glycon site is sensed throughout the active site, whereas the weak binding mostly affects the aglycon site. The interactions with the two active site locations are largely independent of each other and of binding at the secondary binding site.

Ein funktioneller Wechsel zwischen Asperfumen‐ und Fusicoccadien‐Synthase und Eintritt in die Asperfumen‐Biosynthese durch einen vicinalen Deprotonierungs‐Reprotonierungsprozess

AbstractDie Diterpensynthase AfAS wurde aus Aspergillus fumigatiaffinis identifiziert. Ihre Aminosäuresequenz und – einem Strukturmodell zufolge – die Architektur des aktiven Zentrums sind denen der Fusicocca‐2,10(14)‐dien‐Synthase PaFS sehr ähnlich, AfAS produziert jedoch ein strukturell viel komplexeres Diterpen mit einem neuartigen 6–5–5–5 tetracyclischen Gerüst namens Asperfumen. Der Cyclisierungsmechanismus von AfAS wurde durch Isotopenmarkierungsexperimente und DFT‐Rechnungen aufgeklärt. Die Reaktionskaskade verläuft in ihren ersten Schritten über ähnliche Intermediate wie die PaFS‐Kaskade, divergiert dann aber durch einen ungewöhnlichen vicinalen Deprotonierungs‐Reprotonierungsprozess, der eine Skelettumlagerung am Startpunkt der Stufen auslöst, die zum einzigartigen Asperfumen‐Gerüst führen. Das Strukturmodell zeigte nur einen wesentlichen Unterschied zwischen den aktiven Zentren: Der PaFS‐Rest F65 ist in AfAS durch I65 ersetzt. Interessanterweise zeigten ortsspezifische Mutageneseexperimente mit beiden Diterpensynthasen, dass Position 65 als bidirektionaler funktioneller Schalter für die Biosynthese von tetracyclischem Asperfumen im Vergleich zu strukturell weniger komplexen Diterpenen dient.

Enzyme Engineering by Force: DNA Springs for the Modulation of Biocatalytic Trajectories.

The engineering of enzymatic activity generally involves alteration of the protein primary sequences, which introduce structural changes that give rise to functional improvements. Mechanical forces have been used to interrogate protein biophysics, leading to deep mechanistic insights in single-molecule studies. Here, we use simple DNA springs to apply small pulling forces to perturb the active site of a thermostable alcohol dehydrogenase. Methods were developed to enable the study of different spring lengths and spring orientations under bulk catalysis conditions. Tension applied across the active site expanded the binding pocket volume and shifted the preference of the enzyme for longer chain-length substrates, which could be tuned by altering the spring length and the resultant applied force. The substrate specificity changes did not occur when the DNA spring was either severed or rotated by ∼90°. These findings demonstrate an alternative approach in protein engineering, where active site architectures can be dynamically and reversibly remodeled using applied mechanical forces.

Discovery of a new class of bacterial heme-containing C[dbnd]C cleaving oxygenases

Previously, some bacteria were shown to harbour enzymes capable of catalysing the oxidative cleavage of the double bond of t-anethole and related compounds. The cofactor dependence of these enzymes remained enigmatic due to a lack of biochemical information. We report on catalytic and structural details of a representative of this group of oxidative enzymes: t-anethole oxygenase from Stenotrophomonas maltophilia (TAOSm). The bacterial enzyme could be recombinantly expressed and purified, enabling a detailed biochemical study that has settled the dispute on its cofactor dependence. We have established that TAOSm contains a tightly bound b-type heme and merely depends on dioxygen for catalysis. It was found to accept t-anethole, isoeugenol and O-methyl isoeugenol as substrates, all being converted into the corresponding aromatic aldehydes without the need of any cofactor regeneration. The elucidated crystal structure of TAOSm has revealed that it contains a unique active site architecture that is conserved for this distinct class of heme-containing bacterial oxygenases. Similar to other hemoproteins, TAOSm has a histidine (His121) as proximal ligand. Yet, unique for TAOs, an arginine (Arg89) is located at the distal axial position. Site directed mutagenesis confirmed crucial roles for these heme-liganding residues and other residues that form the substrate binding pocket. In conclusion, the results reported here reveal a new class of bacterial heme-containing oxygenases that can be used for the cleavage of alkene double bonds, analogous to ozonolysis in organic chemistry.

In silico studies of the open form of human tissue transglutaminase

Human tissue transglutaminase (tTG) is an intriguing multifunctional enzyme involved in various diseases, including celiac disease and neurological disorders. Although a number of tTG inhibitors have been developed, the molecular determinants governing ligand binding remain incomplete due to the lack of high-resolution structural data in the vicinity of its active site. In this study, we obtained the complete high-resolution model of tTG by in silico methods based on available PDB structures. We discovered significant differences in the active site architecture between our and known tTG models, revealing an additional loop which affects the ligand binding affinity. We assembled a library of new potential tTG inhibitors based on the obtained complete model of the enzyme. Our library substantially expands the spectrum of possible drug candidates targeting tTG and encompasses twelve molecular scaffolds, eleven of which are novel and exhibit higher binding affinity then already known ones, according to our in silico studies. The results of this study open new directions for structure-based drug design of tTG inhibitors, offering the complete protein model and suggesting a wide range of new compounds for further experimental validation.

Creating Asymmetric Fe-N3C-N Sites in Single-Atom Catalysts Boosts Catalytic Performance for Oxygen Reduction Reaction.

Fine tuning of the metal site coordination environment of a single-atom catalyst (SAC) to boost its catalytic activity for oxygen reduction reaction (ORR) is of significance but challenging. Herein, we report a new SAC bearing Fe-N3C-N sites with asymmetric in-plane coordinated Fe-N3C and axial coordinated N atom for ORR, which was obtained by pyrolysis of an iron isoporphyrin on polyvinylimidazole (PVI) coated carbon black. The C@PVI-(NCTPP)Fe-800 catalyst exhibited significantly improved ORR activity (E1/2 = 0.89 V vs RHE) than the counterpart SAC with Fe-N4-N sites in 0.1 M KOH. Significantly, the Zn-air batteries equipped with the C@PVI-(NCTPP)Fe-800 catalyst demonstrated an open-circuit voltage (OCV) of 1.45 V and a peak power density (Pmax) of 130 mW/cm2, outperforming the commercial Pt/C catalyst (OCV = 1.42 V; Pmax = 119 mW/cm2). The density functional theory (DFT) calculations revealed that the d-band center of the asymmetric Fe-N3C-N structure shifted upward, which enhances its electron-donating ability, favors O2 adsorption, and supports O-O bond activation, thus leading to significantly promoted catalytic activity. This research presents an intriguing strategy for the designing of the active site architecture in metal SACs with a structure-function controlled approach, significantly enhancing their catalytic efficiency for the ORR and offering promising prospects in energy-conversion technologies.

Kinetic Characterization and Identification of Key Active Site Residues of the L-Aspartate N-Hydroxylase, CreE.

CreE is a flavin-dependent monooxygenase (FMO) that catalyzes three sequential nitrogen oxidation reactions of L-aspartate to produce nitrosuccinate, contributing to the biosynthesis of the antimicrobial and antiproliferative nautral product, cremeomycin. This compound contains a highly reactive diazo functional group for which the reaction of CreE is essential to its formation. Nitro and diazo functional groups can serve as potent electrophiles, important in some challenging nucleophilic addition reactions. Formation of these reactive groups positions CreE as a promising candidate for biomedical and synthetic applications. Here, we present the catalytic mechanism of CreE and the identification of active site residues critical to binding L-aspartate, aiding in future enzyme engineering efforts. Steady-state analysis demonstrated that CreE is very specific for NADPH over NADH and performs a highly coupled reaction with L-aspartate. Analysis of the rapid-reaction kinetics showed that flavin reduction is very fast, along with the formation of the oxygenating species, the C4a-hydroperoxyflavin. The slowest step observed was the dehydration of the flavin. Structural analysis and site-directed mutagenesis implicated T65, R291, and R440 in the binding L-aspartate. The data presented describes the catalytic mechanism and the active site architecture of this unique FMO.

Efficient calcium sulfite oxidation treatment by a highly active iron-manganese bimetallic metal-organic frameworks (MOFs)

Calcium sulfite (CaSO3) oxidation is the key step in the resource utilization of flue gas desulfurization (FGD) ash waste. However, under natural conditions the oxidation rate of CaSO3 is very low, limiting its conversion into calcium sulfate, an acceptable product for construction material. This study was devoted to the investigation of the synthesis and performance of a bimetallic MOFs with potential catalytic activity, namely, Fe/Mn(BDC)(DMF,F) catalysts. A combination of techniques including SEM, EDS, XRD, FT-IR, etc. were used to characterize the catalysts. The excellent catalytic performance examined in a gas-liquid-solid three-phase oxidation system are due to the special flexible respiratory architecture and homogeneous distribution of active sites, which allows the reactants to fully contact with the catalyst. Mn incorporated in the backbone of the metal-organic framework significantly enriches the catalytic active species that participate in a radical chain reaction with activated SO32- as ·SO3-, which in turn generates the key radical ·SO5- that reacts with SO32- to form SO42-. In the presence of Fe/Mn(BDC)(DMF, F)-13, the oxidation ratio of CaSO3 could reach 90.71%, which is more than 30-fold compared to non-catalytic oxidation and almost 5 times the effects of the single metal Fe(BDC)(DMF, F) catalyst. In addition, the study of kinetics shows that the oxidation rate of Fe/Mn(BDC)(DMF,F)-13 can reach 0.042 mmol·L-1·s-1 with an apparent activation energy of 10.86 kJ/mol. The oxidation rate is mainly affected by reaction temperature, pH value, catalyst concentration, and air flow rate. This study provides a potentially sustainable way for utilization of CaSO3-containing desulfurization ash.

3-Chymotrypsin-like Protease (3CLpro) of SARS-CoV-2: Validation as a Molecular Target, Proposal of a Novel Catalytic Mechanism, and Inhibitors in Preclinical and Clinical Trials.

Proteases represent common targets in combating infectious diseases, including COVID-19. The 3-chymotrypsin-like protease (3CLpro) is a validated molecular target for COVID-19, and it is key for developing potent and selective inhibitors for inhibiting viral replication of SARS-CoV-2. In this review, we discuss structural relationships and diverse subsites of 3CLpro, shedding light on the pivotal role of dimerization and active site architecture in substrate recognition and catalysis. Our analysis of bioinformatics and other published studies motivated us to investigate a novel catalytic mechanism for the SARS-CoV-2 polyprotein cleavage by 3CLpro, centering on the triad mechanism involving His41-Cys145-Asp187 and its indispensable role in viral replication. Our hypothesis is that Asp187 may participate in modulating the pKa of the His41, in which catalytic histidine may act as an acid and/or a base in the catalytic mechanism. Recognizing Asp187 as a crucial component in the catalytic process underscores its significance as a fundamental pharmacophoric element in drug design. Next, we provide an overview of both covalent and non-covalent inhibitors, elucidating advancements in drug development observed in preclinical and clinical trials. By highlighting various chemical classes and their pharmacokinetic profiles, our review aims to guide future research directions toward the development of highly selective inhibitors, underscore the significance of 3CLpro as a validated therapeutic target, and propel the progression of drug candidates through preclinical and clinical phases.

Mechanism of Nitrone Formation by a Flavin-Dependent Monooxygenase.

OxaD is a flavin-dependent monooxygenase (FMO) responsible for catalyzing the oxidation of an indole nitrogen atom, resulting in the formation of a nitrone. Nitrones serve as versatile intermediates in complex syntheses, including challenging reactions like cycloadditions. Traditional organic synthesis methods often yield limited results and involve environmentally harmful chemicals. Therefore, the enzymatic synthesis of nitrone-containing compounds holds promise for more sustainable industrial processes. In this study, we explored the catalytic mechanism of OxaD using a combination of steady-state and rapid-reaction kinetics, site-directed mutagenesis, spectroscopy, and structural modeling. Our investigations showed that OxaD catalyzes two oxidations of the indole nitrogen of roquefortine C, ultimately yielding roquefortine L. The reductive-half reaction analysis indicated that OxaD rapidly undergoes reduction and follows a "cautious" flavin reduction mechanism by requiring substrate binding before reduction can take place. This characteristic places OxaD in class A of the FMO family, a classification supported by a structural model featuring a single Rossmann nucleotide binding domain and a glutathione reductase fold. Furthermore, our spectroscopic analysis unveiled both enzyme-substrate and enzyme-intermediate complexes. Our analysis of the oxidative-half reaction suggests that the flavin dehydration step is the slow step in the catalytic cycle. Finally, through mutagenesis of the conserved D63 residue, we demonstrated its role in flavin motion and product oxygenation. Based on our findings, we propose a catalytic mechanism for OxaD and provide insights into the active site architecture within class A FMOs.

Improving the inhibitory resistance of xylanase FgXyn11C from Fusarium graminearum to SyXIP-I by site-directed mutagenesis

The aim of this study was to improve the inhibitory resistance of xylanase FgXyn11C from Fusarium graminearum to XIP in cereal flour. Site saturation mutagenesis was performed using computer-aided redesign. Firstly, based on multiple primary structure alignments, the amino acid residues in the active site architecture were identified, and specific residue T144 in the thumb region of FgXyn11C was selected for site-saturation mutagenesis. After screening, FgXyn11CT144F was selected as the best mutant, as it displayed the highest enzymatic activity and resistance simultaneously compared to other mutants. The specific activity of FgXyn11CT144F was 208.8 U/mg and it exhibited complete resistance to SyXIP-I. Compared with the wild-type, FgXyn11CT144F displayed similar activity and the most resistant against SyXIP-I. The optimal temperature and pH of the wild-type and purified FgXyn11CT144F were similar at pH 5.0 and 30 °C. Our findings provided preliminary insight into how the specific residue at position 144 in the thumb region of FgXyn11C influenced the enzymatic properties and interacted with SyXIP-I. The inhibition sensitivity of FgXyn11C was reduced through directed evolution, leading to creation of the mutant enzyme FgXyn11CT144F. The FgXyn11CT144F resistance to SyXIP-I has potential application and can also provide references for engineering other resistant xylanases of the GHF11.

Balanced Mass Transfer and Active Sites Density in Hierarchical Porous Catalytic Metal-Organic Framework for Enhancing Redox Reaction in Lithium-Sulfur Batteries.

Developing highly efficient catalysts, characterized by controllable pore architecture and effective utilization of active sites, is paramount in addressing the shuttle effect and sluggish redox kinetics of lithium polysulfides (LiPSs) in lithium-sulfur batteries (LSBs), which, however, remains a formidable challenge. In this study, a hierarchical porous catalytic metal-organic framework (HPC-MOF) with both appropriate porosity and abundant exposed catalytic sites is achieved through time-controlled precise pore engineering. It is revealed that the evolution of the porous structure and catalytic site density is time-dependent during the etching processes. The moderately etched HPC-MOF-M attains heterogeneous pores at various scales, where large apertures ensure fast mass transfer and micropores inherit high-density catalytic sites, enhancing utilization and catalytic kinetics at internal catalytic sites. Capitalizing on these advantages, LSB incorporating the HPC-MOF-M interlayer demonstrates a 164.6% improvement in discharge capability and an 83.3% lower decay rate over long-term cycling at 1.0C. Even under high sulfur loading of 7.1 mg cm-2 and lean electrolyte conditions, the LSB exhibits stable cycling for over 100 cycles. This work highlights the significance of balancing the relationship between mass transfer and catalytic sites through precise chemical regulation of the porous structure in catalytic MOFs, which are anticipated to inspire the development of advanced catalysts for LSBs.