Catalytic Cleft Research Articles

Lasamide, a Potent Human Carbonic Anhydrase Inhibitor from the Market: Inhibition Profiling and Crystallographic Studies.

Lasamide is a synthetic precursor and a contaminant of the diuretic Furosemide manufacturing process and represents a highly valuable building block for fragment-based drug discovery approaches. We assessed the ability of Lasamide to inhibit in vitro the human-expressed Carbonic Anhydrases by means of the stopped-flow technique, and we assessed its binding modes within hCAs II and XII-mimic catalytic clefts by X-ray crystallography. Interestingly, an unprecedented crystal form for the hCA IX mimic H-tag is reported and discussed herein.

Functional and Mechanistic Dissection of Protein Glutaminase PG3 and Its Rational Engineering for Enhanced Modification of Myofibrillar Proteins.

Protein glutaminases (PG; EC = 3.5.1.44) are enzymes known for enhancing protein functionality. In this study, we cloned and expressed the gene chryb3 encoding protein glutaminase PG3, exhibiting 39.4 U/mg specific activity. Mature-PG3 featured a substrate channel surrounded by aromatic and hydrophobic amino acids at positions 38-45 and 78-84, with Val81 playing a pivotal role in substrate affinity. The dynamic opening and closing motions between Gly65, Thr66, and Cys164 at the catalytic cleft greatly influence substrate binding and product release. Redesigning catalytic pocket and cocatalytic region produced combinatorial mutant MT6 showing a 2.69-fold increase in specific activity and a 2.99-fold increase at t65°C1/2. Furthermore, MT6 boosted fish myofibrillar protein (MP) solubility without NaCl. Key residues such as Thr3, Asn54, Val81, Tyr82, Asn107, and Ser108 were vital for PG3-myosin interaction, particularly Asn54 and Asn107. This study sheds light on the catalytic mechanism of PG3 and guided its rational engineering and utilization in low-salt fish MP product production.

Antibiotic resistance challenge: evaluating anthraquinones as rifampicin monooxygenase inhibitors through integrated bioinformatics analysis

ObjectiveAntibiotic resistance poses a pressing and crucial global public health challenge, leading to significant clinical and health-related consequences. Substantial evidence highlights the pivotal involvement of rifampicin monooxygenase (RIFMO) in the context of antibiotic resistance. Hence, inhibiting RIFMO could offer potential in the treatment of various infections. Anthraquinones, a group of organic compounds, have shown promise in addressing tuberculosis. This study employed integrated bioinformatics approaches to evaluate the potential inhibitory effects of a selection of anthraquinones on RIFMO. The findings were subsequently compared with those of rifampicin (RIF), serving as a positive control inhibitor.MethodsThe AutoDock 4.0 tool assessed the binding free energy between 21 anthraquinones and the RIFMO catalytic cleft. The ligands were ranked based on the most favorable scores derived from ΔGbinding. The docking analyses for the highest-ranked anthraquinone and RIF underwent a cross-validation process. This validation procedure utilized the SwissDock server and the Schrödinger Maestro docking software. Molecular dynamics simulations were conducted to scrutinize the stability of the backbone atoms in free RIFMO, RIFMO-RIF, and RIFMO complexed with the top-ranked anthraquinone throughout a 100-ns computer simulation. The Discovery Studio Visualizer tool visualized interactions between RIFMO residues and ligands. An evaluation of the pharmacokinetics and toxicity profiles of the tested compounds was also conducted.ResultsFive anthraquinones were indicated with ΔGbinding scores less than − 10 kcal/mol. Hypericin emerged as the most potent RIFMO inhibitor, boasting a ΔGbinding score and inhibition constant value of − 12.11 kcal/mol and 798.99 pM, respectively. The agreement across AutoDock 4.0, SwissDock, and Schrödinger Maestro results highlighted hypericin’s notable binding affinity to the RIFMO catalytic cleft. The RIFMO-hypericin complex achieved stability after a 70-ns computer simulation, exhibiting a root-mean-square deviation of 0.55 nm. Oral bioavailability analysis revealed that all anthraquinones except hypericin, sennidin A, and sennidin B may be suitable for oral administration. Furthermore, the carcinogenicity prediction analysis indicated a favorable safety profile for all examined anthraquinones.ConclusionInhibiting RIFMO, particularly with anthraquinones such as hypericin, holds promise as a potential therapeutic strategy for infectious diseases.

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.

Docking, MD Simulations, and DFT Calculations: Assessing W254's Function and Sartan Binding in Furin.

Furins are serine endoproteases that are involved in many biological processes, where they play important roles in normal metabolism, in the activation of various pathogens, while they are a target for therapeutic intervention. Dichlorophenyl-pyridine "BOS" compounds are well known drugs that are used as inhibitors of human furin by an induced-fit mechanism, in which tryptophan W254 in the furin catalytic cleft acts as a molecular transition energy gate. The binding of "BOS" drug into the active center of furin has been computationally studied using the density functional theory (DFT) and ONIOM multiscaling methodologies. The binding enthalpies of the W254 with the furin-BOS is -32.8 kcal/mol ("open") and -18.8 kcal/mol ("closed"), while the calculated torsion barrier was found at 30 kcal/mol. It is significantly smaller than the value of previous MD calculations due to the relaxation of the environment, i.e., nearby groups of the W254, leading to the reduction of the energy demands. The significant lower barrier explains the experimental finding that the dihedral barrier of W254 is overcome. Furthermore, sartans were studied to evaluate their potential as furin inhibitors. Sartans are AT1 antagonists, and they effectively inhibit the hypertensive effects induced by the peptide hormone Angiotensin II. Here, they have been docked into the cavity to evaluate their effect on the BOS ligand via docking and molecular dynamics simulations. A consistent binding of sartans within the cavity during the simulation was found, suggesting that they could act as furin inhibitors. Finally, sartans interact with the same amino acids as W254, leading to a competitive binding that may influence the pharmacological efficacy and potential drug interactions of sartans.

Targeting SHP2 Cryptic Allosteric Sites for Effective Cancer Therapy.

SHP2, a pivotal component downstream of both receptor and non-receptor tyrosine kinases, has been underscored in the progression of various human cancers and neurodevelopmental disorders. Allosteric inhibitors have been proposed to regulate its autoinhibition. However, oncogenic mutations, such as E76K, convert SHP2 into its open state, wherein the catalytic cleft becomes fully exposed to its ligands. This study elucidates the dynamic properties of SHP2 structures across different states, with a focus on the effects of oncogenic mutation on two known binding sites of allosteric inhibitors. Through extensive modeling and simulations, we further identified an alternative allosteric binding pocket in solution structures. Additional analysis provides insights into the dynamics and stability of the potential site. In addition, multi-tier screening was deployed to identify potential binders targeting the potential site. Our efforts to identify a new allosteric site contribute to community-wide initiatives developing therapies using multiple allosteric inhibitors to target distinct pockets on SHP2, in the hope of potentially inhibiting or slowing tumor growth associated with SHP2.

Micelles Cascade Assembly to Tandem Porous Catalyst for Waste Plastics Upcycling

AbstractCatalytic upcycling of polyolefins into high‐value chemicals represents the direction in end‐of‐life plastics valorization, but poses great challenges. Here, we report the synthesis of a tandem porous catalyst via a micelle cascade assembly strategy for selectively catalytic cracking of polyethylene into olefins at a low temperature. A hierarchically porous silica layer from mesopore to macropore is constructed on the surface of microporous ZSM‐5 nanosheets through cascade assembly of dynamic micelles. The outer macropore arrays can adsorb bulky polyolefins quickly by the capillary and hydrophobic effects, enhancing the diffusion and access to active sites. The middle mesopores present a nanoconfinement space, pre‐cracking polyolefins into intermediates by weak acid sites, which then transport into zeolites micropores for further cracking by strong Brønsted acid sites. The hierarchically porous and acidic structures, mimicking biomimetic protease catalytic clefts, ideally match the tandem cracking steps of polyolefins, thus suppressing coke formation and facilitating product escape. As a result, light hydrocarbons (C1‐C7) are produced with a yield of 443 mmol gZSM‐5−1, where 74.3 % of them are C3‐C6 olefins, much superior to ZSM‐5 and porous silica catalysts. This tandem porous catalyst exemplifies a superstructure design of catalytic cracking catalysts for industrial and economical upcycling of plastic wastes.

Micelles Cascade Assembly to Tandem Porous Catalyst for Waste Plastics Upcycling.

Catalytic upcycling of polyolefins into high-value chemicals represents the direction in end-of-life plastics valorization, but poses great challenges. Here, we report the synthesis of a tandem porous catalyst via a micelle cascade assembly strategy for selectively catalytic cracking of polyethylene into olefins at a low temperature. A hierarchically porous silica layer from mesopore to macropore is constructed on the surface of microporous ZSM-5 nanosheets through cascade assembly of dynamic micelles. The outer macropore arrays can adsorb bulky polyolefins quickly by the capillary and hydrophobic effects, enhancing the diffusion and access to active sites. The middle mesopores present a nanoconfinement space, pre-cracking polyolefins into intermediates by weak acid sites, which then transport into zeolites micropores for further cracking by strong Brønsted acid sites. The hierarchically porous and acidic structures, mimicking biomimetic protease catalytic clefts, ideally match the tandem cracking steps of polyolefins, thus suppressing coke formation and facilitating product escape. As a result, light hydrocarbons (C1-C7) are produced with a yield of 443 mmol gZSM-5 -1, where 74.3 % of them are C3-C6 olefins, much superior to ZSM-5 and porous silica catalysts. This tandem porous catalyst exemplifies a superstructure design of catalytic cracking catalysts for industrial and economical upcycling of plastic wastes.

Crystal structure of mango α1,3/α1,4-fucosyltransferase elucidates unique elements that regulate Lewis A-dominant oligosaccharide assembly.

In various organisms, α1,3/α1,4-fucosyltransferases (CAZy GT10 family enzymes) mediate the assembly of type I (Galβ1,3GlcNAc) and/or type II (Galβ1,4GlcNAc)-based Lewis structures that are widely distributed in glycoconjugates. Unlike enzymes of other species, plant orthologues show little fucosyltransferase activity for type II-based glycans and predominantly catalyze the assembly of the Lewis A structure [Galβ1,3(Fucα1,4)GlcNAc] on the type I disaccharide unit of their substrates. However, the structural basis underlying this unique substrate selectivity remains elusive. In this study, we investigated the structure-function relationship of MiFUT13A, a mango α1,3/α1,4-fucosyltransferase. The prepared MiFUT13A displayed distinct α1,4-fucosyltransferase activity. Consistent with the enzymatic properties of this molecule, X-ray crystallography revealed that this enzyme has a typical GT-B fold-type structure containing a set of residues that are responsible for its SN2-like catalysis. Site-directed mutagenesis and molecular docking analyses proposed a rational binding mechanism for type I oligosaccharides. Within the catalytic cleft, the pocket surrounding Trp121 serves as a binding site, anchoring the non-reducing terminal β1,3-galactose that belongs to the type I disaccharide unit. Furthermore, Glu177 was postulated to function as a general base catalyst through its interaction with the 4-hydroxy group of the acceptor N-acetylglucosamine residue. Adjacent residues, specifically Thr120, Thr157 and Asp175 were speculated to assist in binding of the reducing terminal residues. Intriguingly, these structural elements were not fully conserved in mammalian orthologue which also shows predominant α1,4-fucosyltransferase activity. In conclusion, we have proposed that MiFUT13A generates the Lewis A structure on type I glycans through a distinct mechanism, divergent from that of mammalian enzymes.

Deciphering the molecular choreography of Janus kinase 2 inhibition via Gaussian accelerated molecular dynamics simulations: a dynamic odyssey.

The Janus kinases (JAK) are crucial targets in drug development for several diseases. However, accounting for the impact of possible structural rearrangements on the binding of different kinase inhibitors is complicated by the extensive conformational variability of their catalytic kinase domain (KD). The dynamic KD contains mainly four prominent mobile structural motifs: the phosphate-binding loop (P-loop), the αC-helix within the N-lobe, the Asp-Phe-Gly (DFG) motif, and the activation loop (A-loop) within the C-lobe. These distinct structural orientations imply a complex signal transmission path for regulating the A-loop's flexibility and conformational preference for optimal JAK function. Nevertheless, the precise dynamical features of the JAK induced by different types of inhibitors still remain elusive. We performed comparative, microsecond-long, Gaussian accelerated molecular dynamics simulations in triplicate of three phosphorylated JAK2 systems: the KD alone, type-I ATP-competitive inhibitor (CI) bound KD in the catalytically active DFG-in conformation, and the type-II inhibitor (AI) bound KD in the catalytically inactive DFG-out conformation. Our results indicate significant conformational variations observed in the A-loop and αC helix motions upon inhibitor binding. Our studies also reveal that the DFG-out inactive conformation is characterized by the closed A-loop rearrangement, open catalytic cleft of N and C-lobe, the outward movement of the αC helix, and open P-loop states. Moreover, the outward positioning of the αC helix impacts the hallmark salt bridge formation between Lys882 and Glu898 in an inactive conformation. Finally, we compared their ligand binding poses and free energy by the MM/PBSA approach. The free energy calculations suggested that the AI's binding affinity is higher than CI against JAK2 due to an increased favorable contribution from the total non-polar interactions and the involvement of the αC helix. Overall, our study provides the structural and energetic insights crucial for developing more promising type I/II JAK2 inhibitors for treating JAK-related diseases.

Plant root associated chitinases: structures and functions.

Chitinases degrade chitin, a linear homopolymer of β-1,4-linked N-acetyl-D-glucosamine (GlcNAc) residues found in the cell walls of fungi and the exoskeletons of arthropods. They are secreted by the roots into the rhizosphere, a complex and dynamic environment where intense nutrient exchange occurs between plants and microbes. Here we modeled, expressed, purified, and characterized Zea mays and Oryza sativa root chitinases, and the chitinase of a symbiotic bacterium, Chitinophaga oryzae 1303 for their activities with chitin, di-, tri-, and tetra-saccharides and Aspergillus niger, with the goal of determining their role(s) in the rhizosphere and better understanding the molecular mechanisms underlying plant-microbe interactions. We show that Zea mays basic endochitinase (ZmChi19A) and Oryza sativa chitinase (OsChi19A) are from the GH19 chitinase family. The Chitinophaga oryzae 1303 chitinase (CspCh18A) belongs to the GH18 family. The three enzymes have similar apparent K M values of (20-40 µM) for the substrate 4-MU-GlcNAc3. They vary in their pH and temperature optima with OsChi19A activity optimal between pH 5-7 and 30-40°C while ZmChi19A and CspCh18A activities were optimal at pH 7-9 and 50-60°C. Modeling and site-directed mutation of ZmChi19A identified the catalytic cleft and the active residues E147 and E169 strategically positioned at ~8.6Å from each other in the folded protein. Cleavage of 4-MU-GlcNAc3 was unaffected by the absence of the CBD but diminished in the absence of the flexible C-terminal domain. However, unlike for the soluble substrate, the CBD and the newly identified flexible C-terminal domain were vital for inhibiting Aspergillus niger growth. The results are consistent with the involvement of the plant chitinases in defense against pathogens like fungi that have chitin exoskeletons. In summary, we have characterized the functional features and structural domains necessary for the activity of two plant root chitinases that are believed to be involved in plant defense and a bacterial chitinase that, along with the plant chitinases, may participate in nutrient recycling in the rhizosphere.

A rumen-derived bifunctional glucanase/mannanase uncanonically releases oligosaccharides with a high degree of polymerization preferentially from branched substrates

Glycoside hydrolases (GHs) are known to depolymerize polysaccharides into oligo-/mono-saccharides, they are extensively used as additives for both animals feed and our food. Here we reported the characterization of IDSGH5-14(CD), a weakly-acidic mesophilic bifunctional mannanase/glucanase of GH5, originally isolated from sheep rumen microbes. Biochemical characterization studies revealed that IDSGH5-14(CD) exhibited preferential hydrolysis of mannan-like and glucan-like substrates. Interestingly, the enzyme exhibited significantly robust catalytic activity towards branched-substrates compared to linear polysaccharides (P < 0.05). Substrate hydrolysis pattern indicated that IDSGH5-14(CD) predominantly liberated oligosaccharides with a degree of polymerization (DP) of 3–7 as the end products, dramatically distinct from canonical endo-acting enzymes. Comparative modeling revealed that IDSGH5-14(CD) was mainly comprised of a (β/α)8-barrel-like structure with a spacious catalytic cleft on surface, facilitating the enzyme to target high-DP or branched oligosaccharides. Molecular dynamics (MD) simulations further suggested that the branched-ligand, 64-α-D-galactosyl-mannohexose, was steadily accommodated within the catalytic pocket via a two-sided clamp formed by the aromatic residues. This study first reports a bifunctional GH5 enzyme that predominantly generates high-DP oligosaccharides, preferentially from branched-substrates. This provides novel insights into the catalytic mechanism and molecular underpinnings of polysaccharide depolymerization, with potential implications for feed additive development and high-DP oligosaccharides preparation.

Structures of Cutibacterium acnes hyaluronate lyases suggest a correlation between active site opened/closed state and conformation of abutting loop.

The structures of hyaluronate lyases from two Cutibacterium acnes strains have been reported recently and show open catalytic clefts. We compared these open structures with more closed structures of homologous lyases and found that the conformation of a loop that abuts the catalytic cleft is seemingly correlated with the opening and closing of the cleft. We illustrate that the loop conformation seen in the open lyase appears incompatible with a closed catalytic cleft, and vice versa; however, mutations designed to disrupt the loop conformation did not significantly affect catalytic activity.

The allosteric mechanism of mTOR activation can inform bitopic inhibitor optimization.

mTOR serine/threonine kinase is a cornerstone in the PI3K/AKT/mTOR pathway. Yet, the detailed mechanism of activation of its catalytic core is still unresolved, likely due to mTOR complexes' complexity. Its dysregulation was implicated in cancer and neurodevelopmental disorders. Using extensive molecular dynamics (MD) simulations and compiled published experimental data, we determine exactly how mTOR's inherent motifs can control the conformational changes in the kinase domain, thus kinase activity. We also chronicle the critical regulation by the unstructured negative regulator domain (NRD). When positioned inside the catalytic cleft (NRD IN state), mTOR tends to adopt a deep and closed catalytic cleft. This is primarily due to the direct interaction with the FKBP-rapamycin binding (FRB) domain which restricts it, preventing substrate access. Conversely, when outside the catalytic cleft (NRD OUT state), mTOR favors an open conformation, exposing the substrate-binding site on the FRB domain. We further show how an oncogenic mutation (L2427R) promotes shifting the mTOR ensemble toward the catalysis-favored state. Collectively, we extend mTOR's "active-site restriction" mechanism and clarify mutation action. In particular, our mechanism suggests that RMC-5552 (RMC-6272) bitopic inhibitors may benefit from adjustment of the (PEG8) linker length when targeting certain mTOR variants. In the cryo-EM mTOR/RMC-5552 structure, the distance between the allosteric and orthosteric inhibitors is ∼22.7 Å. With a closed catalytic cleft, this linker bridges the sites. However, in our activation mechanism, in the open cleft it expands to ∼24.7 Å, offering what we believe to be the first direct example of how discovering an activation mechanism can potentially increase the affinity of inhibitors targeting mutants.

Targeting Matrix Metalloproteinase‐3 for Dental Caries Prevention Using Herbal Isolates: MMP3 Inhibition by Cinnamic Acids

Objectives: Dental caries, a prevalent infectious disease affecting teeth, ranks highest among 328 diseases, according to a 2017 Lancet study. In demineralized human dentin, matrix metalloproteinase‐3 (MMP3) functions as a proteoglycanase, contributing to the degradation of proteoglycan components. This process exposes collagen fibrils, thereby facilitating the demineralization of the dentin matrix. Inhibiting MMP3 shows potential for preventing dental caries.Methods: The binding affinity of 20 cinnamic acid derivatives, namely cynarin, chlorogenic acid, rosmarinic acid, cinnamyl caffeate, phenethyl caffeate, N‐p‐coumaroyltyramine, caffeic acid 3‐glucoside, caffeic acid phenethyl ester, roscovitine, benzyl caffeate, o‐coumaric acid, artepillin C, caffeic acid, methyl caffeate, 2‐methylcinnamic acid, ferulic acid, drupanin, p‐coumaric acid, cinnamic acid, and sinapinic acid, to the MMP3 catalytic cleft, was assessed utilizing AutoDock 4.0. Molecular dynamics simulation was then employed to analyze the stability of backbone atoms in free MMP3, MMP3‐positive control inhibitor, and MMP3 complexed with the top‐ranked cinnamic acid over a 100 ns computer simulation.Results: Four cinnamic acids demonstrated ΔGbinding scores below −10 kcal/mol, with cynarin emerging as the most potent MMP3 inhibitor, featuring a ΔGbinding score and inhibition constant value of −15.57 kcal/mol and 3.83 pM, respectively. The MMP3–cynarin complex exhibited stability after a 50 ns computer simulation, showing a root‐mean‐square deviation of 8 Å.Conclusions: The inhibition of MMP3 by cynarin, chlorogenic acid, rosmarinic acid, and cinnamyl caffeate holds promise as a potential preventive strategy for dental caries.

Characterization of chloroplast ribulose-5-phosphate-3-epimerase from the microalga Chlamydomonas reinhardtii.

Carbon fixation relies on Rubisco and 10 additional enzymes in the Calvin-Benson-Bassham cycle. Epimerization of xylulose-5-phosphate (Xu5P) into ribulose-5-phosphate (Ru5P) contributes to the regeneration of ribulose-1,5-bisphosphate, the substrate of Rubisco. Ribulose-5-phosphate-3-epimerase (RPE, EC 5.1.3.1) catalyzes the formation of Ru5P, but it can also operate in the pentose-phosphate pathway by catalyzing the reverse reaction. Here, we describe the structural and biochemical properties of the recombinant RPE isoform 1 from Chlamydomonas (Chlamydomonas reinhardtii) (CrRPE1). The enzyme is a homo-hexamer that contains a zinc ion in the active site and exposes a catalytic pocket on the top of an α8β8 triose isomerase-type barrel as observed in structurally solved RPE isoforms from both plant and non-plant sources. By optimizing and developing enzyme assays to monitor the reversible epimerization of Ru5P to Xu5P and vice versa, we determined the catalytic parameters that differ from those of other plant paralogs. Despite being identified as a putative target of multiple thiol-based redox modifications, CrRPE1 activity is not affected by both reductive and oxidative treatments, indicating that enzyme catalysis is insensitive to possible redox alterations of cysteine residues. We mapped phosphorylation sites on the crystal structure, and the specific location at the entrance of the catalytic cleft supports a phosphorylation-based regulatory mechanism. This work provides an accurate description of the structural features of CrRPE1 and an in-depth examination of its catalytic and regulatory properties highlighting the physiological relevance of this enzyme in the context of photosynthetic carbon fixation.

Structural Characterization and Ligand-Induced Conformational Changes of SenB, a Se-Glycosyltransferase Involved in Selenoneine Biosynthesis.

Selenium (Se) is an essential micronutrient that is found naturally in proteins, nucleic acids, and natural products. Unlike selenoproteins and selenonucleic acids, little is known about the structures of biosynthetic enzymes that incorporate Se into small molecules. Here, we report the X-ray crystal structure of SenB, the first known Se-glycosyltransferase that was recently found to be involved in the biosynthesis of the Se-containing metabolite selenoneine. SenB catalyzes C-Se bond formation using selenophosphate and an activated uridine diphosphate sugar as a Se and glycosyl donor, respectively, making it the first known selenosugar synthase and one of only four bona fide C-Se bond-forming enzymes discovered to date. Our crystal structure, determined to 2.25 Å resolution, reveals that SenB is a type B glycosyltransferase, displaying the prototypical fold with two globular Rossmann-like domains and a catalytic interdomain cleft. By employing complementary structural biology techniques, we find that SenB undergoes both local and global substrate-induced conformational changes, demonstrating a significant increase in α-helicity and a transition to a more compact conformation. Our results provide the first structure of SenB and set the stage for further biochemical characterization in the future.

An allosteric switch between the activation loop and a c-terminal palindromic phospho-motif controls c-Src function

Autophosphorylation controls the transition between discrete functional and conformational states in protein kinases, yet the structural and molecular determinants underlying this fundamental process remain unclear. Here we show that c-terminal Tyr 530 is a de facto c-Src autophosphorylation site with slow time-resolution kinetics and a strong intermolecular component. On the contrary, activation-loop Tyr 419 undergoes faster kinetics and a cis-to-trans phosphorylation switch that controls c-terminal Tyr 530 autophosphorylation, enzyme specificity, and strikingly, c-Src non-catalytic function as a substrate. In line with this, we visualize by X-ray crystallography a snapshot of Tyr 530 intermolecular autophosphorylation. In an asymmetric arrangement of both catalytic domains, a c-terminal palindromic phospho-motif flanking Tyr 530 on the substrate molecule engages the G-loop of the active kinase adopting a position ready for entry into the catalytic cleft. Perturbation of the phospho-motif accounts for c-Src dysfunction as indicated by viral and colorectal cancer (CRC)-associated c-terminal deleted variants. We show that c-terminal residues 531 to 536 are required for c-Src Tyr 530 autophosphorylation, and such a detrimental effect is caused by the substrate molecule inhibiting allosterically the active kinase. Our work reveals a crosstalk between the activation and c-terminal segments that control the allosteric interplay between substrate- and enzyme-acting kinases during autophosphorylation.

Structures of liganded glycosylphosphatidylinositol transamidase illuminate GPI-AP biogenesis

Many eukaryotic receptors and enzymes rely on glycosylphosphatidylinositol (GPI) anchors for membrane localization and function. The transmembrane complex GPI-T recognizes diverse proproteins at a signal peptide region that lacks consensus sequence and replaces it with GPI via a transamidation reaction. How GPI-T maintains broad specificity while preventing unintentional cleavage is unclear. Here, substrates- and products-bound human GPI-T structures identify subsite features that enable broad proprotein specificity, inform catalytic mechanism, and reveal a multilevel safeguard mechanism against its promiscuity. In the absence of proproteins, the catalytic site is invaded by a locally stabilized loop. Activation requires energetically unfavorable rearrangements that transform the autoinhibitory loop into crucial catalytic cleft elements. Enzyme-proprotein binding in the transmembrane and luminal domains respectively powers the conformational rearrangement and induces a competent cleft. GPI-T thus integrates various weak specificity regions to form strong selectivity and prevent accidental activation. These findings provide important mechanistic insights into GPI-anchored protein biogenesis.

Heterogeneity in M. tuberculosis β-lactamase inhibition by Sulbactam

For decades, researchers have elucidated essential enzymatic functions on the atomic length scale by tracing atomic positions in real-time. Our work builds on possibilities unleashed by mix-and-inject serial crystallography (MISC) at X-ray free electron laser facilities. In this approach, enzymatic reactions are triggered by mixing substrate or ligand solutions with enzyme microcrystals. Here, we report in atomic detail (between 2.2 and 2.7 Å resolution) by room-temperature, time-resolved crystallography with millisecond time-resolution (with timepoints between 3 ms and 700 ms) how the Mycobacterium tuberculosis enzyme BlaC is inhibited by sulbactam (SUB). Our results reveal ligand binding heterogeneity, ligand gating, cooperativity, induced fit, and conformational selection all from the same set of MISC data, detailing how SUB approaches the catalytic clefts and binds to the enzyme noncovalently before reacting to a trans-enamine. This was made possible in part by the application of singular value decomposition to the MISC data using a program that remains functional even if unit cell parameters change up to 3 Å during the reaction.