Covalent Substrate Research Articles

Shifts in Backbone Conformation of Acetylcholinesterases upon Binding of Covalent Inhibitors, Reversible Ligands and Substrates

The influence of ligand binding to human, mouse and Torpedo californica acetylcholinesterase (EC 3.1.1.7; AChE) backbone structures is analyzed in a pairwise fashion by comparison with X-ray structures of unliganded AChEs. Both complexes with reversible ligands (substrates and inhibitors) as well as covalently interacting ligands leading to the formation of covalent AChE conjugates of tetrahedral and of trigonal-planar geometries are considered. The acyl pocket loop (AP loop) in the AChE backbone is recognized as the conformationally most adaptive, but not necessarily sterically exclusive, structural element. Conformational changes of the centrally located AP loop coincide with shifts in C-terminal α-helical positions, revealing interacting components for a potential allosteric interaction within the AChE backbone. The stabilizing power of the aromatic choline binding site, with the potential to attract and pull fitting entities covalently tethered to the active Ser, is recognized. Consequently, the pull can promote catalytic reactions or relieve steric pressure within the impacted space of the AChE active center gorge. These dynamic properties of the AChE backbone inferred from the analysis of static X-ray structures contribute towards a better understanding of the molecular template important in the structure-based design of therapeutically active molecules, including AChE inhibitors as well as reactivators of conjugated, inactive AChE.

Evaluating the covalent binding of carbapenems on BlaC using noncovalent interactions

Carbapenems, as irreversible covalent binders and slow substrates to the class A β-lactamase (BlaC) of Mycobacterium tuberculosis, can inhibit BlaC to hydrolyze the β-lactam drugs which are used to control tuberculosis. Their binding on BlaC involves covalent bonding and noncovalent interaction. We introduce a hypothesis that the noncovalent interactions dominate the difference of binding free energies for covalent ligands based on the assumption that their covalent bonding energies are the same. MM/GBSA binding free energies calculated from the noncovalent interactions provided a threshold with respect to the experimental kinetic data, to select slow carbapenem substrates which were either constructed using the structural units of experimentally identified carbapenems or obtained from the similarity search over the ZINC15 database. Combining molecular docking with consensus scoring and molecular dynamics simulation with MM/GBSA binding free energy calculations, a computational protocol was developed from which several new tight-binding carbapenems were theoretically identified.

Facile synthesis and immobilization of functionalized covalent organic framework-1 for electrochromatographic separation

Inspired by the outstanding functions of 3-aminopropyltriethoxysilane (APTES), which can be used for functionalization of both covalent organic frameworks (COFs) and substrate surfaces, herein, a proof-of-concept demonstration was carried out by one-step synthesis and immobilization of COF-1 in capillary. COF-1 was grown on the inner wall of capillary using APTES, which played a triple role of catalyst, stabilizer, and connecting arm during the process. The immobilization of COF-1 on silicon surface was confirmed by scanning electron microscopy. Moreover, COF-1 modified capillary (COF-1@capillary) column exhibited excellent performance in the electrochromatographic separation of amino acids. High resolution and separation efficiency (225,378 plates/m for 4-methylbiphenyl) were successfully achieved. Separation of methylbenzene, styrene, ethylbenzene, chlorobenzene, 1,2-dichlorobenzene, 1,2,4-trichlorobenzene, 4-methylbiphenyl, naphthalene, and 4-vinylbipheny in the electro-driven mode confirmed the successful growth of COF-1 on the inner wall of capillary. The developed facile method for the immobilization of COF-1 may pave the way for further application prospects of boron-based COFs.

Identification of targets of AMPylating Fic enzymes by co-substrate-mediated covalent capture.

Various pathogenic bacteria use post-translational modifications to manipulate the central components of host cell functions. Many of the enzymes released by these bacteria belong to the large Fic family, which modify targets with nucleotide monophosphates. The lack of a generic method for identifying the cellular targets of Fic family enzymes hinders investigation of their role and the effect of the post-translational modification. Here, we establish an approach that uses reactive co-substrate-linked enzymes for proteome profiling. We combine synthetic thiol-reactive nucleotide derivatives with recombinantly produced Fic enzymes containing strategically placed cysteines in their active sites to yield reactive binary probes for covalent substrate capture. The binary complexes capture their targets from cell lysates and permit subsequent identification. Furthermore, we determined the structures of low-affinity ternary enzyme-nucleotide-substrate complexes by applying a covalent-linking strategy. This approach thus allows target identification of the Fic enzymes from both bacteria and eukarya.

Finetuning hierarchical energy material microstructure via high temperature material synthesis route

Hierarchical energy materials such as graphite are a backbone of various scientifically and commercially important emerging technologies including high-energy density energy storage systems with fast charging capability, multifunctional catalyst systems, selective membrane separation systems, and next-generation nuclear material systems. Consequently, it is extremely crucial to develop an efficient and cost-effective route of bulk hierarchical material synthesis (e.g., carbonaceous materials with a well-controlled fraction of the graphitic content) to cater the extraordinary operational and energy material requirements in a very complex coupled thermophysicochemical environment. Here we present a fabrication of Polyacrylonitrile (PAN) derived carbon films and fibers (~with linear dimension ~100 nm) via electrospinning and spin coating methods ensued by a heat-treatment in the range of 1000–3000 °C under inert atmospheric conditions. Intriguingly, we observed at least a two orders of magnitude enhancement (~134%) in length of graphitic plane accompanied by 36% more graphitization when the carbonization temperature increased from 1000 °C to 3000 °C. Such significant enhancements were attributed to the differences in the fundamental nanomorphology of initial carbonaceous materials and their subsequent kinetic evolution as it was more favorable for underlying graphene layers in films to stack and bond to the adjacent ones without strong rotations as compared to fibers, which were further evident from fewer voids and cracks in the films. The covalent cross-links, substrate effect and physical entanglements of carbon domains in PAN-derived carbon films contributed to a higher graphitic length owing to more shear stress between the graphene layers, compared to fibers and undergoes an enormous transformation from turbostratic structures to ordered state along with nitrogen removing over high temperature heating. This morphology dependent graphitization was also investigated from computational approach and concluded in the similar thoughts. The outcomes from this systematic study can be beneficial to the carbon research community focusing in the morphology dependent applications, for instance catalysis, energy storage, sensors etc.

Flavin-N5 Covalent Intermediate in a Nonredox Dehalogenation Reaction Catalyzed by an Atypical Flavoenzyme.

The flavin-dependent enzyme 2-haloacrylate hydratase (2-HAH) catalyzes the conversion of 2-chloroacrylate, a major component in the manufacture of acrylic polymers, to pyruvate. The enzyme was expressed in Escherichia coli, purified, and characterized. 2-HAH was shown to be monomeric in solution and contained a non-covalent, yet tightly bound, flavin adenine dinucleotide (FAD). Although the catalyzed reaction was redox-neutral, 2-HAH was active only in the reduced state. A covalent flavin-substrate intermediate, consistent with the flavin-acrylate iminium ion, was trapped with cyanoborohydride and characterized by mass spectrometry. Small-angle X-ray scattering was consistent with 2-HAH belonging to the succinate dehydrogenase/fumarate reductase family of flavoproteins. These studies establish 2-HAH as a novel noncanonical flavoenzyme.

Chemical Probes Allow Structural Insight into the Condensation Reaction of Nonribosomal Peptide Synthetases

Nonribosomal peptide synthetases (NRPSs) synthesize a vast variety of small molecules, including antibiotics, antitumors, and immunosuppressants. The NRPS condensation (C) domain catalyzes amide bond formation, the central chemical step in nonribosomal peptide synthesis. The catalytic mechanism and substrate determinants of the reaction are under debate. We developed chemical probes to structurally study the NRPS condensation reaction. These substrate analogs become covalently tethered to a cysteine introduced near the active site, to mimic covalent substrate delivery by carrier domains. They are competent substrates in the condensation reaction and behave similarly to native substrates. Co-crystal structures show C domain-substrate interactions, and suggest that the catalytic histidine's principle role is to position the α-amino group for nucleophilic attack. Structural insight provided by these co-complexes also allowed us to alter the substrate specificity profile of the reaction with a single point mutation.

ChemInform Abstract: The Ever‐expanding Role of Asymmetric Covalent Organocatalysis in Scalable, Natural Product Synthesis

AbstractReview: 155 refs.

Biotin and Lipoic Acid: Synthesis, Attachment, and Regulation.

Two vitamins, biotin and lipoic acid, are essential in all three domains of life. Both coenzymes function only when covalently attached to key metabolic enzymes. There they act as "swinging arms" that shuttle intermediates between two active sites (= covalent substrate channeling) of key metabolic enzymes. Although biotin was discovered over 100 years ago and lipoic acid 60 years ago, it was not known how either coenzyme is made until recently. In Escherichia coli the synthetic pathways for both coenzymes have now been worked out for the first time. The late steps of biotin synthesis, those involved in assembling the fused rings, were well described biochemically years ago, although recent progress has been made on the BioB reaction, the last step of the pathway in which the biotin sulfur moiety is inserted. In contrast, the early steps of biotin synthesis, assembly of the fatty acid-like "arm" of biotin were unknown. It has now been demonstrated that the arm is made by using disguised substrates to gain entry into the fatty acid synthesis pathway followed by removal of the disguise when the proper chain length is attained. The BioC methyltransferase is responsible for introducing the disguise, and the BioH esterase is responsible for its removal. In contrast to biotin, which is attached to its cognate proteins as a finished molecule, lipoic acid is assembled on its cognate proteins. An octanoyl moiety is transferred from the octanoyl acyl carrier protein of fatty acid synthesis to a specific lysine residue of a cognate protein by the LipB octanoyltransferase followed by sulfur insertion at carbons C-6 and C-8 by the LipA lipoyl synthetase. Assembly on the cognate proteins regulates the amount of lipoic acid synthesized, and, thus, there is no transcriptional control of the synthetic genes. In contrast, transcriptional control of the biotin synthetic genes is wielded by a remarkably sophisticated, yet simple, system, exerted through BirA, a dual-function protein that both represses biotin operon transcription and ligates biotin to its cognate proteins.

Fe epilayer magnetic features induced by a covalent, magnetic and metallic substrate

In recent years there has been theoretical and experimental interest in the properties of Fe thin layers grown on metallic substrates. We present here a theoretical study based on ab initio calculations of the structure and magnetic characteristics for a very thin Fe film grown on an As-terminated MnAs substrate, which is metallic. Particular emphasis is put into the relaxation and reconstruction of the interface and on the characterization of the magnetic coupling with the substrate. The absence of Fe interdiffusion and the presence of an intermediate As layer ensure the partial magnetic decoupling between epilayer and substrate.

Controlling substrate specificity and product regio- and stereo-selectivities of P450 enzymes without mutagenesis

P450 enzymes (P450s) are well known for their ability to oxidize unactivated CH bonds with high regio- and stereoselectivity. Hence, there is emerging interest in exploiting P450s as potential biocatalysts. Although bacterial P450s typically show higher activity than their mammalian counterparts, they tend to be more substrate selective. Most drug-metabolizing P450s on the other hand, display remarkable substrate promiscuity, yet product prediction remains challenging. Protein engineering is one established strategy to overcome these issues. A less explored, yet promising alternative involves substrate engineering. This review discusses the use of small molecules for controlling the substrate specificity and product selectivity of P450s. The focus is on two approaches, one taking advantage of non-covalent decoy molecules, and the other involving covalent substrate modifications.

Role of primary bile salts in the regulation of sinusoidal substrate uptake in rat liver

Background Bile salts undergo enterohepatic circulation which is essential for bile salt homeostasis. From the biliary tract, they are excreted into the small intestine, absorbed into the blood and transported back to the liver. The sinusoidal uptake systems of the liver efficiently extract bile salts and other substrates from portal blood. In rat liver, these systems include Ntcp (sodium taurocholate cotransporting polypeptide) and the organic anion transporting polypeptides Oatp1a1, Oatp1a4 and Oatp1b2. While Ntcp represents the major basolateral uptake transporter for conjugated bile salts, the Oatps have broad substrate spectra including estrogenor leukotriene-conjugates. Their transport function is regulated by long-term and short-term mechanisms. Long-term adaptation of bile salt transporters involve changes at the level of gene expression and transporter degradation, while short-term regulation includes covalent transporter modifications, substrate availability and rapid endoand exocytosis of transporter-containing vesicles (reviewed in [1]). Ntcp as well as Oatp1a1 and Oatp1a4 underlie short-term control which has been demonstrated in several in vitro models. Hypoosmolarity or cAMP for instance increase the Ntcp-dependent bile salt uptake (increase of Vmax) within minutes by translocation of intracellular Ntcp to the plasma membrane [2,3]. In contrast, rapid clathrin-dependent endocytosis of Ntcp was demonstrated only recently by activation of PKC with phorbolesters [4,5]. However, to date in vivo-studies on short-term regulation of basolateral transporters are rare. Bile salt inflow to the liver may increase considerably under pathophysiological conditions or postprandial under physiological conditions [6]. To date, the interrelation between substrate load and transport activity is largely unknown. Therefore, the aim of this study was to investigate the role of primary bile salts in short-term feedback regulation of sinusoidal substrate uptake in perfused rat liver.

Tensile failure of bi-materials: High strain-rate simulations of thin adhesive layers on covalent substrates

In the present work, we report on results of simulation-based study of mechanical response behavior and mechanisms of failure of a nanometer-scale polyimide adherent thin-film on a silicon substrate. A transition between adhesive and cohesive modes of failure with the strain rate was shown to occur in the system, subjected to high strain-rate tensile loadings. The physical mechanisms, leading to the bi-material failure, were identified for each failure mode. The study reveals that damage development and propagation are determined by a complex interplay between coupling across the interface and relaxation processes in the adherent layer. The two act in a synergistic manner to generate an instability, leading to either cohesive or adhesive failure.

Zinc oxide composites prepared by in situ process: UV barrier and luminescence properties

This work describes the preparation of composite films of zinc oxide (ZnO) embedded in a covalent copolymer substrate of triethylene glycol dimethacrylate and poly(ethyleneglycol) diacrylate. The ZnO-embedded copolymer was produced in situ from the reaction of a doped precursor zinc bis(2-thenoyltrifluoroacetonate) [Zn(TTA)2] in a hydrazine hydrate reflux system.The in situ production of ZnO nanoparticles with excellent optical qualities embedded in the copolymer substrate was demonstrated to be an effective approach to fabricating new advanced materials for use as a protective barrier that blocks UV rays.

Magnetic exchange coupling in 3d-transition-metal atomic chains adsorbed on Cu2N/Cu(001)

Covalent substrates can give rise to a variety of magnetic interaction mechanisms among adsorbed transition-metal atoms building atomic nanostructures. We show this by calculating the ground state magnetic configuration of monoatomic $3d$ chains deposited on a monolayer of Cu${}_{2}$N grown on Cu(001) as a function of $d$ filling and of adsorption sites of these nanostructures.

Direct Observation of T4 Lysozyme Hinge-Bending Motion by Fluorescence Correlation Spectroscopy

Bacteriophage T4 Lysozyme (T4L) catalyzes the hydrolysis of the peptidoglycan layer of the bacterial cell wall late in the infection cycle. It has long been postulated that equilibrium dynamics enable substrate access to the active site located at the interface between the N- and C-terminal domains. Crystal structures of WT-T4L and point mutants captured a range of conformations that differ by the hinge-bending angle between the two domains. Evidence of equilibrium between open and closed conformations in solution was gleaned from distance measurements between the two domains but the nature of the equilibrium and the timescale of the underlying motion have not been investigated. Here, we used fluorescence fluctuation spectroscopy to directly detect T4L equilibrium conformational fluctuations in solution. For this purpose, Tetramethylrhodamine probes were introduced at pairs of cysteines in regions of the molecule that undergo relative displacement upon transition from open to closed conformations. Correlation analysis of Tetramethylrhodamine intensity fluctuations reveals hinge-bending motion that changes the relative distance and orientation of the N- and C-terminal domains with ≅15 μs relaxation time. That this motion involves interconversion between open and closed conformations was further confirmed by the dampening of its amplitude upon covalent substrate trapping. In contrast to the prevalent two-state model of T4L equilibrium, molecular brightness and number of particles obtained from cumulant analysis suggest that T4L populates multiple intermediate states, consistent with the wide range of hinge-bending angles trapped in the crystal structure of T4L mutants.

YajL, Prokaryotic Homolog of Parkinsonism-associated Protein DJ-1, Functions as a Covalent Chaperone for Thiol Proteome

YajL is the closest Escherichia coli homolog of the Parkinsonism-associated protein DJ-1, a multifunctional oxidative stress response protein whose biochemical function remains unclear. We recently reported the aggregation of proteins in a yajL mutant in an oxidative stress-dependent manner and that YajL exhibits chaperone activity. Here, we show that YajL displays covalent chaperone and weak protein oxidoreductase activities that are dependent on its exposed cysteine 106. It catalyzes reduced RNase oxidation and scrambled RNase isomerization and insulin reduction and forms mixed disulfides with many cellular proteins upon oxidative stress. The formation of mixed disulfides was detected by immunoblotting bacterial extracts with anti-YajL antibodies under nonreducing conditions. Disulfides were purified from bacterial extracts on a YajL affinity column, separated by nonreducing-reducing SDS-PAGE, and identified by mass spectrometry. Covalent YajL substrates included ribosomal proteins, aminoacyl-tRNA synthetases, chaperones, catalases, peroxidases, and other proteins containing cysteines essential for catalysis or FeS cluster binding, such as glyceraldehyde-3-phosphate dehydrogenase, aldehyde dehydrogenase, aconitase, and FeS cluster-containing subunits of respiratory chains. In addition, we show that DJ-1 also forms mixed disulfides with cytoplasmic proteins upon oxidative stress. These results shed light on the oxidative stress-dependent chaperone function of YajL and identify YajL substrates involved in translation, stress protection, protein solubilization, and metabolism. They reveal a crucial role for cysteine 106 and suggest that DJ-1 also functions as a covalent chaperone. These findings are consistent with several defects observed in yajL or DJ-1 mutants, including translational defects, protein aggregation, oxidative stress sensitivity, and metabolic deficiencies.

Structural and mechanistic insight into covalent substrate binding by Escherichia coli dihydroxyacetone kinase

The Escherichia coli dihydroxyacetone (Dha) kinase is an unusual kinase because (i) it uses the phosphoenolpyruvate carbohydrate: phosphotransferase system (PTS) as the source of high-energy phosphate, (ii) the active site is formed by two subunits, and (iii) the substrate is covalently bound to His218(K)* of the DhaK subunit. The PTS transfers phosphate to DhaM, which in turn phosphorylates the permanently bound ADP coenzyme of DhaL. This phosphoryl group is subsequently transferred to the Dha substrate bound to DhaK. Here we report the crystal structure of the E. coli Dha kinase complex, DhaK-DhaL. The structure of the complex reveals that DhaK undergoes significant conformational changes to accommodate binding of DhaL. Combined mutagenesis and enzymatic activity studies of kinase mutants allow us to propose a catalytic mechanism for covalent Dha binding, phosphorylation, and release of the Dha-phosphate product. Our results show that His56(K) is involved in formation of the covalent hemiaminal bond with Dha. The structure of H56N(K) with noncovalently bound substrate reveals a somewhat different positioning of Dha in the binding pocket as compared to covalently bound Dha, showing that the covalent attachment to His218(K) orients the substrate optimally for phosphoryl transfer. Asp109(K) is critical for activity, likely acting as a general base activating the γ-OH of Dha. Our results provide a comprehensive picture of the roles of the highly conserved active site residues of dihydroxyacetone kinases.

Asymmetric Dynamics of the Acyl-Carrier Protein Inside the Fungal Fatty-Acid Mega-Synthase

Fatty-acid mega-synthases (FAS) are large multifunctional protein complexes responsible for the synthesis of fatty acids. In fungi, FAS features two distinct reaction chambers with three-fold symmetry, each of which includes all enzymes necessary to catalyze the iterative elongation of fatty acids, together with three acyl-carrier proteins (ACP), used for covalent substrate shuttling. Flexible linkers double-tether the ACPs to FAS scaffold to facilitate the delivery of chemical intermediates. However, the actual shuttling mechanism is still unknown; several contribution factors have been proposed, e.g. linker length and elasticity, electrostatics complementarity between the ACPs and the catalytic centers, etc.To assess these and other factors, we have analyzed the dynamics of ACP within the FAS reaction chamber, using multi-scale molecular dynamics (MD) simulations. We have adopted a novel model, which comprises a coarse-grained (CG), semi-rigid-body representation of the ACPs; a CG, flexible representation of the ACP-chamber linkers; a grid-based representation of the FAS chamber; and an implicit description of solvent.It was found that ACP dynamics is not hindered by the linker length nor its flexibility. Indeed, each ACP domain is able to visit all catalytic sites. Nonetheless, the probability of ACP encounters with the catalytic sites was found to differ between adjacent sub-chambers. It was found that this asymmetry arises from the steric hindrance imposed on the ACPs by the linkers. In conclusion, the dynamics of ACP within FAS appears to be essentially stochastic, and not limited by the native linker length; instead, it is modulated by volume-exclusion effects due to ‘molecular crowding’and by electrostatic steering towards the chamber walls. It follows that residence times at each catalytic site will be primarily determined by their individual binding affinities for the ACP domain and its substrate.

X-ray Structures of the Three Lactococcus lactis Dihydroxyacetone Kinase Subunits and of a Transient Intersubunit Complex

Bacterial dihydroxyacetone (Dha) kinases do not exchange the ADP for ATP but utilize a subunit of the phosphoenolpyruvate carbohydrate phosphotransferase system for in situ rephosphorylation of a permanently bound ADP-cofactor. Here we report the 2.1-angstroms crystal structure of the transient complex between the phosphotransferase subunit DhaM of the phosphotransferase system and the nucleotide binding subunit DhaL of the Dha kinase of Lactococcus lactis, the 1.1-angstroms structure of the free DhaM dimer, and the 2.5-angstroms structure of the Dha-binding DhaK subunit. Conserved salt bridges and an edge-to-plane stacking contact between two tyrosines serve to orient DhaL relative to the DhaM dimer. The distance between the imidazole Nepsilon2 of the DhaM His-10 and the beta-phosphate oxygen of ADP, between which the gamma-phosphate is transferred, is 4.9 angstroms. An invariant arginine, which is essential for activity, is appropriately positioned to stabilize the gamma-phosphate in the transition state. The (betaalpha)4alpha fold of DhaM occurs a second time as a subfold in the DhaK subunit. By docking DhaL-ADP to this subfold, the nucleotide bound to DhaL and the C1-hydroxyl of Dha bound to DhaK are positioned for in-line transfer of phosphate.