Substrate recognition by the 4-hydroxytryptamine kinase PsiK in psilocybin biosynthesis.

Psilocybin, the natural hallucinogen from Psilocybe (magic) mushrooms, is a highly promising drug candidate for the treatment of depression and several other mental health conditions. Biosynthesis of psilocybin from the amino acid l-tryptophan involves four strictly sequential modifications. The third of these, ATP-dependent phosphorylation of the intermediate 4-hydroxytryptamine, is catalysed by PsiK. Here we present a crystallographic analysis and a structure-based mutagenesis study of this kinase, providing insight into its mode of substrate recognition. The results of our work will support future bioengineering efforts aimed at generating variants of psilocybin with enhanced therapeutic properties.

Sequence diversity in the monooxygenases involved in oxime production in plant defense and signaling: a conservative revision in the nomenclature of the highly complex CYP79 family.

Cytochrome P450 monooxygenases of the CYP79 family catalyze conversion of specific amino acids into oximes feeding into a variety of metabolic plant pathways. Here we present an extensive phylogenetic tree of the CYP79 family built on carefully curated sequences collected across the entire plant kingdom. Based on a monophyletic origin of the P450s, a set of evolutionarily distinct branches was identified. Founded on the functionally characterized CYP79 sequences, sequence features of the individual substrate recognition sites (SRSs) were analyzed. Co-evolving amino acid residues were identified using co-evolutionary sequence analysis. SRS4 possesses a specific sequence pattern when tyrosine is a substrate. Except for the CYP79Cs and CYP79Fs, substrate preferences toward specific amino acids could not be assigned to specific subfamilies. The highly diversified CYP79 tree, reflecting recurrent independent evolution of CYP79s, may relate to the different roles of oximes in different plant species. The sequence differences across individual CYP79 subfamilies may facilitate the in vivo orchestration of channeled metabolic pathways based on altered surface charge domains of the CYP79 protein. Alternatively, they may serve to optimize dynamic interactions with oxime metabolizing enzymes to enable optimal ecological interactions. The outlined detailed curation of the CYP79 sequences used for building the phylogenetic tree made it appropriate to make a conservative phylogenetic tree-based revision of the naming of the sequences within this highly complex cytochrome P450 family. The same approach may be used in other complex P450 subfamilies. The detailed phylogeny of the CYP79 family will enable further exploration of the evolution of function in these enzymes.

Elucidating the Substrate Envelope of Enterovirus 68-3C Protease: Structural Basis of Specificity and Potential Resistance.

Enterovirus-D68 (EV68) has emerged as a global health concern over the last decade with severe symptomatic infections resulting in long-lasting neurological deficits and death. Unfortunately, there are currently no FDA-approved antiviral drugs for EV68 or any other non-polio enterovirus. One particularly attractive class of potential drugs are small molecules inhibitors, which can target the conserved active site of EV68-3C protease. For other viral proteases, we have demonstrated that the emergence of drug resistance can be minimized by designing inhibitors that leverage the evolutionary constraints of substrate specificity. However, the structural characterization of EV68-3C protease bound to its substrates has been lacking. Here, we have determined the substrate specificity of EV68-3C protease through molecular modeling, molecular dynamics (MD) simulations, and co-crystal structures. Molecular models enabled us to successfully characterize the conserved hydrogen-bond networks between EV68-3C protease and the peptides corresponding to the viral cleavage sites. In addition, co-crystal structures we determined have revealed substrate-induced conformational changes of the protease which involved new interactions, primarily surrounding the S1 pocket. We calculated the substrate envelope, the three-dimensional consensus volume occupied by the substrates within the active site. With the elucidation of the EV68-3C protease substrate envelope, we evaluated how 3C protease inhibitors, AG7088 and SG-85, fit within the active site to predict potential resistance mutations.

Molecular insights into the hydrolysis and transglycosylation of a deep-sea Planctomycetota-derived GH16 family laminarinase.

The biochemical and structural characteristics of PtLam, a laminarinase from deep-sea Planctomycetota, have been extensively elucidated, unveiling the fundamental molecular mechanisms governing substrate recognition and enzymatic catalysis. PtLam functions as an exo-laminarinase with the ability to sequentially hydrolyze laminarin, cleaving glucose units individually. Notably, PtLam exhibits proficient transglycosylation capabilities, utilizing various sugar alcohols as acceptors, with lyxose, in particular, yielding exclusively transglycosylated products. Structural analysis of both apo-PtLam and its laminarin oligosaccharide-bound complex revealed significant conformational alterations in active residues upon substrate binding. Moreover, pivotal residues involved in substrate recognition were identified, with subsequent mutation assays indicating the contribution of positive subsites in modulating exo-hydrolysis and transglycosidic activities. These results enhance our comprehension of laminarin cycling mechanisms by marine Planctomycetota, while also providing essential enzyme components for laminarin hetero-oligosaccharide synthesis.IMPORTANCEThe ubiquitous Planctomycetota, with distinctive physiological traits, exert a significant influence on global carbon and nitrogen fluxes. Their intimate association with algae suggests a propensity for efficient polysaccharide degradation; however, research on glycoside hydrolases derived from Planctomycetota remains scarce. Herein, we unveil the GH16 family laminarinase PtLam from deep-sea Planctomycetota, shedding light on its catalytic mechanisms underlying hydrolysis and transglycosylation. Our findings elucidate the enzymatic pathways governing the marine laminarin cycle orchestrated by Planctomycetota, thereby fostering the exploration of novel polysaccharide hydrolases with promising practical implications.

Production of hybrid macrolide antibiotics by exploiting the specific substrate recognition characteristics of multifunctional cytochrome P450 enzyme MycG.

Macrolide antibiotics are biosynthesized via enzymatic modifications, including glycosylation, methylation, and oxidation, after the core macro-lactone ring is generated by a polyketide synthase system. This study explored the diversification of macrolides by combining biosynthetic enzymes and reports an approach to produce unnatural hybrid macrolide antibiotics. The cytochrome (CYP) P450 monooxygenase MycG exhibits bifunctional activity, catalyzing late-stage hydroxylation at C-14 followed by epoxidation at C-12/13 during mycinamicin biosynthesis. The mycinose sugar of mycinamicin serves as a key molecular recognition element for binding to MycG. Thus, we subjected the hybrid macrolide antibiotic 23-O-mycinosyl-20-deoxo-20-dihydro-12,13-deepoxyrosamicin (IZI) to MycG, and confirmed that MycG catalyzed hydroxylation at C-22 and epoxidation at C-12/13 in IZI. In addition, the introduction of mycinose biosynthesis-related genes and mycG into rosamicin-producing Micromonospora rosaria enabled the fermentative production of 22-hydroxylated and 12,13-epoxidized forms of IZI. Interestingly, MycG catalyzed the sequential oxidation of hydroxylation and epoxidation in mycinamicin biosynthesis, but only single reactions in IZI. These findings highlight the potential for expanding the application of the multifunctional P450 monooxygenase MycG for the production of unnatural compounds.

General ADP-Ribosylation Mechanism Based on the Structure of ADP-Ribosyltransferase-Substrate Complexes.

ADP-ribosylation is a ubiquitous modification of proteins and other targets, such as nucleic acids, that regulates various cellular functions in all kingdoms of life. Furthermore, these ADP-ribosyltransferases (ARTs) modify a variety of substrates and atoms. It has been almost 60 years since ADP-ribosylation was discovered. Various ART structures have been revealed with cofactors (NAD+ or NAD+ analog). However, we still do not know the molecular mechanisms of ART. It needs to be better understood how ART specifies the target amino acids or bases. For this purpose, more information is needed about the tripartite complex structures of ART, the cofactors, and the substrates. The tripartite complex is essential to understand the mechanism of ADP-ribosyltransferase. This review updates the general ADP-ribosylation mechanism based on ART tripartite complex structures.

Type 1 secretion necessitates a tight interplay between all domains of the ABC transporter.

Type I secretion systems (T1SS) facilitate the secretion of substrates in one step across both membranes of Gram-negative bacteria. A prime example is the hemolysin T1SS which secretes the toxin HlyA. Secretion is energized by the ABC transporter HlyB, which forms a complex together with the membrane fusion protein HlyD and the outer membrane protein TolC. HlyB features three domains: an N-terminal C39 peptidase-like domain (CLD), a transmembrane domain (TMD) and a C-terminal nucleotide binding domain (NBD). Here, we created chimeric transporters by swapping one or more domains of HlyB with the respective domain(s) of RtxB, a HlyB homolog from Kingella kingae. We tested all chimeric transporters for their ability to secrete pro-HlyA when co-expressed with HlyD. The CLD proved to be most critical, as a substitution abolished secretion. Swapping only the TMD or NBD reduced the secretion efficiency, while a simultaneous exchange abolished secretion. These results indicate that the CLD is the most critical secretion determinant, while TMD and NBD might possess additional recognition or interaction sites. This mode of recognition represents a hierarchical and extreme unusual case of substrate recognition for ABC transporters and optimal secretion requires a tight interplay between all domains.

Sugar binding of sodium-glucose cotransporters analyzed by voltage-clamp fluorometry.

Sugar absorption is crucial for life and relies on glucose transporters, including sodium-glucose cotransporters (SGLTs). Although the structure of SGLTs has been resolved, the substrate selectivity of SGLTs across diverse isoforms has not been determined owing to the complex substrate-recognition processes and limited analysis methods. Therefore, this study used voltage-clamp fluorometry (VCF) to explore the substrate-binding affinities of human SGLT1 in Xenopus oocytes. VCF analysis revealed high-affinity binding of D-glucose and D-galactose, which are known transported substrates. D-fructose, which is not a transported substrate, also bound to SGLT1, suggesting potential recognition despite the lack of transport activity. VCF analysis using the T287N mutant of the substrate-binding pocket, which has reduced D-glucose transport capacity, showed that its D-galactose-binding affinity exceeded its D-glucose-binding affinity. This suggests that the change in the VCF signal was due to substrate binding to the binding pocket. Both D-fructose and L-sorbose showed similar binding affinities, indicating that SGLT1 preferentially binds to pyranose-form sugars, including D-fructopyranose. Electrophysiological analysis confirmed that D-fructose binding did not affect the SGLT1 transport function. The significance of the VCF assay lies in its ability to measure sugar-protein interactions in living cells, thereby bridging the gap between structural analyses and functional characterizations of sugar transporters. Our findings also provide insights into SGLT substrate selectivity and the potential for developing medicines with reduced side effects by targeting non-glucose sugars with low bioreactivity.

Engineering non-conservative substrate recognition sites of extradiol dioxygenase: Computation guided design to diversify and accelerate degradation of aromatic compounds.

Extradiol dioxygenases (EDOs) catalyzing meta-cleavage of catecholic compounds promise an effective way to detoxify aromatic pollutants. This work reported a novel scenario to engineer our recently identified Type I EDO from Tcu3516 for a broader substrate scope and enhanced activity, which was based on 2,3-dihydroxybiphenyl (2,3-DHB)-liganded molecular docking of Tcu3516 and multiple sequence alignment with other 22 Type I EDOs. 11 non-conservative residues of Tcu3516 within 6 Å distance to the 2,3-DHB ligand center were selected as potential hotspots and subjected to semi-rational design using 6 catecholic analogues as substrates; the mutants V186L and V212N returned with progressive evolution in substrate scope and catalytic activity. Both mutants were combined with D285A for construction of double mutants and final triple mutant V186L/V212N/D285A. Except for 2,3-DHB (the mutant V186L/D285A gave the best catalytic performance), the triple mutant prevailed all other 5 catecholic compounds for their degradation; affording the catalytic efficiency kcat/Km value increase by 10-30 folds, protein Tm (structural rigidity) increase by 15 °C and the half-life time enhancement by 10 times compared to the wild type Tcu3516. The molecular dynamic simulation suggested that a stabler core and a more flexible entrance are likely accounting for enhanced catalytic activity and stability of enzymes.

Transport mechanism and pharmacology of the human GlyT1.

The glycine transporter 1 (GlyT1) plays a crucial role in the regulation of both inhibitory and excitatory neurotransmission by removing glycine from the synaptic cleft. Given its close association with glutamate/glycine co-activated NMDA receptors (NMDARs), GlyT1 has emerged as a central target for the treatment of schizophrenia, which is often linked to hypofunctional NMDARs. Here, we report the cryo-EM structures of GlyT1 bound with substrate glycine and drugs ALX-5407, SSR504734, and PF-03463275. These structures, captured at three fundamental states of the transport cycle-outward-facing, occluded, and inward-facing-enable us to illustrate a comprehensive blueprint of the conformational change associated with glycine reuptake. Additionally, we identified three specific pockets accommodating drugs, providing clear insights into the structural basis of their inhibitory mechanism and selectivity. Collectively, these structures offer significant insights into the transport mechanism and recognition of substrate and anti-schizophrenia drugs, thus providing a platform to design small molecules to treat schizophrenia.

Functional and structural characterisation of RimL from Bacillus cereus, a new Nα-acetyltransferase of ribosomal proteins that was wrongly assigned as an aminoglycosyltransferase.

Enzymes of the GNAT (GCN5-relate N-acetyltransferases) superfamily are important regulators of cell growth and development. They are functionally diverse and share low amino acid sequence identity, making functional annotation difficult. In this study, we report the function and structure of a new ribosomal enzyme, Nα-acetyl transferase from Bacillus cereus (RimLBC), a protein that was previously wrongly annotated as an aminoglycosyltransferase. Firstly, extensive comparative amino acid sequence analyses suggested RimLBC belongs to a cluster of proteins mediating acetylation of the ribosomal protein L7/L12. To assess if this was the case, several well established substrates of aminoglycosyltransferases were screened. The results of these studies did not support an aminoglycoside acetylating function for RimLBC. To gain further insight into RimLBC biological role, a series of studies that included MALDI-TOF, isothermal titration calorimetry, NMR, X-ray protein crystallography, and site-directed mutagenesis confirmed RimLBC affinity for Acetyl-CoA and that the ribosomal protein L7/L12 is a substrate of RimLBC. Last, we advance a mechanistic model of RimLBC mode of recognition of its protein substrates. Taken together, our studies confirmed RimLBC as a new ribosomal Nα-acetyltransferase and provide structural and functional insights into substrate recognition by Nα-acetyltransferases and protein acetylation in bacteria.

Intersubunit communication in glycogen phosphorylase influences substrate recognition at the catalytic sites.

Glycogen phosphorylase (GP) is biologically active as a dimer of identical subunits, each activated by phosphorylation of the serine-14 residue. GP exists in three interconvertible forms, namely GPa (di-phosphorylated form), GPab (mono-phosphorylated form), and GPb (non-phosphorylated form); however, information on GPab remains scarce. Given the prevailing view that the two GP subunits collaboratively determine their catalytic characteristics, it is essential to conduct GPab characterization to gain a comprehensive understanding of glycogenolysis regulation. Thus, in the present study, we prepared rabbit muscle GPab from GPb, using phosphorylase kinase as the catalyst, and identified it using a nonradioactive phosphate-affinity gel electrophoresis method. Compared with the half-half GPa/GPb mixture, the as-prepared GPab showed a unique AMP-binding affinity. To further investigate the intersubunit communication in GP, its catalytic site was probed using pyridylaminated-maltohexaose (a maltooligosaccharide-based substrate comprising the essential dextrin structure for GP; abbreviated as PA-0) and a series of specifically modified PA-0 derivatives (substrate analogs lacking part of the essential dextrin structure). By comparing the initial reaction rates toward the PA-0 derivative (Vderivative) and PA-0 (VPA-0), we demonstrated that the Vderivative/VPA-0 ratio for GPab was significantly different from that for the half-half GPa/GPb mixture. This result indicates that the interaction between the two GP subunits significantly influences substrate recognition at the catalytic sites, thereby providing GPab its unique substrate recognition profile.

Research Progress on hCNT3 Structure/Function and Nucleoside Anticancer Drugs.

Membrane protein human concentrative nucleoside transporter 3 (hCNT3) can not only transport extracellular nucleosides into the cell but also transport various nucleoside-derived anticancer drugs to the focus of infection for therapeutic effects. Typical nucleoside anticancer drugs, including fludarabine, cladabine, decitabine, and clofarabine, are recognized by hCNT3 and then delivered to the lesion site for their therapeutic effects. hCNT3 is highly conserved during the evolution from lower to higher vertebrates, which contains scaffold and transport domains in structure and delivers substrates by coupling with Na+ and H+ ions in function. In the process of substrate delivery, the transport domain rises from the lower side of transmembrane 9 (TM9) in the inward conformation to the upper side of the outward conformation, accompanied by the collaborative motion of TM7b/ TM4b and hairpin 1b (HP1b)/ HP2b. With the report of a series of three-dimensional structures of homologous CNTs, the structural characteristics and biological functions of hCNT3 have attracted increasing attention from pharmacists and biologists. Our research group has also recently designed an anticancer lead compound with high hCNT3 transport potential based on the structure of 5-fluorouracil. In this work, the sequence evolution, conservation, molecular structure, cationic chelation, substrate recognition, elevator motion pattern and nucleoside derivative drugs of hCNT3 were reviewed, and the differences in hCNT3 transport mode and nucleoside anticancer drug modification were summarized, aiming to provide theoretical guidance for the subsequent molecular design of novel anticancer drugs targeting hCNT3.

Phosphorylation Dynamics in a flg22-Induced, G Protein-Dependent Network Reveals the AtRGS1 Phosphatase.

The microbe-associated molecular pattern flg22 is recognized in a flagellin-sensitive 2-dependent manner in root tip cells. Here, we show a rapid and massive change in protein abundance and phosphorylation state of the Arabidopsis root cell proteome in WT and a mutant deficient in heterotrimeric G-protein-coupled signaling. flg22-induced changes fall on proteins comprising a subset of this proteome, the heterotrimeric G protein interactome, and on highly-populated hubs of the immunity network. Approximately 95% of the phosphorylation changes in the heterotrimeric G-protein interactome depend, at least partially, on a functional G protein complex. One member of this interactome is ATBα, a substrate-recognition subunit of a protein phosphatase 2A complex and an interactor to Arabidopsis thaliana Regulator of G Signaling 1 protein (AtRGS1), a flg22-phosphorylated, 7-transmembrane spanning modulator of the nucleotide-binding state of the core G-protein complex. A null mutation of ATBα strongly increases basal endocytosis of AtRGS1. AtRGS1 steady-state protein level is lower in the atbα mutant in a proteasome-dependent manner. We propose that phosphorylation-dependent endocytosis of AtRGS1 is part of the mechanism to degrade AtRGS1, thus sustaining activation of the heterotrimeric G protein complex required for the regulation of system dynamics in innate immunity. The PP2A(ATBα) complex is a critical regulator of this signaling pathway.

Structural mechanism of Escherichia coli cyanase.

Cyanase plays a vital role in the detoxification of cyanate and supplies a continuous nitrogen source for soil microbes by converting cyanate to ammonia and carbon dioxide in a bicarbonate-dependent reaction. The structures of cyanase complexed with dianion inhibitors, in conjunction with biochemical studies, suggest putative binding sites for substrates. However, the substrate-recognition and reaction mechanisms of cyanase remain unclear. Here, crystal structures of cyanase from Escherichia coli were determined in the native form and in complexes with cyanate, bicarbonate and intermediates at 1.5-1.9 Šresolution using synchrotron X-rays and an X-ray free-electron laser. Cyanate and bicarbonate interact with the highly conserved Arg96, Ser122 and Ala123 in the active site. In the presence of a mixture of cyanate and bicarbonate, three different electron densities for intermediates were observed in the cyanase structures. Moreover, the observed electron density could explain the dynamics of the substrate or product. In addition to conformational changes in the substrate-binding pocket, dynamic movement of Leu151 was observed, which functions as a gate for the passage of substrates or products. These findings provide a structural mechanism for the substrate-binding and reaction process of cyanase.

tRNA m1G9 modification depends on substrate-specific RNA conformational changes induced by the methyltransferase Trm10.

The methyltransferase Trm10 modifies a subset of tRNAs on the base N1 position of the ninth nucleotide in the tRNA core. Trm10 is conserved throughout Eukarya and Archaea, and mutations in the human gene (TRMT10A) have been linked to neurological disorders such as microcephaly and intellectual disability, as well as defects in glucose metabolism. Of the 26 tRNAs in yeast with guanosine at position 9, only 13 are substrates for Trm10. However, no common sequence or other posttranscriptional modifications have been identified among these substrates, suggesting the presence of some other tRNA feature(s) that allow Trm10 to distinguish substrate from nonsubstrate tRNAs. Here, we show that substrate recognition by Saccharomyces cerevisiae Trm10 is dependent on both intrinsic tRNA flexibility and the ability of the enzyme to induce specific tRNA conformational changes upon binding. Using the sensitive RNA structure-probing method SHAPE, conformational changes upon binding to Trm10 in tRNA substrates, but not nonsubstrates, were identified and mapped onto a model of Trm10-bound tRNA. These changes may play an important role in substrate recognition by allowing Trm10 to gain access to the target nucleotide. Our results highlight a novel mechanism of substrate recognition by a conserved tRNA modifying enzyme. Further, these studies reveal a strategy for substrate recognition that may be broadly employed by tRNA-modifying enzymes which must distinguish between structurally similar tRNA species.

Interaction of Substrates with γ-Secretase at the Level of Individual Transmembrane Helices-A Methodological Approach.

Intramembrane proteases, such as γ secretase, typically recruit multiple substrates from an excess of single-span membrane proteins. It is currently unclear to which extent substrate recognition depends on specific interactions of their transmembrane domains (TMDs) with TMDs of a protease. Here, we investigated a large number of potential pairwise interactions between TMDs of γ secretase and a diverse set of its substrates using two different configurations of BLaTM, a genetic reporter system. Our results reveal significant interactions between TMD2 of presenilin, the enzymatic subunit of γ secretase, and the TMD of the amyloid precursor protein, as well as of several other substrates. Presenilin TMD2 is a prime candidate for substrate recruitment, as has been shown from previous studies. In addition, the amyloid precursor protein TMD enters interactions with presenilin TMD 4 as well as with the TMD of nicastrin. Interestingly, the Gly-rich interfaces between the amyloid precursor protein TMD and presenilin TMDs 2 and 4 are highly similar to its homodimerization interface. In terms of methodology, the economics of the newly developed library-based method could prove to be a useful feature in related future work for identifying heterotypic TMD-TMD interactions within other biological contexts.

Introduction of pseudo-base benzimidazole derivatives into nucleosides via base exchange by a nucleoside metabolic enzyme.

In alternate organic synthesis, biocatalysis using enzymes provides a more stereoselective and cost-effective approach. Synthesis of unnatural nucleosides by nucleoside base exchange reactions using nucleoside-metabolizing enzymes has previously shown that the 5-position recognition of pyrimidine bases on nucleoside substrates is loose and can be used to introduce functional molecules into pyrimidine nucleosides. Here we explored the incorporation of purine pseudo bases into nucleosides by the base exchange reaction of pyrimidine nucleoside phosphorylase (PyNP), demonstrating that an imidazole five-membered ring is an essential structure for the reaction. In the case of benzimidazole, the base exchange proceeded to give the deoxyribose form in 96 % yield, and the ribose form in 23 % yield. The reaction also proceeded with 1H-imidazo[4,5-b]phenazine, a benzimidazole analogue with an additional ring, although the yield of nucleoside was only 31 %. Docking simulations between 1H and imidazo[4,5-b]phenazine nucleoside and the active site of PyNP (PDB 1BRW) supported our observation that 1H-imidazo[4,5-b]phenazine can be used as a substrate by PyNP. Thus, the enzymatic substitution reaction using PyNP can be used to incorporate many purine pseudo bases and benzimidazole derivatives with various functional groups into nucleoside structures, which have potential utility as diagnostic or therapeutic agents.

A biosynthetic aspartate N-hydroxylase performs successive oxidations by holding intermediates at a site away from the catalytic center.

Nitrosuccinate is a biosynthetic building block in many microbial pathways. The metabolite is produced by dedicated L-aspartate hydroxylases that use NADPH and molecular oxygen as co-substrates. Here, we investigate the mechanism underlying the unusual ability of these enzymes to perform successive rounds of oxidative modifications. The crystal structure of Streptomyces sp. V2 L-aspartate N-hydroxylase outlines a characteristic helical domain wedged between two dinucleotide-binding domains. Together with NADPH and FAD, a cluster of conserved arginine residues forms the catalytic core at the domain interface. Aspartate is found to bind in an entry chamber that is close to but not in direct contact with the flavin. It is recognized by an extensive H-bond network that explains the enzyme's strict substrate-selectivity. A mutant designed to create steric and electrostatic hindrance to substrate binding disables hydroxylation without perturbing the NADPH oxidase side-activity. Critically, the distance between the FAD and the substrate is far too long to afford N-hydroxylation by the C4a-hydroperoxyflavin intermediate whose formation is confirmed by our work. We conclude that the enzyme functions through a catch-and-release mechanism. L-aspartate slides into the catalytic center only when the hydroxylating apparatus is formed. It is then re-captured by the entry chamber where it waits for the next round of hydroxylation. By iterating these steps, the enzyme minimizes the leakage of incompletely oxygenated products and ensures that the reaction carries on until nitrosuccinate is formed. This unstable product can then be engaged by a successive biosynthetic enzyme or undergoes spontaneous decarboxylation to produce 3-nitropropionate, a mycotoxin.

Structure-Based Virtual Screening and Discovery of New Bi-functional DAPK1 Inhibitors.

Recently, a new signaling complex Death-Associated Protein Kinase 1 (DAPK1)-N-methyl D-aspartate receptor subtype 2B (NR2B) engaged in the neuronal death cascade was identified where it was found that after stroke injury, N-methyl-D-aspartate glutamate (NMDA) receptors interact with DAPK1 through NR2B subunit and lead to excitotoxicity via overactivation of NMDA receptors. In this study, we used ZINC-12 database to find out potential inhibitor of DAPK1 and found some natural compounds showing good binding affinity towards DAPK1. These natural compounds showed interactions with ATP-binding site residues as well as substrate-recognition motifs. Thus, it has been concluded that the ligands those are showing interactions with both the sites could be considered as potential inhibitors for DAPK1.