Initial Steps in Methanobactin Biosynthesis: Substrate Binding by the Mixed-Valent Diiron Enzyme MbnBC.

The MbnBC enzyme complex converts cysteine residues in a peptide substrate, MbnA, to oxazolone/thioamide groups during the biosynthesis of copper chelator methanobactin (Mbn). MbnBC belongs to the mixed-valent diiron oxygenase (MVDO) family, of which members use an Fe(II)Fe(III) cofactor to react with dioxygen for substrate modification. Several crystal structures of the inactive Fe(III)Fe(III) form of MbnBC alone and in complex with MbnA have been reported, but a mechanistic understanding requires determination of the oxidation states of the crystallographically observed Fe ions in the catalytically active Fe(II)Fe(III) state, along with the site of MbnA binding. Here, we have used electron nuclear double resonance (ENDOR) spectroscopy to determine such structural and electronic properties of the active site, in particular, the mode of substrate binding to the MV state, information not accessible by X-ray crystallography alone. The oxidation states of the two Fe ions were determined by 15N ENDOR analysis. The presence and locations of both bridging and terminal exogenous solvent ligands were determined using 1H and 2H ENDOR. In addition, 2H ENDOR using an isotopically labeled MbnA substrate indicates that MbnA binds to the Fe(III) ion of the cluster via the sulfur atom of its N-terminal modifiable cysteine residue, with displacement of a coordinated solvent ligand as shown by complementary 1H ENDOR. These results, which underscore the utility of ENDOR in studying MVDOs, provide a molecular picture of the initial steps in Mbn biosynthesis.

PAS domain of flagellar histidine kinase FlrB has a unique architecture and binds heme as a sensory ligand in an unconventional fashion.

Phosphorylation of the σ54-dependent transcription activator FlrC by the sensor histidine kinase FlrB is essential for flagellar synthesis of Vibrio cholerae. Despite that, the structure, sensory signal, and mechanistic basis of function of FlrB were elusive. Here, we report the crystal structure of the sensory PAS domain of FlrB in its functional dimeric state that exhibits a unique architecture. Series of biochemical/biophysical experiments on different constructs and mutants established that heme binds hydrophobically as sensory ligand in the shallow ligand-binding cleft of FlrB-PAS without axial coordination. Intriguingly, ATP binding to the C-terminal ATP-binding (CA) domain assists PAS domain to bind heme, vis-à-vis, heme binding to the PAS facilitates ATP binding to the CA domain. We hypothesize that synergistic binding of heme and ATP triggers conformational signaling in FlrB, leading to downstream flagellar gene transcription. Enhanced swimming motility of V. cholerae with increased heme uptake supports this proposition.

An Adenosylcobalamin Specific Whole-Cell Biosensor.

Vitamin B12 (cobalamin) is essential for human health and its deficiency results in anemia and neurological damage. Vitamin B12 exists in different forms with various bioactivity but most sensors are unable to discriminate between them. Here, a whole-cell agglutination assay that is specific for adenosylcobalamin (AboB12), which is one of two bioactive forms, is reported. This biosensor consists of Escherichia coli that express the AdoB12 specific binding domain of CarH at their surface. In the presence of AdoB12, CarH forms tetramers, which leads to specific bacterial cell-cell adhesions and agglutination. These CarH tetramers disassemble upon green light illumination such that reversion of the bacterial aggregation can serve as internal quality control. The agglutination assay has a detection limit of 500 nм AdoB12, works in protein-poor biofluids such as urine, and has high specificity to AdoB12 over other forms of vitamin B12 as also demonstrated with commercially available supplements. This work is a proof of concept for a cheap and easy-to-readout AdoB12 sensor that can be implemented at the point-of-care to monitor high-dose vitamin B12 supplementation.

The role of CopA in Streptococcus pyogenes copper homeostasis and virulence.

Maintenance of intracellular metal homeostasis during interaction with host niches is critical to the success of bacterial pathogens. To prevent infection, the mammalian innate immune response employs metal-withholding and metal-intoxication mechanisms to limit bacterial propagation. The first-row transition metal ion copper serves critical roles at the host-pathogen interface and has been associated with antimicrobial activity since antiquity. Despite lacking any known copper-utilizing proteins, streptococci have been reported to accumulate significant levels of copper. Here, we report that loss of CopA, a copper-specific exporter, confers increased sensitivity to copper in Streptococcus pyogenes strain HSC5, with prolonged exposure to physiological levels of copper resulting in reduced viability during stationary phase cultivation. This defect in stationary phase survival was rescued by supplementation with exogeneous amino acids, indicating the pathogen had altered nutritional requirements during exposure to copper stress. Furthermore, S. pyogenes HSC5 ΔcopA was substantially attenuated during murine soft-tissue infection, demonstrating the importance of copper efflux at the host-pathogen interface. Collectively, these data indicate that copper can severely reduce the viability of stationary phase S. pyogenes and that active efflux mechanisms are required to survive copper stress in vitro and during infection.

DosR Regulates the Transcription of the Arginine Biosynthesis Gene Cluster by Binding to the Regulatory Sequences in Mycobacterium bovis Bacille Calmette-Guerin.

l-Arginine serves as a carbon and nitrogen source and is critical for Mycobacterium tuberculosis (Mtb) survival in the host. Generally, ArgR acts as a repressor regulating arginine biosynthesis by binding to the promoter of the argCJBDFGH gene cluster. In this study, we report that the dormancy regulator DosR is a novel arginine regulator binding to the promoter region of argC (rv1652), which regulates arginine synthesis. Phosphorylation modification promoted DosR binding to a region upstream of the promoter. Cofactors, including arginine and metal ions, had an inhibitory effect on this association. Furthermore, DosR regulatory function relies on the interaction of the 167, 181, 182, and 197 amino acid residues with an inverse complementary sequence. Arginine also binds to DosR and directly affects its DNA-binding ability. Together, the results demonstrate that DosR acts as a novel transcriptional regulator of arginine synthesis in Mycobacterium bovis bacille Calmette-Guerin.

Vitamin K3 inhibits FtsZ assembly, disrupts the Z-ring in Streptococcus pneumoniae and displays anti-pneumococcal activity.

The respiratory pathogen, Streptococcus pneumoniae has acquired multiple-drug resistance over the years. An attractive strategy to combat pneumococcal infection is to target cell division to inhibit the proliferation of S. pneumoniae. This work presents Vitamin K3 as a potential anti-pneumococcal drug that targets FtsZ, the master coordinator of bacterial cell division. Vitamin K3 strongly inhibited S. pneumoniae proliferation with a minimum inhibitory concentration (MIC) and a minimum bactericidal concentration (MBC) of 6 µg/ml. Vitamin K3 disrupted the Z-ring localization in both S. pneumoniae and Bacillus subtilis within 30 min of treatment, while the membrane integrity and nucleoid segregation remain unchanged. Several complementary experiments showed that Vitamin K3 inhibits the assembly of purified S. pneumoniae FtsZ (SpnFtsZ) and induces conformational changes in the protein. Interestingly, Vitamin K3 interfered with GTP binding onto FtsZ and increased the GTPase activity of FtsZ polymers. The intrinsic tryptophan fluorescence of SpnFtsZ revealed that Vitamin K3 delays the nucleation of FtsZ polymers and reduces the rate of polymerization. In the presence of a non-hydrolyzable analog of GTP, Vitamin K3 did not show inhibition of FtsZ polymerization. These results indicated that Vitamin K3 induces conformational changes in FtsZ that increase GTP hydrolysis and thereby, destabilize the FtsZ polymers. Together, our data provide evidence that Vitamin K3 derives its potent anti-pneumococcal activity by inhibiting FtsZ assembly.

A Novel Alginate Lyase: Identification, Characterization, and Potential Application in Alginate Trisaccharide Preparation.

Alginate oligosaccharides (AOS) have many biological activities and significant applications in prebiotics, nutritional supplements, and plant growth development. Alginate lyases have unique advantages in the preparation of AOS. However, only a limited number of alginate lyases have been so far reported to have potentials in the preparation of AOS with specific degrees of polymerization. Here, an alginate-degrading strain Pseudoalteromonasarctica M9 was isolated from Sargassum, and five alginate lyases were predicted in its genome. These putative alginate lyases were expressed and their degradation products towards sodium alginate were analyzed. Among them, AlyM2 mainly generated trisaccharides, which accounted for 79.9% in the products. AlyM2 is a PL6 lyase with low sequence identity (≤28.3%) to the characterized alginate lyases and may adopt a distinct catalytic mechanism from the other PL6 alginate lyases based on sequence alignment. AlyM2 is a bifunctional endotype lyase, exhibiting the highest activity at 30 °C, pH 8.0, and 0.5 M NaCl. AlyM2 predominantly produces trisaccharides from homopolymeric M block (PM), homopolymeric G block (PG), or sodium alginate, with a trisaccharide production of 588.4 mg/g from sodium alginate, indicating its promising potential in preparing trisaccharides from these polysaccharides.

Ursolic acid as a potential inhibitor of Mycobacterium tuberculosis cytochrome bc1 oxidase-a molecular modelling perspective.

The escalating burden of tuberculosis disease and drastic effects of current medicine has stimulated a search for alternative drugs. A medicinal plant Warburgia salutaris has been reported to possess inhibitory properties against M. tuberculosis. In this study, we apply computational methods to investigate the probability of W. salutaris compounds as potential inhibitors of M. tuberculosis QcrB protein. We performed molecular docking, molecular dynamics simulations, radius of gyration, principal component analysis (PCA), and molecular mechanics-generalized born surface area (MM-GBSA) binding-free energy calculations in explicit solvent to achieve our objective. The results suggested that ursolic acid (UA) and ursolic acid acetate (UAA) could serve as preferred potential inhibitors of mycobacterial QcrB compared to lansoprazole sulphide (LSPZ) and telacebec (Q203)-UA and UAA have a higher binding affinity to QcrB compared to LSPZ and Q203 drugs. UA binding affinity is attributed to hydrogen bond formation with Val120, Arg364 and Arg366, and largely resonated from van der Waals forces resulting from UA interactions with hydrophobic amino acids in its vicinity. UAA binds to the porphyrin ring binding site with higher binding affinity compared to LSPZ. The binding affinity results primarily from van der Waals forces between UAA and hydrophobic residues of QcrB in the porphyrin ring binding site where UAA binds competitively. UA and UAA formed stable complexes with the protein with reduced overall residue mobility, consequently supporting the magnitude of binding affinity of the respective ligands. UAA could potentially compete with the porphyrin ring for the binding site and deprive the mycobacterial cell from oxygen, consequently disturbing mycobacterial oxygen-dependent metabolic processes. Therefore, discovery of a compound that competes with porphyrin ring for the binding site may be useful in QcrB pharmocological studies. UA proved to be a superior compound, although its estimated toxicity profile revealed UA to be hepatotoxic within acceptable parameters. Although preliminary findings of this report still warrant experimental validation, they could serve as a baseline for the development of new anti-tubercular drugs from natural resources that target QcrB.

Quorum Sensing Inhibitory Potential and Molecular Docking Studies of Phyllanthus emblica Phytochemicals Against Pseudomonas aeruginosa.

Phyllanthus emblica is a traditional medicinal plant that is endowed with curative properties including anti-bacterial, anti-fungal, anti-viral, and analgesic properties. Bacteria make use of cell-cell signaling system known as quorum sensing (QS) and respond to their own population. In most gram-negative bacteria, the transcriptional regulators belonging to the Lux R protein play a crucial role in the QS mechanism by detecting the presence of signaling molecules known as N-acyl homoserine lactones (AHLs). In this present work, the anti-quorum sensing activity of Phyllanthus emblica was evaluated against Pseudomonas aeruginosa. Anti-quorum sensing efficacy of Phyllanthus emblica was estimated with reference to QS bio-monitoring strain Chromobacterium violaceum. The binding efficacy of the phytochemicals of Phyllanthus emblica against CviR protein from Chromobacterium violaceum and LasR protein from Phyllanthus emblica were studied.

Microbial arginine deiminase: A multifaceted green catalyst in biomedical sciences.

Arginine deiminase is a well-recognized guanidino-modifying hydrolase that catalyzes the conversion of L-arginine to citrulline and ammonia. Their biopotential to regress tumors via amino acid deprivation therapy (AADT) has been well established. PEGylated formulation of recombinant Mycoplasma ADI is in the last-phase clinical trials against various arginine-auxotrophic cancers like hepatocellular carcinoma, melanoma, and mesothelioma. Recently, ADIs have attained immense importance in several other biomedical applications, namely treatment of Alzheimer's, as an antiviral drug, bioproduction of nutraceutical L-citrulline and bio-analytics involving L-arginine detection. Considering the wide applications of this biodrug, the demand for ADI is expected to escalate several-fold in the coming years. However, the sustainable production aspects of the enzyme with improved pharmacokinetics is still limited, creating bottlenecks for effective biopharmaceutical development. To circumvent the lacunae in enzyme production with appropriate paradigms of 'quality-by-design' an explicit overview of its properties with 'biobetter' formulations strategies are required. Present review provides an insight into all the potential biomedical applications of ADI along with the improvements required for its reach to clinics. Recent research advances with special emphasis on the development of ADI as a 'biobetter' enzyme have also been comprehensively elaborated.

Rosmarinic Acid and Sodium Citrate Have a Synergistic Bacteriostatic Effect against Vibrio Species by Inhibiting Iron Uptake.

BACKGROUND: New strategies are needed to combat multidrug-resistant bacteria. The restriction of iron uptake by bacteria is a promising way to inhibit their growth. We aimed to suppress the growth of Vibrio bacterial species by inhibiting their ferric ion-binding protein (FbpA) using food components. METHODS: Twenty spices were selected for the screening of FbpA inhibitors. The candidate was applied to antibacterial tests, and the mechanism was further studied. RESULTS: An active compound, rosmarinic acid (RA), was screened out. RA binds competitively and more tightly than Fe3+ to VmFbpA, the FbpA from V. metschnikovii, with apparent KD values of 8 µM vs. 17 µM. Moreover, RA can inhibit the growth of V. metschnikovii to one-third of the control at 1000 µM. Interestingly, sodium citrate (SC) enhances the growth inhibition effect of RA, although SC only does not inhibit the growth. The combination of RA/SC completely inhibits the growth of not only V. metschnikovii at 100/100 µM but also the vibriosis-causative pathogens V. vulnificus and V. parahaemolyticus, at 100/100 and 1000/100 µM, respectively. However, RA/SC does not affect the growth of Escherichia coli. CONCLUSIONS: RA/SC is a potential bacteriostatic agent against Vibrio species while causing little damage to indigenous gastrointestinal bacteria.

Molecular Insights into Binding Mode and Interactions of Structure-Based Virtually Screened Inhibitors for Pseudomonas aeruginosa Multiple Virulence Factor Regulator (MvfR).

Multi-drug resistance (MDR) bacterial pathogens pose a threat to global health and warrant the discovery of new therapeutic molecules, particularly those that can neutralize their virulence and stop the evolution of new resistant mechanisms. The superbug nosocomial pathogen, Pseudomonas aeruginosa, uses a multiple virulence factor regulator (MvfR) to regulate the expression of multiple virulence proteins during acute and persistent infections. The present study targeted MvfR with the intention of designing novel anti-virulent compounds, which will function in two ways: first, they will block the virulence and pathogenesis P. aeruginosa by disrupting the quorum-sensing network of the bacteria, and second, they will stop the evolution of new resistant mechanisms. A structure-based virtual screening (SBVS) method was used to screen druglike compounds from the Asinex antibacterial library (~5968 molecules) and the comprehensive marine natural products database (CMNPD) (~32 thousand compounds), against the ligand-binding domain (LBD) of MvfR, to identify molecules that show high binding potential for the relevant pocket. In this way, two compounds were identified: Top-1 (4-((carbamoyloxy)methyl)-10,10-dihydroxy-2,6-diiminiodecahydropyrrolo[1,2-c]purin-9-yl sulfate) and Top-2 (10,10-dihydroxy-2,6-diiminio-4-(((sulfonatocarbamoyl)oxy)methyl)decahydropyrrolo[1,2-c]purin-9-yl sulfate), in contrast to the co-crystallized M64 control. Both of the screened leads were found to show deep pocket binding and interactions with several key residues through a network of hydrophobic and hydrophilic interactions. The docking results were validated by a long run of 200 ns of molecular dynamics simulation and MM-PB/GBSA binding free energies. All of these analyses confirmed the presence of strong complex formation and rigorous intermolecular interactions. An additional analysis of normal mode entropy and a WaterSwap assay were also performed to complement the aforementioned studies. Lastly, the compounds were found to show an acceptable range of pharmacokinetic properties, making both compounds potential candidates for further experimental studies to decipher their real biological potency.

Identification of diphenyl furan derivatives via high throughput and computational studies as ArgA inhibitors of Mycobacterium tuberculosis.

Microbial amino acid biosynthetic pathways are underexploited for the development of anti-bacterial agents. N-acetyl glutamate synthase (ArgA) catalyses the first committed step in L-arginine biosynthesis and is essential for M. tuberculosis growth. Here, we have purified and optimized assay conditions for the acetylation of l-glutamine by ArgA. Using the optimized conditions, high throughput screening was performed to identify ArgA inhibitors. We identified 2,5-Bis (2-chloro-4-guanidinophenyl) furan, a dicationic diaryl furan derivatives, as ArgA inhibitor, with a MIC99 values of 1.56 µM against M. tuberculosis. The diaryl furan derivative displayed bactericidal killing against both M. bovis BCG and M. tuberculosis. Inhibition of ArgA by the lead compound resulted in transcriptional reprogramming and accumulation of reactive oxygen species. The lead compound and its derivatives showed micromolar binding with ArgA as observed in surface plasmon resonance and tryptophan quenching experiments. Computational and dynamic analysis revealed that these scaffolds share similar binding site residues with L-arginine, however, with slight variations in their interaction pattern. Partial restoration of growth upon supplementation of liquid cultures with either L-arginine or N-acetyl cysteine suggests a multi-target killing mechanism for the lead compound. Taken together, we have identified small molecule inhibitors against ArgA enzyme from M. tuberculosis.

The minor pilin PilV provides a conserved adhesion site throughout the antigenically variable meningococcal type IV pilus.

Neisseria meningitidis utilizes type IV pili (T4P) to adhere to and colonize host endothelial cells, a process at the heart of meningococcal invasive diseases leading to meningitis and sepsis. T4P are polymers of an antigenically variable major pilin building block, PilE, plus several core minor pilins that initiate pilus assembly and are thought to be located at the pilus tip. Adhesion of N. meningitidis to human endothelial cells requires both PilE and a conserved noncore minor pilin PilV, but the localization of PilV and its precise role in this process remains to be clarified. Here, we show that both PilE and PilV promote adhesion to endothelial vessels in vivo. The substantial adhesion defect observed for pilV mutants suggests it is the main adhesin. Consistent with this observation, superresolution microscopy showed the abundant distribution of PilV throughout the pilus. We determined the crystal structure of PilV and modeled it within the pilus filament. The small size of PilV causes it to be recessed relative to adjacent PilE subunits, which are dominated by a prominent hypervariable loop. Nonetheless, we identified a conserved surface-exposed adhesive loop on PilV by alanine scanning mutagenesis. Critically, antibodies directed against PilV inhibit N. meningitidis colonization of human skin grafts. These findings explain how N. meningitidis T4P undergo antigenic variation to evade the humoral immune response while maintaining their adhesive function and establish the potential of this highly conserved minor pilin as a vaccine and therapeutic target for the prevention and treatment of N. meningitidis infections.

The phage defence island of a multidrug resistant plasmid uses both BREX and type IV restriction for complementary protection from viruses.

Bacteria have evolved a multitude of systems to prevent invasion by bacteriophages and other mobile genetic elements. Comparative genomics suggests that genes encoding bacterial defence mechanisms are often clustered in 'defence islands', providing a concerted level of protection against a wider range of attackers. However, there is a comparative paucity of information on functional interplay between multiple defence systems. Here, we have functionally characterised a defence island from a multidrug resistant plasmid of the emerging pathogen Escherichia fergusonii. Using a suite of thirty environmentally-isolated coliphages, we demonstrate multi-layered and robust phage protection provided by a plasmid-encoded defence island that expresses both a type I BREX system and the novel GmrSD-family type IV DNA modification-dependent restriction enzyme, BrxU. We present the structure of BrxU to 2.12 Å, the first structure of the GmrSD family of enzymes, and show that BrxU can utilise all common nucleotides and a wide selection of metals to cleave a range of modified DNAs. Additionally, BrxU undergoes a multi-step reaction cycle instigated by an unexpected ATP-dependent shift from an intertwined dimer to monomers. This direct evidence that bacterial defence islands can mediate complementary layers of phage protection enhances our understanding of the ever-expanding nature of phage-bacterial interactions.

In Silico Identification of Possible Inhibitors for Protein Kinase B (PknB) of Mycobacterium tuberculosis.

With tuberculosis still being one of leading causes of death in the world and the emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb), researchers have been seeking to find further therapeutic strategies or more specific molecular targets. PknB is one of the 11 Ser/Thr protein kinases of Mtb and is responsible for phosphorylation-mediated signaling, mainly involved in cell wall synthesis, cell division and metabolism. With the amount of structural information available and the great interest in protein kinases, PknB has become an attractive target for drug development. This work describes the optimization and application of an in silico computational protocol to find new PknB inhibitors. This multi-level computational approach combines protein-ligand docking, structure-based virtual screening, molecular dynamics simulations and free energy calculations. The optimized protocol was applied to screen a large dataset containing 129,650 molecules, obtained from the ZINC/FDA-Approved database, Mu.Ta.Lig Virtual Chemotheca and Chimiothèque Nationale. It was observed that the most promising compounds selected occupy the adenine-binding pocket in PknB, and the main interacting residues are Leu17, Val26, Tyr94 and Met155. Only one of the compounds was able to move the active site residues into an open conformation. It was also observed that the P-loop and magnesium position loops change according to the characteristics of the ligand. This protocol led to the identification of six compounds for further experimental testing while also providing additional structural information for the design of more specific and more effective derivatives.

A universal pocket in fatty acyl-AMP ligases ensures redirection of fatty acid pool away from coenzyme A-based activation.

Fatty acyl-AMP ligases (FAALs) channelize fatty acids towards biosynthesis of virulent lipids in mycobacteria and other pharmaceutically or ecologically important polyketides and lipopeptides in other microbes. They do so by bypassing the ubiquitous coenzyme A-dependent activation and rely on the acyl carrier protein-tethered 4'-phosphopantetheine (holo-ACP). The molecular basis of how FAALs strictly reject chemically identical and abundant acceptors like coenzyme A (CoA) and accept holo-ACP unlike other members of the ANL superfamily remains elusive. We show that FAALs have plugged the promiscuous canonical CoA-binding pockets and utilize highly selective alternative binding sites. These alternative pockets can distinguish adenosine 3',5'-bisphosphate-containing CoA from holo-ACP and thus FAALs can distinguish between CoA and holo-ACP. These exclusive features helped identify the omnipresence of FAAL-like proteins and their emergence in plants, fungi, and animals with unconventional domain organizations. The universal distribution of FAALs suggests that they are parallelly evolved with FACLs for ensuring a CoA-independent activation and redirection of fatty acids towards lipidic metabolites.

Delivery of membrane impermeable molecules to primary mouse T lymphocytes.

The pore-forming toxin streptolysin-O (SLO) enables intracellular delivery of molecules up to 100 kDa and has been used for short-term delivery of membrane-impermeable substances to assess their effects on cellular activities. A limitation of this technique is the loss of intracellular components and the potential unpredicted alterations of cellular metabolism and signaling. This protocol, optimized for primary mouse T lymphocytes, describes steps for SLO-mediated cell membrane permeabilization and substance supplementation, followed by immunoblotting and immunofluorescent microscopy for assessing cellular effects. For complete details on the use and execution of this protocol, please refer to Xu et al., 2021a, Xu et al., 2021b.

Structures and function of the amino acid polymerase cyanophycin synthetase.

Cyanophycin is a natural biopolymer produced by a wide range of bacteria, consisting of a chain of poly-L-Asp residues with L-Arg residues attached to the ß-carboxylate sidechains by isopeptide bonds. Cyanophycin is synthesized from ATP, aspartic acid and arginine by a homooligomeric enzyme called cyanophycin synthetase (CphA1). CphA1 has domains that are homologous to glutathione synthetases and muramyl ligases, but no other structural information has been available. Here, we present cryo-electron microscopy and X-ray crystallography structures of cyanophycin synthetases from three different bacteria, including cocomplex structures of CphA1 with ATP and cyanophycin polymer analogs at 2.6 Å resolution. These structures reveal two distinct tetrameric architectures, show the configuration of active sites and polymer-binding regions, indicate dynamic conformational changes and afford insight into catalytic mechanism. Accompanying biochemical interrogation of substrate binding sites, catalytic centers and oligomerization interfaces combine with the structures to provide a holistic understanding of cyanophycin biosynthesis.

Urease Inhibitory Kinetic Studies of Various Extracts and Pure Compounds from Cinnamomum Genus.

Urease is an enzyme that plays a significant role in the hydrolysis of urea into carbonic acid and ammonia via the carbamic acid formation. The resultant increase in pH leads to the onset of various pathologies such as gastric cancer, urolithiasis, hepatic coma, hepatic encephalopathy, duodenal ulcers and peptic ulcers. Urease inhibitors can reduce the urea hydrolysis rate and development of various diseases. The Cinnamomum genus is used in a large number of traditional medicines. It is well established that stem bark of Cinnamomum cassia exhibits antiulcerogenic potential. The present study evaluated the inhibitory effect of seven extracts of Cinnamomum camphora, Cinnamomum verum and two pure compounds Camphene and Cuminaldehyde on urease enzyme. Kinetic studies of potential inhibitors were carried out. Methanol extract (IC50 980 µg/mL) of C. camphora and a monoterpene Camphene (IC50 0.147 µg/mL) possess significant inhibitory activity. The Lineweaver Burk plot analysis suggested the competitive inhibition by methanol extract, hexane fraction and Camphene. The Gas Chromatography-Mass Spectroscopy (GC-MS) analysis of hexane fraction revealed the contribution of various terpenes. The present study targets terpenes as a new class of inhibitors that have potential therapeutic value for further development as novel drugs.