scholarly journals In Silico Characterization and Homology Modeling of a Cyanobacterial Phosphoenolpyruvate Carboxykinase Enzyme

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Aubrey A. Smith ◽  
Amanda Caruso
Keyword(s):  
Active Site ◽  
Aquatic Ecosystems ◽  
Metal Ion ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Inorganic Carbon ◽  
Carbon Fixation ◽  
Homology Model ◽  
Active Site Residues ◽  
Cyanobacterial Species ◽  
In Silico Characterization

ATP-dependent phosphoenolpyruvate carboxykinase (PEPCK) is a key catabolic enzyme found in various species of bacteria, plants, and yeast. PEPCK may play a role in carbon fixation in aquatic ecosystems consisting of photosynthetic cyanobacteria. RuBisCO-based CO2 fixation is prevalent in cyanobacteria through C3 intermediates; however, a significant amount of carbon flows into C4 acids during cyanobacterial photosynthesis. This indicates that a C4 mechanism for inorganic carbon fixation is prevalent in cyanobacteria with PEPCK as an important β-carboxylation enzyme. Newly available genomic information has confirmed the existence of putative PEPCK genes in a number of cyanobacterial species. This project represents the first structural and physicochemical study of cyanobacterial PEPCKs. Biocomputational analyses of cyanobacterial PEPCKs were performed and a homology model of Cyanothece sp. PCC 7424 PEPCK was generated. The modeled enzyme consists of an N-terminal and C-terminal domains with a mixed α/β topology with the active site located in a deep cleft between the two domains. Active site residues and those involved in metal ion coordination were found to be conserved in the cyanobacterial enzymes. An active site lid which is known to close upon substrate binding was also predicted. Amino acid stretches that are unique to cyanobacterial PEPCKs were also identified.

scholarly journals Crystal structures of murine norovirus-1 RNA-dependent RNA polymerase

2011 ◽  
Vol 92 (7) ◽  
pp. 1607-1616 ◽  
Author(s):  
Ji-Hye Lee ◽  
Intekhab Alam ◽  
Kang Rok Han ◽  
Sunyoung Cho ◽  
Sungho Shin ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Crystal Structures ◽  
Active Site ◽  
Metal Ion ◽  
Health Concern ◽  
Murine Norovirus ◽  
Active Site Residues ◽  
Rna Dependent Rna Polymerase ◽  
Close Proximity ◽  
Functional Features

Norovirus is one of the leading agents of gastroenteritis and is a major public health concern. In this study, the crystal structures of recombinant RNA-dependent RNA polymerase (RdRp) from murine norovirus-1 (MNV-1) and its complex with 5-fluorouracil (5FU) were determined at 2.5 Å resolution. Crystals with C2 symmetry revealed a dimer with half a dimer in the asymmetrical unit, and the protein exists predominantly as a monomer in solution, in equilibrium with a smaller population of dimers, trimers and hexamers. MNV-1 RdRp exhibited polymerization activity with a right-hand fold typical of polynucleotide polymerases. The metal ion modelled in close proximity to the active site was found to be coordinated tetrahedrally to the carboxyl groups of aspartate clusters. The orientation of 5FU observed in three molecules in the asymmetrical unit was found to be slightly different, but it was stabilized by a network of favourable interactions with the conserved active-site residues Arg185, Asp245, Asp346, Asp347 and Arg395. The information gained on the structural and functional features of MNV-1 RdRp will be helpful in understanding replication of norovirus and in designing novel therapeutic agents against this important pathogen.

Inorganic carbon fixation in ice-covered lakes of the McMurdo Dry Valleys

2019 ◽  
Vol 31 (3) ◽  
pp. 123-132 ◽  
Author(s):  
Trista J. Vick-Majors ◽  
John C. Priscu
Keyword(s):  
Aquatic Ecosystems ◽  
Inorganic Carbon ◽  
Carbon Fixation ◽  
Dry Valleys ◽  
Ammonia Oxidizers ◽  
Fixed Carbon ◽  
Carbon Supply ◽  
Gain Energy ◽  
Photosynthetic Microorganisms ◽  
Dark Carbon Fixation

AbstractInorganic carbon fixation, usually mediated by photosynthetic microorganisms, is considered to form the base of the food chain in aquatic ecosystems. In high-latitude lakes, lack of sunlight owing to seasonal solar radiation limits the activity of photosynthetic plankton during the polar winter, causing respiration-driven demand for carbon to exceed supply. Here, we show that inorganic carbon fixation in the dark, driven by organisms that gain energy from chemical reactions rather than sunlight (chemolithoautotrophs), provides a significant influx of fixed carbon to two permanently ice-covered lakes (Fryxell and East Bonney). Fryxell, which has higher biomass per unit volume of water, had higher rates of inorganic dark carbon fixation by chemolithoautotrophs than East Bonney (trophogenic zone average 1.0 µg C l−1 d−1vs 0.08 µg C l−1 d−1, respectively). This contribution from dark carbon fixation was partly due to the activity of ammonia oxidizers, which are present in both lakes. Despite the potential importance of new carbon input by chemolithoautotrophic activity, both lakes remain net heterotrophic, with respiratory demand for carbon exceeding supply. Dark carbon fixation increased the ratio of new carbon supply to respiratory demand from 0.16 to 0.47 in Fryxell, and from 0.14 to 0.22 in East Bonney.

Navigating Research Toward the Re-emerging Nipah Virus- A New Piece to the Puzzle

2019 ◽  
Vol 25 (12) ◽  
pp. 1392-1401
Author(s):  
Pritika Ramharack ◽  
Nikita Devnarain ◽  
Letitia Shunmugam ◽  
Mahmoud E.S. Soliman
Keyword(s):  
Active Site ◽  
Hydrophobic Interactions ◽  
Nipah Virus ◽  
Homology Model ◽  
Future Research ◽  
Active Site Residues ◽  
Comprehensive Overview ◽  
Computational Tools ◽  
Plot Analysis ◽  
Potential Inhibitors

Background: The recent Nipah virus (NiV) outbreak in India has caused a state of chaos, with potential to become the next international pandemic. There is still a great deal to learn about NiV for the development of a potent treatment against it. The NiV non-structural proteins play important roles in the lifecycle of the virus, with the RNA-dependent RNA-polymerase (RdRp) being a vital component in viral replication. In this study, we not only provide a comprehensive overview of all the literature concerning NiV, we also propose a model of the NiV RdRp and screen for potential inhibitors of the viral enzyme. Objectives: In this study, computational tools were utilized in the design of a NiV RdRp homology model. The active site of RdRp was then identified and potential inhibitors of the protein were discovered with the use of pharmacophore-based screening. Methods: In this study, computational tools were utilized in the design of a NiV RdRp homology model. The active site of RdRp was then identified and potential inhibitors of the protein were discovered with the use of pharmacophore-based screening. Results: Ramachandran plot analysis revealed a favourable model. Upon binding of nucleoside analog, 4’- Azidocytidine, active site residues Trp1714 and Ser1713 took part in stabilizing hydrogen bonds, while Thr1716, Ser1478, Ser1476 and Glu1465 contributed to hydrophobic interactions. Pharmacophore based screening yielded 18 hits, of which ZINC00085930 demonstrated the most optimal binding energy (-8.1 kcal/mol), validating its use for further analysis as an inhibitor of NiV. Conclusion: In this study we provide a critical guide, elucidating on the in silico requirements of the drug design and discovery process against NiV. This material lays a foundation for future research into the design and development of drugs that inhibit NiV.

scholarly journals Ferrochelatase: the convergence of the porphyrin biosynthesis and iron transport pathways

2011 ◽  
Vol 15 (05n06) ◽  
pp. 350-356 ◽  
Author(s):  
Gregory A. Hunter ◽  
Salam Al-Karadaghi ◽  
Gloria C. Ferreira
Keyword(s):  
Active Site ◽  
Ferrous Iron ◽  
Metal Ion ◽  
Iron Transport ◽  
Enzyme Commission Number ◽  
Metal Ion Binding ◽  
Active Site Residues ◽  
Transport Pathways ◽  
Living Organisms

Ferrochelatase (also known as PPIX ferrochelatase; Enzyme Commission number 4.9.9.1.1) catalyzes the insertion of ferrous iron into PPIX to form heme. This reaction unites the biochemically synchronized pathways of porphyrin synthesis and iron transport in nearly all living organisms. The ferrochelatases are an evolutionarily diverse family of enzymes with no more than six active site residues known to be perfectly conserved. The availability of over thirty different crystal structures, including many with bound metal ions or porphyrins, has added tremendously to our understanding of ferrochelatase structure and function. It is generally believed that ferrous iron is directly channeled to ferrochelatase in vivo, but the identity of the suspected chaperone remains uncertain despite much recent progress in this area. Identification of a conserved metal ion binding site at the base of the active site cleft may be an important clue as to how ferrochelatases acquire iron, and catalyze desolvation during transport to the catalytic site to complete heme synthesis.

scholarly journals Insight into the Function of Active Site Residues in the Catalytic Mechanism of Human Ferrochelatase

2021 ◽  
Author(s):  
Amy E. Medlock ◽  
Wided Najahi-Missaoui ◽  
Mesafint T. Shiferaw ◽  
Angela N. Albetel ◽  
William N. Lanzilotta ◽  
...  
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Catalytic Mechanism ◽  
Structural Data ◽  
Active Site Residues ◽  
Proton Abstraction ◽  
Product Release ◽  
Iron Delivery ◽  
High Level

Ferrochelatase catalyzes the insertion of ferrous iron into a porphyrin macrocycle to produce the essential cofactor, heme. In humans this enzyme not only catalyzes the terminal step, but also serves a regulatory step in the heme synthesis pathway. Over a dozen crystal structures of human ferrochelatase have been solved and many variants have been characterized kinetically. In addition, hydrogen deuterium exchange, resonance Raman, molecular dynamics, and high level quantum mechanic studies have added to our understanding of  the catalytic cycle of the enzyme. However, an understanding of how the metal ion is delivered and the specific role that active site residues play in catalysis remain open questions. Data are consistent with metal binding and insertion occurring from the side opposite from where pyrrole proton abstraction takes place. To better understand iron delivery and binding as well as the role of conserved residues in the active site, we have constructed and characterized a series of enzyme variants. Crystallographic studies as well as rescue and kinetic analysis of variants were performed. Data from these studies are consistent with the M76 residue playing a role in active site metal binding and formation of a weak iron protein ligand being necessary for product release. Additionally, structural data support a role for E343 in proton abstraction and product release in coordination with a peptide loop composed of Q302, S303 and K304 that act a metal sensor.

Structural insights into chaperone-activity enhancement by a K354E mutation in tomato acidic leucine aminopeptidase

2016 ◽  
Vol 72 (5) ◽  
pp. 694-702 ◽  
Author(s):  
Kevin T. DuPrez ◽  
Melissa A. Scranton ◽  
Linda L. Walling ◽  
Li Fan
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Leucine Aminopeptidase ◽  
Gel Filtration ◽  
Chaperone Activity ◽  
Peptidase Activity ◽  
Wild Type ◽  
Active Site Residues ◽  
In The Wild ◽  
Hydrophobic Areas

Tomato plants express acidic leucine aminopeptidase (LAP-A) in response to various environmental stressors. LAP-A not only functions as a peptidase for diverse peptide substrates, but also displays chaperone activity. A K354E mutation has been shown to abolish the peptidase activity but to enhance the chaperone activity of LAP-A. To better understand this moonlighting function of LAP-A, the crystal structure of the K354E mutant was determined at 2.15 Å resolution. The structure reveals that the K354E mutation destabilizes an active-site loop and causes significant rearrangement of active-site residues, leading to loss of the catalytic metal-ion coordination required for the peptidase activity. Although the mutant was crystallized in the same hexameric form as wild-type LAP-A, gel-filtration chromatography revealed an apparent shift from the hexamer to lower-order oligomers for the K354E mutant, showing a mixture of monomers to trimers in solution. In addition, surface-probing assays indicated that the K354E mutant has more accessible hydrophobic areas than wild-type LAP-A. Consistently, computational thermodynamic estimations of the interfaces between LAP-A monomers suggest that increased exposure of hydrophobic surfaces occurs upon hexamer breakdown. These results suggest that the K354E mutation disrupts the active-site loop, which also contributes to the hexameric assembly, and destabilizes the hexamers, resulting in much greater hydrophobic areas accessible for efficient chaperone activity than in the wild-type LAP-A.

scholarly journals Crystallographic Comparison of Manganese- and Iron-Dependent Homoprotocatechuate 2,3-Dioxygenases

2004 ◽  
Vol 186 (7) ◽  
pp. 1945-1958 ◽  
Author(s):  
Matthew W. Vetting ◽  
Lawrence P. Wackett ◽  
Lawrence Que ◽  
John D. Lipscomb ◽  
Douglas H. Ohlendorf
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Ion Selectivity ◽  
Axial Ligand ◽  
Activation Process ◽  
Type I ◽  
Active Site Residues ◽  
Metal Ion Selectivity ◽  
Mean Square Difference ◽  
Extradiol Dioxygenases

ABSTRACT The X-ray crystal structures of homoprotocatechuate 2,3-dioxygenases isolated from Arthrobacter globiformis and Brevibacterium fuscum have been determined to high resolution. These enzymes exhibit 83% sequence identity, yet their activities depend on different transition metals, Mn2+ and Fe2+, respectively. The structures allow the origins of metal ion selectivity and aspects of the molecular mechanism to be examined in detail. The homotetrameric enzymes belong to the type I family of extradiol dioxygenases (vicinal oxygen chelate superfamily); each monomer has four βαβββ modules forming two structurally homologous N-terminal and C-terminal barrel-shaped domains. The active-site metal is located in the C-terminal barrel and is ligated by two equatorial ligands, H214NE1 and E267OE1; one axial ligand, H155NE1; and two to three water molecules. The first and second coordination spheres of these enzymes are virtually identical (root mean square difference over all atoms, 0.19 Å), suggesting that the metal selectivity must be due to changes at a significant distance from the metal and/or changes that occur during folding. The substrate (2,3-dihydroxyphenylacetate [HPCA]) chelates the metal asymmetrically at sites trans to the two imidazole ligands and interacts with a unique, mobile C-terminal loop. The loop closes over the bound substrate, presumably to seal the active site as the oxygen activation process commences. An “open” coordination site trans to E267 is the likely binding site for O2. The geometry of the enzyme-substrate complexes suggests that if a transiently formed metal-superoxide complex attacks the substrate without dissociation from the metal, it must do so at the C-3 position. Second-sphere active-site residues that are positioned to interact with the HPCA and/or bound O2 during catalysis are identified and discussed in the context of current mechanistic hypotheses.

The conserved active site histidine-glutamate pair of ferrochelatase coordinately catalyzes porphyrin metalation

2016 ◽  
Vol 20 (01n04) ◽  
pp. 556-569 ◽  
Author(s):  
Gregory A. Hunter ◽  
Sai Lakshmana Vankayala ◽  
Mallory E. Gillam ◽  
Fiona L. Kearns ◽  
H. Lee Woodcock ◽  
...  
Keyword(s):  
Active Site ◽  
Ferrous Iron ◽  
Metal Ion ◽  
Conformational Transition ◽  
Protoporphyrin Ix ◽  
Insertion Reaction ◽  
Active Site Residues ◽  
Protonation State ◽  
Dynamic Simulations ◽  
Ion Insertion

Ferrochelatase catalyzes the insertion of ferrous iron into protoporphyrin IX to generate heme. Despite recent research on the reaction mechanism of ferrochelatase, the precise roles and localization of individual active site residues in catalysis, particularly those involved in the insertion of the ferrous iron into the protoporphyrin IX substrate, remain controversial. One outstanding question is from which side of the macrocycle of the bound porphyin substrate is the ferrous iron substrate inserted. Pre-steady state kinetic experiments done under single-turnover conditions conclusively demonstrate that metal ion insertion is pH-dependent, and that the conserved active site His-Glu pair coordinately catalyzes the metal ion insertion reaction. Further, p[Formula: see text] calculations and molecular dynamic simulations indicate that the active site His is deprotonated and the protonation state of the Glu relates to the conformational state of ferrochelatase. Specifically, the conserved Glu in the open conformation of ferrochelatase is deprotonated, while it remains protonated in the closed conformation. These findings support not only the role of the His-Glu pair in catalyzing metal ion insertion, as these residues need to be deprotonated to bind the incoming metal ion, but also the importance of the relationship between the protonation state of the Glu residue and the conformation of ferrochelatase. Finally, the results of this study are consistent with our previous proposal that the unwinding of the [Formula: see text]-helix, the major structural determinant of the closed to open conformational transition in ferrochelatase, is associated with the Glu residue binding the Fe[Formula: see text] substrate from a mitochondrial Fe[Formula: see text] donor.

scholarly journals Computational Elucidation of Structural Basis for Ligand Binding withLeishmania donovaniAdenosine Kinase

2013 ◽  
Vol 2013 ◽  
pp. 1-14 ◽  
Author(s):  
Rajiv K. Kar ◽  
Md. Yousuf Ansari ◽  
Priyanka Suryadevara ◽  
Bikash R. Sahoo ◽  
Ganesh C. Sahoo ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Active Site ◽  
Protein Complex ◽  
Leishmania Donovani ◽  
Homology Model ◽  
Adenosine Kinase ◽  
Dynamics Simulation ◽  
Structural Basis ◽  
Active Site Residues ◽  
Drug Candidates

Enzyme adenosine kinase is responsible for phosphorylation of adenosine to AMP and is crucial for parasites which are purine auxotrophs. The present study describes development of robust homology model ofLeishmania donovaniadenosine kinase to forecast interaction phenomenon with inhibitory molecules using structure-based drug designing strategy. Docking calculation using reported organic small molecules and natural products revealed key active site residues such as Arg131 and Asp16 for ligand binding, which is consistent with previous studies. Molecular dynamics simulation of ligand protein complex revealed the importance of hydrogen bonding with active site residues and solvent molecules, which may be crucial for successful development of drug candidates. Precise role of Phe168 residue in the active site was elucidated in this report that provided stability to ligand-protein complex via aromatic-πcontacts. Overall, the present study is believed to provide valuable information to design a new compound with improved activity for antileishmanial therapeutics development.

scholarly journals Compilation of Potential Protein Targets for SARS-CoV-2: Preparation of Homology Model and Active Site Determination for Future Rational Antiviral Design

2020 ◽  
Author(s):  
Sourav Pal ◽  
Dr. Arindam Talukdar
Keyword(s):  
Active Site ◽  
Drug Targets ◽  
Active Sites ◽  
Structural Alignment ◽  
Homology Model ◽  
Active Site Residues ◽  
Protein Targets ◽  
Rational Development ◽  
Novel Coronavirus ◽  
Approved Drugs

<p>The recent pandemic due to the novel coronavirus SARS-CoV-2 (COVID-19) is causing significant mortality worldwide. However, there is a lack of specific drugs which can either prevent or treat the patient suffering from COVID-19. To understand the SARS-CoV-2 receptor recognition causing infectivity and pathogenesis, we have compiled a list of 20 probable drug targets on host and virus based on viral life cycle along with their PDB IDs for the rational development of future antivirals. We have prepared nine homology model for vital proteins for which no crystal structure is reported, which includes protein from host, viral membrane proteins and essential non-structural proteins (NSPs) of virus. The generated models were validated followed by Ramachandran plot along with their sequence and structural alignment. The active site residues of all the protein models are calculated by utilizing COACH meta-server and also cross verified with the CASTp webservers. All the active sites of the homology build proteins were evaluated after superimposition of the closely related X-ray crystallized structure bound with the co-crystal ligands. These information present in the manuscript can be used for the discovery effort towards new antivirals as well as repurposing FDA approved drugs against SARS-CoV-2.</p><br>

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