Proton Relay Network in P450cam is Formed Upon Docking of its Redox Partner Putidaredoxin (Pdx)

2019 ◽  
Author(s):  
Ilke Ugur Marion ◽  
Prasanna Chandrasekhar
Keyword(s):  
Secondary Structure ◽  
Cytochromes P450 ◽  
Direct Interaction ◽  
Relay Networks ◽  
Relay Network ◽  
Wild Type ◽  
Redox Partner ◽  
Proton Relay ◽  
Cleavage Step ◽  
First Time

Cytochromes P450 are versatile heme-based enzymes responsible for vital life processes. Of these, P450cam (substrate camphor) has been most studied. Despite this, precise mechanisms of the key O-O cleavage step remain elusive to date; effects observed in various enzyme mutants remain unexplained. We have carried out extended (up to 1000 ns) MM-MD and follow-on QM/MM computations, both on the well-studied FeOO state and, for the first time, on Cpd(0). Our simulations include (all camphor-bound) : (1) WT (wild type), FeOO state. (2) WT, Cpd(0). (3) Pdx-docked-WT, FeOO state. (4) Pdx-docked WT, Cpd(0). (5) Pdx-docked T252A mutant, Cpd(0). Among our key findings, for the first time to our knowledge: (1) Effect of Pdx docking goes far beyond that indicated in prior studies: it leads to specific alterations in secondary structure that create the crucial proton relay network. (2) The specific proton relay networks we identify are FeOO(H)---T252---nH2O---D251 in WT and FeOO(H)---nH2O---D251 in T252A mutant; both occur with Pdx docking. (3) Direct interaction of D251 with -FeOOH is, respectively, rare/frequent in WT/T252A mutant. (4) T252 is in the proton relay network. (5) Positioning of camphor is crucial: when camphor is part of H-bonding network, coupling is facilitated.<br>

Proton Relay Network in P450cam is Formed Upon Docking of its Redox Partner Putidaredoxin (Pdx)

2019 ◽  
Author(s):  
Ilke Ugur Marion ◽  
Prasanna Chandrasekhar
Keyword(s):  
Secondary Structure ◽  
Cytochromes P450 ◽  
Direct Interaction ◽  
Relay Networks ◽  
Relay Network ◽  
Wild Type ◽  
Redox Partner ◽  
Proton Relay ◽  
Cleavage Step ◽  
First Time

Cytochromes P450 are versatile heme-based enzymes responsible for vital life processes. Of these, P450cam (substrate camphor) has been most studied. Despite this, precise mechanisms of the key O-O cleavage step remain elusive to date; effects observed in various enzyme mutants remain unexplained. We have carried out extended (up to 1000 ns) MM-MD and follow-on QM/MM computations, both on the well-studied FeOO state and, for the first time, on Cpd(0). Our simulations include (all camphor-bound) : (1) WT (wild type), FeOO state. (2) WT, Cpd(0). (3) Pdx-docked-WT, FeOO state. (4) Pdx-docked WT, Cpd(0). (5) Pdx-docked T252A mutant, Cpd(0). Among our key findings, for the first time to our knowledge: (1) Effect of Pdx docking goes far beyond that indicated in prior studies: it leads to specific alterations in secondary structure that create the crucial proton relay network. (2) The specific proton relay networks we identify are FeOO(H)---T252---nH2O---D251 in WT and FeOO(H)---nH2O---D251 in T252A mutant; both occur with Pdx docking. (3) Direct interaction of D251 with -FeOOH is, respectively, rare/frequent in WT/T252A mutant. (4) T252 is in the proton relay network. (5) Positioning of camphor is crucial: when camphor is part of H-bonding network, coupling is facilitated.<br>

scholarly journals A requirement for an active proton delivery network supports a compound I-mediated C–C bond cleavage in CYP51 catalysis

2020 ◽  
Vol 295 (29) ◽  
pp. 9998-10007 ◽  
Author(s):  
Tatiana Y. Hargrove ◽  
Zdzislaw Wawrzak ◽  
F. Peter Guengerich ◽  
Galina I. Lepesheva
Keyword(s):  
Bond Cleavage ◽  
Cytochromes P450 ◽  
Relay Network ◽  
Compound I ◽  
X Ray ◽  
Proton Relay ◽  
Multistep Reactions ◽  
Biochemical Analyses ◽  
Ferric Peroxo ◽  
Delivery Network

CYP51 enzymes (sterol 14α-demethylases) are cytochromes P450 that catalyze multistep reactions. The CYP51 reaction occurs in all biological kingdoms and is essential in sterol biosynthesis. It removes the 14α-methyl group from cyclized sterol precursors by first forming an alcohol, then an aldehyde, and finally eliminating formic acid with the introduction of a Δ14–15 double bond in the sterol core. The first two steps are typical hydroxylations, mediated by an electrophilic compound I mechanism. The third step, C–C bond cleavage, has been proposed to involve either compound I (i.e. FeO3+) or, alternatively, a proton transfer-independent nucleophilic ferric peroxo anion (compound 0, i.e. Fe3+O2–). Here, using comparative crystallographic and biochemical analyses of WT human CYP51 (CYP51A1) and its D231A/H314A mutant, whose proton delivery network is destroyed (as evidenced in a 1.98-Å X-ray structure in complex with lanosterol), we demonstrate that deformylation of the 14α-carboxaldehyde intermediate requires an active proton relay network to drive the catalysis. These results indicate a unified, compound I-based mechanism for all three steps of the CYP51 reaction, as previously established for CYP11A1 and CYP19A1. We anticipate that our approach can be applied to mechanistic studies of other P450s that catalyze multistep reactions, such as C–C bond cleavage.

scholarly journals Engineered Phage Endolysin Eliminates Gardnerella Biofilm without Damaging Beneficial Bacteria in Bacterial Vaginosis Ex Vivo

Pathogens ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 54
Author(s):  
Christine Landlinger ◽  
Lenka Tisakova ◽  
Vera Oberbauer ◽  
Timo Schwebs ◽  
Abbas Muhammad ◽  
...  
Keyword(s):  
Bacterial Vaginosis ◽  
Bactericidal Activity ◽  
High Selectivity ◽  
Ex Vivo ◽  
Vaginal Microbiome ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Promising Alternative ◽  
First Time

Bacterial vaginosis is characterized by an imbalance of the vaginal microbiome and a characteristic biofilm formed on the vaginal epithelium, which is initiated and dominated by Gardnerella bacteria, and is frequently refractory to antibiotic treatment. We investigated endolysins of the type 1,4-beta-N-acetylmuramidase encoded on Gardnerella prophages as an alternative treatment. When recombinantly expressed, these proteins demonstrated strong bactericidal activity against four different Gardnerella species. By domain shuffling, we generated several engineered endolysins with 10-fold higher bactericidal activity than any wild-type enzyme. When tested against a panel of 20 Gardnerella strains, the most active endolysin, called PM-477, showed minimum inhibitory concentrations of 0.13–8 µg/mL. PM-477 had no effect on beneficial lactobacilli or other species of vaginal bacteria. Furthermore, the efficacy of PM-477 was tested by fluorescence in situ hybridization on vaginal samples of fifteen patients with either first time or recurring bacterial vaginosis. In thirteen cases, PM-477 killed the Gardnerella bacteria and physically dissolved the biofilms without affecting the remaining vaginal microbiome. The high selectivity and effectiveness in eliminating Gardnerella, both in cultures of isolated strains as well as in clinically derived samples of natural polymicrobial biofilms, makes PM-477 a promising alternative to antibiotics for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.

scholarly journals Seed morphology uncovers 1500 years of vine agrobiodiversity before the advent of the Champagne wine

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Vincent Bonhomme ◽  
Jean-Frédéric Terral ◽  
Véronique Zech-Matterne ◽  
Sarah Ivorra ◽  
Thierry Lacombe ◽  
...  
Keyword(s):  
Middle Ages ◽  
Climatic Changes ◽  
Seed Morphology ◽  
Crucial Aspect ◽  
Wild Type ◽  
Grape Seeds ◽  
The Social ◽  
Reference Collection ◽  
Grape Varieties ◽  
First Time

AbstractA crucial aspect of viticulture is finally unveiled as the historical dynamics of its agrobiodiversity are described in the Champagne region for the first time. Outline analyses were carried out to compare the morphology of archaeological grape seeds from Troyes and Reims (first c. AD to fifteenth c. AD) with that of a reference collection of modern seeds, including wild vines and traditional grape varieties, believed to be ancient and characteristic of the French vine heritage. This allows us to document the chronological dynamics of the use of the wild Vitis type and of the diversity of the varieties used, based on morphological disparity. After showing the existence of morphological types corresponding to geographical groups, we highlight a geochronological dynamic. Our results show that the wild type is used throughout the series, up to the Middle Ages. In addition, domestic forms, morphologically related to southern varietal groups, are very early involved in the Champagne grape agrodiversity. The groups corresponding to the typical grape varieties of today do not appear until the second millennium. These previously unsuspected dynamics are discussed in light of the social, societal and climatic changes documented for the period.

scholarly journals A Novel and Efficient High-Yield Method for Preparing Bacterial Ghosts

Toxins ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 420
Author(s):  
Yi Ma ◽  
Liu Cui ◽  
Meng Wang ◽  
Qiuli Sun ◽  
Kaisheng Liu ◽  
...  
Keyword(s):  
Large Scale ◽  
Expression System ◽  
High Yield ◽  
Wild Type ◽  
High Quality ◽  
Efficient Protection ◽  
Bacterial Ghosts ◽  
Cell Envelopes ◽  
Extracellular Structures ◽  
First Time

Bacterial ghosts (BGs) are empty cell envelopes possessing native extracellular structures without a cytoplasm and genetic materials. BGs are proposed to have significant prospects in biomedical research as vaccines or delivery carriers. The applications of BGs are often limited by inefficient bacterial lysis and a low yield. To solve these problems, we compared the lysis efficiency of the wild-type protein E (EW) from phage ΦX174 and the screened mutant protein E (EM) in the Escherichia coli BL21(DE3) strain. The results show that the lysis efficiency mediated by protein EM was improved. The implementation of the pLysS plasmid allowed nearly 100% lysis efficiency, with a high initial cell density as high as OD600 = 2.0, which was higher compared to the commonly used BG preparation method. The results of Western blot analysis and immunofluorescence indicate that the expression level of protein EM was significantly higher than that of the non-pLysS plasmid. High-quality BGs were observed by SEM and TEM. To verify the applicability of this method in other bacteria, the T7 RNA polymerase expression system was successfully constructed in Salmonella enterica (S. Enterica, SE). A pET vector containing EM and pLysS were introduced to obtain high-quality SE ghosts which could provide efficient protection for humans and animals. This paper describes a novel and commonly used method to produce high-quality BGs on a large scale for the first time.

scholarly journals Large-Scale Synonymous Substitutions in Cucumber Mosaic Virus RNA 3 Facilitate Amino Acid Mutations in the Coat Protein

2018 ◽  
Vol 92 (22) ◽  
Author(s):  
Tomofumi Mochizuki ◽  
Rie Ohara ◽  
Marilyn J. Roossinck
Keyword(s):  
Amino Acid ◽  
Secondary Structure ◽  
Viral Genome ◽  
Large Scale ◽  
Viral Rna ◽  
Wild Type ◽  
Competitive Fitness ◽  
Content Type ◽  
Synonymous Substitutions ◽  
Cp Gene

ABSTRACTThe effect of large-scale synonymous substitutions in a small icosahedral, single-stranded RNA viral genome on virulence, viral titer, and protein evolution were analyzed. The coat protein (CP) gene of the Fny stain of cucumber mosaic virus (CMV) was modified. We created four CP mutants in which all the codons of nine amino acids in the 5′ or 3′ half of the CP gene were replaced by either the most frequently or the least frequently used synonymous codons in monocot plants. When the dicot host (Nicotiana benthamiana) was inoculated with these four CP mutants, viral RNA titers in uninoculated symptomatic leaves decreased, while all mutants eventually showed mosaic symptoms similar to those for the wild type. The codon adaptation index of these four CP mutants against dicot genes was similar to those of the wild-type CP gene, indicating that the reduction of viral RNA titer was due to deleterious changes of the secondary structure of RNAs 3 and 4. When two 5′ mutants were serially passaged inN. benthamiana, viral RNA titers were rapidly restored but competitive fitness remained decreased. Although no nucleic acid changes were observed in the passaged wild-type CMV, one to three amino acid changes were observed in the synonymously mutated CP of each passaged virus, which were involved in recovery of viral RNA titer of 5′ mutants. Thus, we demonstrated that deleterious effects of the large-scale synonymous substitutions in the RNA viral genome facilitated the rapid amino acid mutation(s) in the CP to restore the viral RNA titer.IMPORTANCERecently, it has been known that synonymous substitutions in RNA virus genes affect viral pathogenicity and competitive fitness by alteration of global or local RNA secondary structure of the viral genome. We confirmed that large-scale synonymous substitutions in the CP gene of CMV resulted in decreased viral RNA titer. Importantly, when viral evolution was stimulated by serial-passage inoculation, viral RNA titer was rapidly restored, concurrent with a few amino acid changes in the CP. This novel finding indicates that the deleterious effects of large-scale nucleic acid mutations on viral RNA secondary structure are readily tolerated by structural changes in the CP, demonstrating a novel part of the adaptive evolution of an RNA viral genome. In addition, our experimental system for serial inoculation of large-scale synonymous mutants could uncover a role for new amino acid residues in the viral protein that have not been observed in the wild-type virus strains.

scholarly journals Baculovirus Transregulator IE1 Requires a Dimeric Nuclear Localization Element for Nuclear Import and Promoter Activation

2002 ◽  
Vol 76 (18) ◽  
pp. 9505-9515 ◽  
Author(s):  
Victoria A. Olson ◽  
Justin A. Wetter ◽  
Paul D. Friesen
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Direct Interaction ◽  
Productive Infection ◽  
Homologous Region ◽  
Wild Type ◽  
Nuclear Entry ◽  
Early Protein ◽  
Biochemical Fractionation ◽  
Localization Element

ABSTRACT Immediate-early protein IE1 is a principal regulator of viral transcription and a contributor to origin-specific DNA replication of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Since these viral functions involve interaction of dimeric IE1 with palindromic homologous region (hr) enhancer-origin elements of the AcMNPV genome within the nucleus, it is presumed that proper nuclear transport of IE1 is essential for productive infection. To investigate the mechanisms of IE1 nuclear import, we analyzed the effect of site-directed mutations on IE1 subcellular distribution. As demonstrated by fluorescence microscopy and biochemical fractionation of plasmid-transfected cells, wild-type IE1 localized predominantly to the nucleus. Substitution or deletion of amino acid residues within a positively charged domain (residues 534 to 538) adjacent to IE1's oligomerization motif impaired nuclear import and caused loss of transactivation. Moreover, upon coexpression, these import-defective mutations prevented nuclear entry of wild-type IE1. In contrast, double-mutated IE1 defective for both nuclear import and dimerization failed to block nuclear entry or transactivation by wild-type IE1. Thus, import-defective IE1 dominantly interfered with wild-type IE1 by direct interaction and cytosolic trapping. Collectively, our data indicate that the small basic domain encompassing residues R537 and R538 constitutes a novel nuclear localization element that functions only upon IE1 dimerization. These findings support a model wherein IE1 oligomerizes within the cytosol as a prerequisite for nuclear entry and subsequent high-affinity interaction with the symmetrical binding sites comprising AcMNPV hr enhancer-origin elements.

Enzyme activity enhancement of chondroitinase ABC I from Proteus vulgaris by site-directed mutagenesis

RSC Advances ◽  
2015 ◽  
Vol 5 (93) ◽  
pp. 76040-76047 ◽  
Author(s):  
Zhenya Chen ◽  
Ye Li ◽  
Yue Feng ◽  
Liang Chen ◽  
Qipeng Yuan
Keyword(s):  
Active Site ◽  
Simulation Analysis ◽  
Docking Simulation ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Proteus Vulgaris ◽  
Molecular Docking Simulation ◽  
Activity Enhancement ◽  
First Time

Arg660 was found as a new active site and Asn795Ala and Trp818Ala mutants showed higher activities than the wild type based on molecular docking simulation analysis for the first time.

Abstract P283: Parkin Mediates Mitophagy in Cardioprotection

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Allen M Andres ◽  
Chengqun Huang ◽  
Eric P Ratliff ◽  
Genaro Hernandez ◽  
Pamela Lee ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Wild Type ◽  
Simulated Ischemia ◽  
Selective Targeting ◽  
Cardioprotective Effects ◽  
Mitochondrial Turnover ◽  
First Time

Autophagy-dependent mitochondrial turnover in response to cellular stress is necessary for maintaining cellular homeostasis. However, the mechanisms that govern the selective targeting of damaged mitochondria are poorly understood. Parkin, an E3 ubiquitin ligase, has been shown to be essential for the selective clearance of damaged mitochondria. Parkin is expressed in the heart, yet its function has not been investigated in the context of cardioprotection. We previously reported that autophagy is required for cardioprotection by ischemic preconditioning (IPC). In the present study, we used simulated ischemia in vitro and IPC in hearts (in vivo and ex vivo) to investigate the role of Parkin in mediating cardioprotection. In HL-1 cells, simulated ischemia induced Parkin translocation to mitochondria and mitochondrial elimination. Mitochondrial loss was blunted in Atg5-deficient cells, revealing the requirement for autophagy in mitochondrial elimination. Consistent with previous reports implicating p62/SQSTM1 in mitophagy, we found that downregulation of p62 attenuated mitophagy and exacerbated cell death in HL-1 cardiomyocytes subjected to simulated ischemia. While wild type mice showed p62 translocation to mitochondria after IPC, Parkin knockout mice exhibited attenuated translocation of p62 to mitochondria. Importantly, ablation of Parkin in mice abolished the cardioprotective effects of IPC. These results reveal for the first time the crucial role of Parkin and mitophagy in cardioprotection.

scholarly journals WT and A53T α-synuclein systems: Melting Diagram and its new interpretation

2019 ◽  
Author(s):  
M. Bokor ◽  
Á. Tantos ◽  
P. Tompa ◽  
K.-H. Han ◽  
K. Tompa
Keyword(s):  
Secondary Structure ◽  
Structural Disorder ◽  
Water Molecules ◽  
Hydration Water ◽  
Wild Type ◽  
Water Binding ◽  
Potential Barriers ◽  
Intrinsically Disordered ◽  
Mobile Water ◽  
Binding Interfaces

AbstractParkinson’s disease is connected with abnormal α-synuclein (αS) aggregation. Energetics of potential barriers governing motions of hydration water is examined. Information about the distributions and heights of potential barriers is gained by a thermodynamical approach. The ratios of the heterogeneous water-binding interfaces measure proteins’ structural disorder. All αS forms possess secondary structural elements though they are intrinsically disordered. Monomers are functional at the lowest potential barriers, where mobile hydration water exists, with monolayer coverage of mobile hydration. The αS monomer contains 33% secondary structure and is more compact than a random coil. A53T αS monomer has a more open structure than the wild type. Monomers realize all possible hydrogen bonds. Half of the mobile hydration water amount for monomers is missing in αS oligomers and αS amyloids. Oligomers are ordered by 66%. Mobile water molecules in the first hydration shell of amyloids are the weakest bound compared to other forms. Wild type and A53T amyloids show identical, low-level hydration, and are considered as disordered to 75%.Statement of SignificanceAggregation of α-synuclein into oligomers, amyloid fibrils is a hallmark of Parkinson’s disease. A thermodynamic approach provides information on the heterogeneity of protein-water bonds in the wild type and A53T mutant monomers, oligomers, amyloids. This information can be related to ratios of heterogeneous water-binding interfaces, which measure the proteins’ structural disorder. Both α-synuclein monomers are intrinsically disordered. The monomers nevertheless have 33% secondary structure. They are functional as long as mobile water molecules surround them. They realize every possible H-bonds with water. Oligomers are like globular proteins with 66% ordered structure. Amyloids are disordered to 75% and are poorly hydrated with loosely bound water. Their hydration is identical. Oligomers, amyloids have only half as much hydrating mobile water as monomers.

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