scholarly journals Crystal structure of cis-aconitate decarboxylase reveals the impact of naturally occurring human mutations on itaconate synthesis

2019 ◽  
Vol 116 (41) ◽  
pp. 20644-20654 ◽  
Author(s):  
Fangfang Chen ◽  
Peer Lukat ◽  
Azeem Ahmed Iqbal ◽  
Kyrill Saile ◽  
Volkhard Kaever ◽  
...  
Keyword(s):  
Itaconic Acid ◽  
Active Site ◽  
Disease Risk ◽  
Population Level ◽  
Therapeutic Interventions ◽  
Active Site Residues ◽  
Biotechnological Production ◽  
Naturally Occurring ◽  
The Impact

cis-Aconitate decarboxylase (CAD, also known as ACOD1 or Irg1) converts cis-aconitate to itaconate and plays central roles in linking innate immunity with metabolism and in the biotechnological production of itaconic acid by Aspergillus terreus. We have elucidated the crystal structures of human and murine CADs and compared their enzymological properties to CAD from A. terreus. Recombinant CAD is fully active in vitro without a cofactor. Murine CAD has the highest catalytic activity, whereas Aspergillus CAD is best adapted to a more acidic pH. CAD is not homologous to any known decarboxylase and appears to have evolved from prokaryotic enzymes that bind negatively charged substrates. CADs are homodimers, the active center is located in the interface between 2 distinct subdomains, and structural modeling revealed conservation in zebrafish and Aspergillus. We identified 8 active-site residues critical for CAD function and rare naturally occurring human mutations in the active site that abolished CAD activity, as well as a variant (Asn152Ser) that increased CAD activity and is common (allele frequency 20%) in African ethnicity. These results open the way for 1) assessing the potential impact of human CAD variants on disease risk at the population level, 2) developing therapeutic interventions to modify CAD activity, and 3) improving CAD efficiency for biotechnological production of itaconic acid.

scholarly journals Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 21-29 ◽  
Author(s):  
David R H Evans ◽  
Brian A Hemmings
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Protein Function ◽  
Mutational Analysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Active Site Residues ◽  
Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

scholarly journals ADP-Ribose-1"-Monophosphatase: a Conserved Coronavirus Enzyme That Is Dispensable for Viral Replication in Tissue Culture

2005 ◽  
Vol 79 (20) ◽  
pp. 12721-12731 ◽  
Author(s):  
Ákos Putics ◽  
Witold Filipowicz ◽  
Jonathan Hall ◽  
Alexander E. Gorbalenya ◽  
John Ziebuhr
Keyword(s):  
Active Site ◽  
Biological Significance ◽  
Virus Titer ◽  
Site Directed Mutagenesis ◽  
Replicase Gene ◽  
Active Site Residues ◽  
Nonstructural Proteins ◽  
Trna Splicing

ABSTRACT Replication of the ∼30-kb plus-strand RNA genome of coronaviruses and synthesis of an extensive set of subgenome-length RNAs are mediated by the replicase-transcriptase, a membrane-bound protein complex containing several cellular proteins and up to 16 viral nonstructural proteins (nsps) with multiple enzymatic activities, including protease, polymerase, helicase, methyltransferase, and RNase activities. To get further insight into the replicase gene-encoded functions, we characterized the coronavirus X domain, which is part of nsp3 and has been predicted to be an ADP-ribose-1"-monophosphate (Appr-1"-p) processing enzyme. Bacterially expressed forms of human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome-coronavirus X domains were shown to dephosphorylate Appr-1"-p, a side product of cellular tRNA splicing, to ADP-ribose in a highly specific manner. The enzyme had no detectable activity on several other nucleoside phosphates. Guided by the crystal structure of AF1521, an X domain homolog from Archaeoglobus fulgidus, potential active-site residues of the HCoV-229E X domain were targeted by site-directed mutagenesis. The data suggest that the HCoV-229E replicase polyprotein residues, Asn 1302, Asn 1305, His 1310, Gly 1312, and Gly 1313, are part of the enzyme's active site. Characterization of an Appr-1"-pase-deficient HCoV-229E mutant revealed no significant effects on viral RNA synthesis and virus titer, and no reversion to the wild-type sequence was observed when the mutant virus was passaged in cell culture. The apparent dispensability of the conserved X domain activity in vitro indicates that coronavirus replicase polyproteins have evolved to include nonessential functions. The biological significance of the novel enzymatic activity in vivo remains to be investigated.

scholarly journals Exploiting correlated molecular-dynamics networks to counteract enzyme activity–stability trade-off

2018 ◽  
Vol 115 (52) ◽  
pp. E12192-E12200 ◽  
Author(s):  
Haoran Yu ◽  
Paul A. Dalby
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Active Site ◽  
Dynamic Networks ◽  
Aromatic Aldehydes ◽  
Active Site Residues ◽  
Trade Off ◽  
Highly Correlated ◽  
Dynamics Simulations ◽  
The Impact

The directed evolution of enzymes for improved activity or substrate specificity commonly leads to a trade-off in stability. We have identified an activity–stability trade-off and a loss in unfolding cooperativity for a variant (3M) of Escherichia coli transketolase (TK) engineered to accept aromatic substrates. Molecular dynamics simulations of 3M revealed increased flexibility in several interconnected active-site regions that also form part of the dimer interface. Mutating the newly flexible active-site residues to regain stability risked losing the new activity. We hypothesized that stabilizing mutations could be targeted to residues outside of the active site, whose dynamics were correlated with the newly flexible active-site residues. We previously stabilized WT TK by targeting mutations to highly flexible regions. These regions were much less flexible in 3M and would not have been selected a priori as targets using the same strategy based on flexibility alone. However, their dynamics were highly correlated with the newly flexible active-site regions of 3M. Introducing the previous mutations into 3M reestablished the WT level of stability and unfolding cooperativity, giving a 10.8-fold improved half-life at 55 °C, and increased midpoint and aggregation onset temperatures by 3 °C and 4.3 °C, respectively. Even the activity toward aromatic aldehydes increased up to threefold. Molecular dynamics simulations confirmed that the mutations rigidified the active-site via the correlated network. This work provides insights into the impact of rigidifying mutations within highly correlated dynamic networks that could also be useful for developing improved computational protein engineering strategies.

scholarly journals Self-Directedness and Cooperativeness, Psychosocial Dysfunction and Suffering in ESSENCE

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Danilo Garcia ◽  
Henrik Anckarsäter ◽  
Sebastian Lundström
Keyword(s):  
Developmental Coordination Disorder ◽  
Population Level ◽  
Autism Spectrum ◽  
Severe Form ◽  
Therapeutic Interventions ◽  
Psychosocial Dysfunction ◽  
Hyperactivity Disorder ◽  
Character Inventory ◽  
Novel Method ◽  
The Impact

Background. The acronym ESSENCE (Early Symptomatic Syndromes Eliciting Neurodevelopmental Clinical Examinations) highlights that children seeking clinical treatment are often multiply impaired, thus requiring treatment from several specialties. The aim was to map and relate, on a population level, ESSENCE to two salient predictors of health and adaptation to adversities, namely, Self-Directedness and Cooperativeness and also to dysfunction and suffering.Methods. Participants were twins(N=1892)aged 9 or 12 whose parents were interviewed with the Autism-Tics, ADHD and other Comorbidities inventory (A-TAC), and the Junior Temperament and Character Inventory (J-TCI). The A-TAC was first used to discern four ESSENCE-related screening diagnoses: autism spectrum disorders, attention deficit hyperactivity disorder, learning disabilities, and developmental coordination disorder; second, to quantify dysfunction and suffering in important social areas.Results. ESSENCE symptoms were continuously and categorically associated with deficiency in Self-Directedness and Cooperativeness and higher ratings of dysfunction and suffering. The impact of ESSENCE symptoms on these measures of mental health was found in a milder form in about 16% of all children and in a severe form in about 2%.Conclusion. Therapeutic interventions focusing on Self-Directedness and Cooperativeness might provide a novel method for child psychiatry in its approach to ESSENCE.

scholarly journals Mutational Analysis of Active Site Residues Essential for Sensing of Organic Hydroperoxides by Bacillus subtilis OhrR

2007 ◽  
Vol 189 (19) ◽  
pp. 7069-7076 ◽  
Author(s):  
Sumarin Soonsanga ◽  
Mayuree Fuangthong ◽  
John D. Helmann
Keyword(s):  
Hydrogen Bond ◽  
Bacillus Subtilis ◽  
Conformational Changes ◽  
Active Site ◽  
Mutational Analysis ◽  
High Sensitivity ◽  
Oxidative Modification ◽  
Active Site Residues

ABSTRACT Bacillus subtilis OhrR is the prototype for the one-Cys family of organic peroxide-sensing regulatory proteins. Mutational analyses indicate that the high sensitivity of the active site cysteine (C15) to peroxidation requires three Tyr residues. Y29 and Y40 from the opposing subunit of the functional dimer hydrogen bond with the reactive Cys thiolate, and substitutions at these positions reduce or eliminate the ability of OhrR to respond to organic peroxides. Y19 is also critical for peroxide sensing, and the Ala substitution mutant (OhrR Y19A) is less susceptible to oxidation at the active site C15 in vivo. The Y19A protein also displays decreased sensitivity to peroxide-mediated oxidation in vitro. Y19 is in van der Waals contact with two residues critical for protein function, F16 and R23. The latter residue makes critical contact with the DNA backbone in the OhrR-operator complex. These results indicate that the high sensitivity of the OhrR C15 residue to oxidation requires interactions with the opposed Tyr residues. Oxidative modification of C15 likely disrupts the C15-Y29′-Y40′ hydrogen bond network and thereby initiates conformational changes that reduce the ability of OhrR to bind to its operator site.

scholarly journals Lethal Factor Active-Site Mutations Affect Catalytic Activity In Vitro

1998 ◽  
Vol 66 (5) ◽  
pp. 2374-2378 ◽  
Author(s):  
S. E. Hammond ◽  
P. C. Hanna
Keyword(s):  
Active Site ◽  
Lethal Factor ◽  
Peptide Sequencing ◽  
The Novel ◽  
Active Site Residues ◽  
Zinc Metalloprotease ◽  
Proteolytic Activities ◽  
Substitution Mutations ◽  
Catalytic Functions

ABSTRACT The lethal factor (LF) protein of Bacillus anthracislethal toxin contains the thermolysin-like active-site and zinc-binding consensus motif HEXXH (K. R. Klimpel, N. Arora, and S. H. Leppla, Mol. Microbiol. 13:1093–1100, 1994). LF is hypothesized to act as a Zn2+ metalloprotease in the cytoplasm of macrophages, but no proteolytic activities have been previously shown on any target substrate. Here, synthetic peptides are hydrolyzed by LF in vitro. Mass spectroscopy and peptide sequencing of isolated cleavage products separated by reverse-phase high-pressure liquid chromatography indicate that LF seems to prefer proline-containing substrates. Substitution mutations within the consensus active-site residues completely abolish all in vitro catalytic functions, as does addition of 1,10-phenanthroline, EDTA, and certain amino acid hydroxamates, including the novel zinc metalloprotease inhibitor ZINCOV. In contrast, the protease inhibitors bestatin and lysine CMK, previously shown to block LF activity on macrophages, did not block LF activity in vitro. These data provide the first direct evidence that LF may act as an endopeptidase.

scholarly journals Enantioseparation, in vitro testing, and structural characterization of triple-binding reactivators of organophosphate-inhibited cholinesterases

2020 ◽  
Vol 477 (15) ◽  
pp. 2771-2790 ◽  
Author(s):  
Nikola Maraković ◽  
Anamarija Knežević ◽  
Igor Rončević ◽  
Xavier Brazzolotto ◽  
Zrinka Kovarik ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Proton Transfer ◽  
Structural Characterization ◽  
Active Site ◽  
In Vitro Testing ◽  
Active Site Residues

The enantiomers of racemic 2-hydroxyimino-N-(azidophenylpropyl)acetamide-derived triple-binding oxime reactivators were separated, and tested for inhibition and reactivation of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibited with tabun (GA), cyclosarin (GF), sarin (GB), and VX. Both enzymes showed the greatest affinity toward the methylimidazole derivative (III) of 2-hydroxyimino-N-(azidophenylpropyl)acetamide (I). The crystal structure was determined for the complex of oxime III within human BChE, confirming that all three binding groups interacted with active site residues. In the case of BChE inhibited by GF, oximes I (kr = 207 M−1 min−1) and III (kr = 213 M−1 min−1) showed better reactivation efficiency than the reference oxime 2-PAM. Finally, the key mechanistic steps in the reactivation of GF-inhibited BChE with oxime III were modeled using the PM7R6 method, stressing the importance of proton transfer from Nε of His438 to Oγ of Ser203 for achieving successful reactivation.

scholarly journals Exploring thermosonication as non-chemical disinfection technology for strawberries

2021 ◽  
Author(s):  
Iolanda Nicolau-Lapeña ◽  
Ingrid Aguiló-Aguayo ◽  
Marina Anguera ◽  
Inmaculada Viñas ◽  
Maribel Abadias
Keyword(s):  
Fruit Quality ◽  
Listeria Innocua ◽  
Naturally Occurring ◽  
Effects Of Temperature ◽  
Yeasts And Molds ◽  
The Impact ◽  
Detrimental Impact ◽  
Chemical Disinfection ◽  
Main Factor

AbstractThe scope of this work was to study the efficacy of the combination of sonication at 35 or 130 kHz with three temperature treatments: 20, 50 and 55 ºC, on the population of artificially inoculated Listeria innocua in strawberries, and on their overall quality. Prior in vitro results showed that temperature was the main factor in decreasing L. innocua population: a maximum of 3.8 log reductions was obtained with sonication at 130 kHz and 55 ºC for 15 min Treatments combining—or not—sonication at 130 kHz with mild temperatures (50 and 55 ºC) for 5 or 10 min were able to decrease about 3 log units of artificially inoculated L. innocua in strawberries and about 2 log units of total aerobic mesophilic and yeasts and molds populations naturally occurring in strawberries. Thermosonication treatments did not exert a detrimental impact on fruit quality, except for those at the higher temperatures and times, which caused a change in color to more purplish and a little softening of the strawberries, which were proposed to be assessed for further processing other than fresh commercialization. Overall, the impact of sonication in fresh strawberries needs to be further investigated to find the adequate conditions to enhance the effects of temperature itself.

scholarly journals Utility of polygenic embryo screening for disease depends on the selection strategy

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Todd Lencz ◽  
Daniel Backenroth ◽  
Einat Granot-Hershkovitz ◽  
Adam Green ◽  
Kyle Gettler ◽  
...  
Keyword(s):  
Complex Disease ◽  
Disease Risk ◽  
Threshold Model ◽  
Absolute Risk ◽  
Risk Scores ◽  
Selection Strategy ◽  
Case Control Studies ◽  
Disease Case ◽  
The Impact

Polygenic risk scores (PRSs) have been offered since 2019 to screen in vitro fertilization embryos for genetic liability to adult diseases, despite a lack of comprehensive modeling of expected outcomes. Here we predict, based on the liability threshold model, the expected reduction in complex disease risk following polygenic embryo screening for a single disease. A strong determinant of the potential utility of such screening is the selection strategy, a factor that has not been previously studied. When only embryos with a very high PRS are excluded, the achieved risk reduction is minimal. In contrast, selecting the embryo with the lowest PRS can lead to substantial relative risk reductions, given a sufficient number of viable embryos. We systematically examine the impact of several factors on the utility of screening, including: variance explained by the PRS, number of embryos, disease prevalence, parental PRSs, and parental disease status. We consider both relative and absolute risk reductions, as well as population-averaged and per-couple risk reductions, and also examine the risk of pleiotropic effects. Finally, we confirm our theoretical predictions by simulating ‘virtual’ couples and offspring based on real genomes from schizophrenia and Crohn’s disease case-control studies. We discuss the assumptions and limitations of our model, as well as the potential emerging ethical concerns.

scholarly journals Structures of Arabidopsis thaliana oxygen-sensing plant cysteine oxidases 4 and 5 enable targeted manipulation of their activity

2020 ◽  
Vol 117 (37) ◽  
pp. 23140-23147 ◽  
Author(s):  
Mark D. White ◽  
Laura Dalle Carbonare ◽  
Mikel Lavilla Puerta ◽  
Sergio Iacopino ◽  
Martin Edwards ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Active Site ◽  
Higher Plants ◽  
Structural Information ◽  
Oxygen Sensing ◽  
Hypoxia Tolerance ◽  
Central Metal ◽  
Active Site Residues

In higher plants, molecular responses to exogenous hypoxia are driven by group VII ethylene response factors (ERF-VIIs). These transcriptional regulators accumulate in the nucleus under hypoxia to activate anaerobic genes but are destabilized in normoxic conditions through the action of oxygen-sensing plant cysteine oxidases (PCOs). The PCOs catalyze the reaction of oxygen with the conserved N-terminal cysteine of ERF-VIIs to form cysteine sulfinic acid, triggering degradation via the Cys/Arg branch of the N-degron pathway. The PCOs are therefore a vital component of the plant oxygen signaling system, connecting environmental stimulus with cellular and physiological response. Rational manipulation of PCO activity could regulate ERF-VII levels and improve flood tolerance, but requires detailed structural information. We report crystal structures of the constitutively expressed PCO4 and PCO5 from Arabidopsis thaliana to 1.24 and 1.91 Å resolution, respectively. The structures reveal that the PCOs comprise a cupin-like scaffold, which supports a central metal cofactor coordinated by three histidines. While this overall structure is consistent with other thiol dioxygenases, closer inspection of the active site indicates that other catalytic features are not conserved, suggesting that the PCOs may use divergent mechanisms to oxidize their substrates. Conservative substitution of two active site residues had dramatic effects on PCO4 function both in vitro and in vivo, through yeast and plant complementation assays. Collectively, our data identify key structural elements that are required for PCO activity and provide a platform for engineering crops with improved hypoxia tolerance.

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