Coupled structural transitions enable highly cooperative regulation of human CTPS2 filaments

Mapping Intimacies ◽

10.1101/770594 ◽

2019 ◽

Author(s):

Eric M. Lynch ◽

Justin M. Kollman

Keyword(s):

Enzyme Activity ◽

Active Sites ◽

Large Scale ◽

Conformational State ◽

Ctp Synthase ◽

Widespread Phenomenon ◽

And Control ◽

Allosteric Regulators ◽

Low Activity

Many enzymes assemble into defined oligomers, providing a mechanism for cooperatively regulating enzyme activity. Recent studies in tissues, cells, and in vitro have described a mode of regulation in which enzyme activity is modulated by polymerization into large-scale filaments1–5. Enzyme polymerization is often driven by binding to substrates, products, or allosteric regulators, and tunes enzyme activity by locking the enzyme in high or low activity states1–5. Here, we describe a unique, ultrasensitive form of polymerization-based regulation employed by human CTP synthase 2 (CTPS2). High-resolution cryoEM structures of active and inhibited CTPS2 filaments reveal the molecular basis of this regulation. Rather than selectively stabilizing a single conformational state, CTPS2 filaments dynamically switch between active and inactive filament forms in response to changes in substrate and product levels. Linking the conformational state of many CTPS2 subunits in a filament results in highly cooperative regulation, greatly exceeding the limits of cooperativity for the CTPS2 tetramer alone. The structures also reveal a link between conformational state and control of ammonia channeling between the enzyme’s two active sites. This filament-based mechanism of enhanced cooperativity demonstrates how the widespread phenomenon of enzyme polymerization can be adapted to achieve different regulatory outcomes.

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Biochemical and genetic analysis of Ecm14, a conserved fungal pseudopeptidase

10.21203/rs.3.rs-52558/v2 ◽

2020 ◽

Author(s):

R Christian McDonald ◽

Matthew J Schott ◽

Temitope A Idowu ◽

Peter J Lyons

Keyword(s):

Enzyme Activity ◽

Large Scale ◽

Structure And Function ◽

Activation Mechanism ◽

Marker Genes ◽

Synthetic Lethal ◽

Fungal Process ◽

And Function

Abstract Background. Like most major enzyme families, the M14 family of metallocarboxypeptidases (MCPs) contains a number of pseudoenzymes predicted to lack enzyme activity and with poorly characterized molecular function. The genome of the yeast Saccharomyces cerevisiae encodes one member of the M14 MCP family, a pseudoenzyme named Ecm14 proposed to function in the extracellular matrix. In order to better understand the function of such pseudoenzymes, we studied the structure and function of Ecm14 in S. cerevisiae. Results. A phylogenetic analysis of Ecm14 in fungi found it to be conserved throughout the ascomycete phylum, with a group of related pseudoenzymes found in basidiomycetes. To investigate the structure and function of this conserved protein, His6-tagged Ecm14 was overexpressed in Sf9 cells and purified. The prodomain of Ecm14 was cleaved in vivo and in vitro by endopeptidases, suggesting an activation mechanism; however, no activity was detectable using standard carboxypeptidase substrates. In order to determine the function of Ecm14 using an unbiased screen, we undertook a synthetic lethal assay. Upon screening approximately 27,000 yeast colonies, twenty-two putative synthetic lethal clones were identified. Further analysis showed many to be synthetic lethal with auxotrophic marker genes and requiring multiple mutations, suggesting that there are few, if any, single S. cerevisiae genes that present synthetic lethal interactions with ecm14Δ. Conclusions. We show in this study that Ecm14, although lacking detectable enzyme activity, is a conserved carboxypeptidase-like protein that is secreted from cells and is processed to a mature form by the action of an endopeptidase. Our study and datasets from other recent large-scale screens suggest a role for Ecm14 in processes such as vesicle-mediated transport and aggregate invasion, a fungal process that has been selected against in modern laboratory strains of S. cerevisiae.

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Common regulatory control of CTP synthase enzyme activity and filament formation

Molecular Biology of the Cell ◽

10.1091/mbc.e14-04-0912 ◽

2014 ◽

Vol 25 (15) ◽

pp. 2282-2290 ◽

Cited By ~ 54

Author(s):

Chalongrat Noree ◽

Elena Monfort ◽

Andrew K. Shiau ◽

James E. Wilhelm

Keyword(s):

Enzyme Activity ◽

Feedback Inhibition ◽

Regulatory Control ◽

Filament Formation ◽

Filament Structure ◽

Ctp Synthase ◽

Multiple Regions ◽

Ideal Model System ◽

Widespread Phenomenon ◽

Regulatory Sites

The ability of enzymes to assemble into visible supramolecular complexes is a widespread phenomenon. Such complexes have been hypothesized to play a number of roles; however, little is known about how the regulation of enzyme activity is coupled to the assembly/disassembly of these cellular structures. CTP synthase is an ideal model system for addressing this question because its activity is regulated via multiple mechanisms and its filament-forming ability is evolutionarily conserved. Our structure–function studies of CTP synthase in Saccharomyces cerevisiae reveal that destabilization of the active tetrameric form of the enzyme increases filament formation, suggesting that the filaments comprise inactive CTP synthase dimers. Furthermore, the sites responsible for feedback inhibition and allosteric activation control filament length, implying that multiple regions of the enzyme can influence filament structure. In contrast, blocking catalysis without disrupting the regulatory sites of the enzyme does not affect filament formation or length. Together our results argue that the regulatory sites that control CTP synthase function, but not enzymatic activity per se, are critical for controlling filament assembly. We predict that the ability of enzymes to form supramolecular structures in general is closely coupled to the mechanisms that regulate their activity.

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Fe-Based Single-Atom Nanozyme with Superior Peroxidase-Mimicking Activity for Enhanced Ultrasensitive Biosensing

Journal of Nanoscience and Nanotechnology ◽

10.1166/jnn.2021.19533 ◽

2021 ◽

Vol 21 (12) ◽

pp. 6126-6134

Author(s):

Lili Chi ◽

Yuetong Zhang ◽

Yusheng Hua ◽

Qiqi Xu ◽

Mingzhu Lv ◽

...

Keyword(s):

Active Sites ◽

Large Scale ◽

Low Cost ◽

Single Atom ◽

Specificity And Sensitivity ◽

Large Scale Preparation ◽

Species Formation ◽

Biomedical Diagnosis ◽

Scale Preparation

Nanomaterials with intrinsic enzyme-mimicking characteristics, refered to as nanozymes, have become a hot research topic owing to their unique advantages of comparative low cost, high stability and large-scale preparation. Among them, Single-atom nanozymes (SAzymes), as novel nanozymes with abundant atomically dispersed active sites, have caused specific attention in the development of nanozymes for their remarkable catalytic activities, maximum atomic utilization and excellent selectivity, the homogeneous catalytic sites and clear catalytic mechanisms. Herein, a novel single-atom nanozyme based on Fe(III)-doped polydiaminopyridine nanofusiforms (Fe-PDAP SAzyme) was successfully proposed via facile oxidation polymerization strategy. With well-defined coordination structure and abundant Fe-Nx active sites similar to natural metalloproteases, the Fe-PDAP SAzyme exhibits superior peroxidase-like activity by efficiently decomposing H2O2 for hydroxyl radical (.OH) species formation. Based on their superior peroxidase-like activity, colorimetric biosensing of H2O2 and glucose in vitro was performed by using a typical 3,3,5,5-tetramethylbenzidine through a multienzyme biocatalytic cascade platform, exhibiting the superior specificity and sensitivity. This work not only provides a novel promising SAzyme-based biosensor but also paves an avenue for evaluating enzyme activity and broadens the application of other nanozyme-based biosensors in the fields of biomedical diagnosis.

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Cyclic AMP phosphodiesterase activity in the hearts of trained rats

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y83-152 ◽

1983 ◽

Vol 61 (9) ◽

pp. 1017-1024

Author(s):

Warren K. Palmer ◽

Sylvia Doukas

Keyword(s):

Enzyme Activity ◽

Cyclic Amp ◽

Total Concentration ◽

Chelating Agent ◽

Phosphodiesterase Activity ◽

Cyclic Amp Phosphodiesterase ◽

Rat Hearts ◽

Elevated Activity ◽

And Control

Running exercise trained rats at either 60 or 76% of their [Formula: see text] caused myocardial cyclic AMP phosphodiesterase (PDE) activity to be increased above control levels for at least 24 h following work. Neither training nor the exercise had any effect on the total concentration of calmodulin in heart tissues. The affinity of PDE for cyclic AMP was not changed by the exercise or training. The chelating agent, EGTA, had the same influence on PDE activity regardless of whether it was present in assays of control or exercised heart extract. Km and EGTA results suggest that calcium-bound calmodulin does not account for the higher PDE activity in the hearts of exercised rats. Supernatants from hearts homogenized in the presence of charcoal, to remove nucleotides from the extract, did not eliminate the exercise-associated increase in PDE activity. These results suggest that the elevated activity was not caused by an in vitro nucleotide activation. Preincubation of the enzyme from exercised and control rat hearts with snake venom activated PDE when assays were performed with the low concentration of cyclic AMP (1 μM). Moreover, the activity reached in the extract of exercisers (23.3 pmol∙100 μL−1∙min−1) was significantly greater than the activity found in control hearts (17.59 pmol∙100 μL−1∙min−1). Exercise increases PDE activity in the myocardium of trained rats. The results presented suggest that the increased PDE activity resulting from exercise is not dependent upon exercise intensity when the work is in excess of 60% of [Formula: see text]. In addition, the data obtained using indirect probes suggest that the increased enzyme activity was not caused by metabolites, endogenous nucleotides, or calmodulin changes in the hearts of exercised animals.

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Large scale meta-analysis of preclinical toxicity data for target characterisation and hypotheses generation

PLoS ONE ◽

10.1371/journal.pone.0252533 ◽

2021 ◽

Vol 16 (6) ◽

pp. e0252533

Author(s):

Jordi Munoz-Muriedas

Keyword(s):

Animal Studies ◽

Large Scale ◽

Meta Analysis ◽

Preclinical Studies ◽

Technological Advances ◽

Consistent Manner ◽

Critical Components ◽

Meta Analyses ◽

And Control

Recent technological advances in the field of big data have increased our capabilities to query large databases and combine information from different domains and disciplines. In the area of preclinical studies, initiatives like SEND (Standard for Exchange of Nonclinical Data) will also contribute to collect and present nonclinical data in a consistent manner and increase analytical possibilities. With facilitated access to preclinical data and improvements in analytical algorithms there will surely be an expectation for organisations to ensure all the historical data available to them is leveraged to build new hypotheses. These kinds of analyses may soon become as important as the animal studies themselves, in addition to being critical components to achieve objectives aligned with 3Rs. This article proposes the application of meta-analyses at large scale in corporate databases as a tool to exploit data from both preclinical studies and in vitro pharmacological activity assays to identify associations between targets and tissues that can be used as seeds for the development of causal hypotheses to characterise of targets. A total of 833 in-house preclinical toxicity studies relating to 416 compounds reported to be active (pXC50 ≥ 5.5) against a panel of 96 selected targets of interest for potential off-target non desired effects were meta-analysed, aggregating the data in tissue–target pairs. The primary outcome was the odds ratio (OR) of the number of animals with observed events (any morphology, any severity) in treated and control groups in the tissue analysed. This led to a total of 2139 meta-analyses producing a total of 364 statistically significant associations (random effects model), 121 after adjusting by multiple comparison bias. The results show the utility of the proposed approach to leverage historical corporate data and may offer a vehicle for researchers to share, aggregate and analyse their preclinical toxicological data in precompetitive environments.

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Biochemical and genetic analysis of Ecm14, a conserved fungal pseudopeptidase

BMC Molecular and Cell Biology ◽

10.1186/s12860-020-00330-w ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

R. Christian McDonald ◽

Matthew J. Schott ◽

Temitope A. Idowu ◽

Peter J. Lyons

Keyword(s):

Enzyme Activity ◽

Large Scale ◽

Structure And Function ◽

Activation Mechanism ◽

Marker Genes ◽

Synthetic Lethal ◽

Fungal Process ◽

And Function

Abstract Background Like most major enzyme families, the M14 family of metallocarboxypeptidases (MCPs) contains a number of pseudoenzymes predicted to lack enzyme activity and with poorly characterized molecular function. The genome of the yeast Saccharomyces cerevisiae encodes one member of the M14 MCP family, a pseudoenzyme named Ecm14 proposed to function in the extracellular matrix. In order to better understand the function of such pseudoenzymes, we studied the structure and function of Ecm14 in S. cerevisiae. Results A phylogenetic analysis of Ecm14 in fungi found it to be conserved throughout the ascomycete phylum, with a group of related pseudoenzymes found in basidiomycetes. To investigate the structure and function of this conserved protein, His6-tagged Ecm14 was overexpressed in Sf9 cells and purified. The prodomain of Ecm14 was cleaved in vivo and in vitro by endopeptidases, suggesting an activation mechanism; however, no activity was detectable using standard carboxypeptidase substrates. In order to determine the function of Ecm14 using an unbiased screen, we undertook a synthetic lethal assay. Upon screening approximately 27,000 yeast colonies, twenty-two putative synthetic lethal clones were identified. Further analysis showed many to be synthetic lethal with auxotrophic marker genes and requiring multiple mutations, suggesting that there are few, if any, single S. cerevisiae genes that present synthetic lethal interactions with ecm14Δ. Conclusions We show in this study that Ecm14, although lacking detectable enzyme activity, is a conserved carboxypeptidase-like protein that is secreted from cells and is processed to a mature form by the action of an endopeptidase. Our study and datasets from other recent large-scale screens suggest a role for Ecm14 in processes such as vesicle-mediated transport and aggregate invasion, a fungal process that has been selected against in modern laboratory strains of S. cerevisiae.

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Functional and computational identification of a rescue mutation near the active site of an mRNA methyltransferase

Scientific Reports ◽

10.1038/s41598-020-79026-2 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Pierre-Yves Colin ◽

Paul A. Dalby

Keyword(s):

Active Site ◽

Active Sites ◽

Large Scale ◽

Interaction Network ◽

Catalytic Efficiency ◽

Transferase Activity ◽

Viral Mrna ◽

In Vitro Production ◽

Cell Immunity

AbstractRNA-based drugs are an emerging class of therapeutics combining the immense potential of DNA gene-therapy with the absence of genome integration-associated risks. While the synthesis of such molecules is feasible, large scale in vitro production of humanised mRNA remains a biochemical and economical challenge. Human mRNAs possess two post-transcriptional modifications at their 5′ end: an inverted methylated guanosine and a unique 2′O-methylation on the ribose of the penultimate nucleotide. One strategy to precisely methylate the 2′ oxygen is to use viral mRNA methyltransferases that have evolved to escape the host’s cell immunity response following virus infection. However, these enzymes are ill-adapted to industrial processes and suffer from low turnovers. We have investigated the effects of homologous and orthologous active-site mutations on both stability and transferase activity, and identified new functional motifs in the interaction network surrounding the catalytic lysine. Our findings suggest that despite their low catalytic efficiency, the active-sites of viral mRNA methyltransferases have low mutational plasticity, while mutations in a defined third shell around the active site have strong effects on folding, stability and activity in the variant enzymes, mostly via network-mediated effects.

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Analysis of the interaction between human steroid 21-hydroxylase and various monoclonal antibodies using comparative structural modelling

Acta Endocrinologica ◽

10.1530/eje.1.02044 ◽

2005 ◽

Vol 153 (6) ◽

pp. 949-961 ◽

Cited By ~ 3

Author(s):

Ricardo Núñez Miguel ◽

Shu Chen ◽

Laleh Nikfarjam ◽

Shiro Kominami ◽

Byron Carpenter ◽

...

Keyword(s):

Enzyme Activity ◽

Monoclonal Antibodies ◽

Protein Binding ◽

Active Sites ◽

Structural Models ◽

Structural Modelling ◽

Redox Protein ◽

Comparative Model ◽

And Control ◽

The Relationship

Objective: To study the interaction between human steroid 21-hydroxylase (21-OH) and monoclonal antibodies (MAbs) to 21-OH directed to 3 different epitopes recognised by 21-OH autoantibodies characteristic of autoimmune Addison’s disease. Design: Build comparative structural models of 21-OH, 21-OH MAbs and complexes of 21-OH–21-OH MAbs and study the effects of 21-OH MAbs on 21-OH enzyme activity. Then, analyse the relationship between sites important for binding of 21-OH MAbs and 21-OH autoantibodies and sites important for 21-OH enzyme activity. Methods: Variable (V) regions of 21-OH MAbs (M21-OH1, M21-OH3, M21-OH5) were sequenced and models of the MAbs built using structures of antibodies in the database as templates. A comparative model of 21-OH was built using the crystal structure of rabbit cytochrome p450 2c5/3LVdH as template. 21-OH enzyme activity was measured in terms of conversion of [3H]progesterone to deoxycorticosterone and the effect of purified MAb IgGs on 21-OH enzyme activity was assessed. Results: M21-OH1, M21-OH3 and control MAb had no effect on 21-OH enzyme activity with 88.8% ± 24% (n = 6), 86.7% ± 7.6% (n = 6) and 86.5% ± 10.6% (n = 6) of activity remaining in the presence of the respective IgGs. This was consistent with the epitopes for M21-OH1 and M21-OH3 being located distant from 21-OH enzyme active sites in our 21-OH model. The epitope for M21-OH5 which inhibited 21-OH enzyme activity (48.5 ± 8.3% activity remaining; P < 0.001 compared with control MAb IgG) was found close to the redox protein binding site in our 21-OH model. Conclusions: A comparative model of 21-OH has been produced. Analysis of experimental data in the context of the model suggests that M21-OH5 inhibits 21-OH enzyme activity through interference with redox protein binding.

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Biochemical and genetic analysis of Ecm14, a conserved fungal pseudopeptidase

10.21203/rs.3.rs-52558/v1 ◽

2020 ◽

Author(s):

R Christian McDonald ◽

Matthew J Schott ◽

Temitope A Idowu ◽

Peter J Lyons

Keyword(s):

Enzyme Activity ◽

Large Scale ◽

Structure And Function ◽

Activation Mechanism ◽

Marker Genes ◽

Synthetic Lethal ◽

Fungal Process ◽

And Function

Abstract Background. Like most major enzyme families, the M14 family of metallocarboxypeptidases (MCPs) contains a number of pseudoenzymes predicted to lack enzyme activity and with poorly characterized molecular function. The genome of the yeast S. cerevisiae encodes one member of the M14 MCP family, a pseudoenzyme named Ecm14 proposed to function in the extracellular matrix. In order to better understand the function of such pseudoenzymes, we studied the structure and function of Ecm14 in S. cerevisiae. Results. A phylogenetic analysis of Ecm14 in fungi found it to be conserved throughout the ascomycete phylum, with a group of related pseudoenzymes found in basidiomycetes. To investigate the structure and function of this conserved protein, his6-tagged Ecm14 was overexpressed in Sf9 cells and purified. The prodomain of Ecm14 was cleaved in vivo and in vitro by endopeptidases, suggesting an activation mechanism; however, no activity was detectable using standard carboxypeptidase substrates. In order to determine the function of Ecm14 using an unbiased screen, we undertook a synthetic lethal assay. Upon screening approximately 27,000 yeast colonies, twenty-two putative synthetic lethal clones were identified. Further analysis showed many to be synthetic lethal with auxotrophic marker genes and requiring multiple mutations, suggesting that there are few, if any, single S. cerevisiae genes that present synthetic lethal interactions with ecm14. Conclusions. We show in this study that Ecm14, although lacking detectable enzyme activity, is a conserved carboxypeptidase-like protein that is secreted from cells and is processed to a mature form by the action of an endopeptidase. Our study and datasets from other recent large-scale screens suggest a role for Ecm14 in processes such as vesicle-mediated transport and aggregate invasion, a fungal process that has been selected against in modern laboratory strains of S. cerevisiae.

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