scholarly journals Targeting Methyltransferases in Human Pathogenic Bacteria: Insights into Thymidylate Synthase (TS) and Flavin-Dependent TS (FDTS)

Molecules ◽  
2019 ◽  
Vol 24 (8) ◽  
pp. 1638 ◽  
Author(s):  
Cecilia Pozzi ◽  
Ludovica Lopresti ◽  
Giusy Tassone ◽  
Stefano Mangani
Keyword(s):  
Thymidylate Synthase ◽  
Pathogenic Bacteria ◽  
De Novo ◽  
Functional Characterization ◽  
Mechanisms Of Action ◽  
Human Pathogenic Bacteria ◽  
Thymidylate Synthases ◽  
Open Debate ◽  
Definition Of

In cells, thymidylate synthases provide the only de novo source of 2′-deoxythymidine-5′-monophosphate (dTMP), required for DNA synthesis. The activity of these enzymes is pivotal for cell survival and proliferation. Two main families of thymidylate synthases have been identified in bacteria, folate-dependent thymidylate synthase (TS) and flavin-dependent TS (FDTS). TS and FDTS are highly divergent enzymes, characterized by exclusive catalytic mechanisms, involving different sets of cofactors. TS and FDTS mechanisms of action have been recently revised, providing new perspectives for the development of antibacterial drugs targeting these enzymes. Nonetheless, some catalytic details still remain elusive. For bacterial TSs, half-site reactivity is still an open debate and the recent evidences are somehow controversial. Furthermore, different behaviors have been identified among bacterial TSs, compromising the definition of common mechanisms. Moreover, the redox reaction responsible for the regeneration of reduced flavin in FDTSs is not completely clarified. This review describes the recent advances in the structural and functional characterization of bacterial TSs and FDTSs and the current understanding of their mechanisms of action. Furthermore, the recent progresses in the development of inhibitors targeting TS and FDTS in human pathogenic bacteria are summarized.

scholarly journals Functional characterization of 3D protein structures informed by human genetic diversity

2019 ◽  
Vol 116 (18) ◽  
pp. 8960-8965 ◽  
Author(s):  
Michael Hicks ◽  
Istvan Bartha ◽  
Julia di Iulio ◽  
J. Craig Venter ◽  
Amalio Telenti
Keyword(s):  
Large Scale ◽  
Functional Characterization ◽  
Protein Structures ◽  
Large Scale Data ◽  
Allosteric Sites ◽  
Missense Variation ◽  
Binding Pockets ◽  
Definition Of ◽  
Scale Data

Sequence variation data of the human proteome can be used to analyze 3D protein structures to derive functional insights. We used genetic variant data from nearly 140,000 individuals to analyze 3D positional conservation in 4,715 proteins and 3,951 homology models using 860,292 missense and 465,886 synonymous variants. Sixty percent of protein structures harbor at least one intolerant 3D site as defined by significant depletion of observed over expected missense variation. Structural intolerance data correlated with deep mutational scanning functional readouts for PPARG, MAPK1/ERK2, UBE2I, SUMO1, PTEN, CALM1, CALM2, and TPK1 and with shallow mutagenesis data for 1,026 proteins. The 3D structural intolerance analysis revealed different features for ligand binding pockets and orthosteric and allosteric sites. Large-scale data on human genetic variation support a definition of functional 3D sites proteome-wide.

scholarly journals Structural and Functional Characterization of the Human Thymidylate Synthase (hTS) Interface Variant R175C, New Perspectives for the Development of hTS Inhibitors

Molecules ◽  
2019 ◽  
Vol 24 (7) ◽  
pp. 1362
Author(s):  
Cecilia Pozzi ◽  
Stefania Ferrari ◽  
Rosaria Luciani ◽  
Maria Costi ◽  
Stefano Mangani
Keyword(s):  
Thymidylate Synthase ◽  
Active Site ◽  
Functional Characterization ◽  
Binding Mode ◽  
Anticancer Chemotherapy ◽  
Innovative Strategies ◽  
X Ray Crystallography ◽  
Site Targeting ◽  
New Strategies

Human thymidylate synthase (hTS) is pivotal for cell survival and proliferation, indeed it provides the only synthetic source of dTMP, required for DNA biosynthesis. hTS represents a validated target for anticancer chemotherapy. However, active site-targeting drugs towards hTS have limitations connected to the onset of resistance. Thus, new strategies have to be applied to effectively target hTS without inducing resistance in cancer cells. Here, we report the generation and the functional and structural characterization of a new hTS interface variant in which Arg175 is replaced by a cysteine. Arg175 is located at the interface of the hTS obligate homodimer and protrudes inside the active site of the partner subunit, in which it provides a fundamental contribution for substrate binding. Indeed, the R175C variant results catalytically inactive. The introduction of a cysteine at the dimer interface is functional for development of new hTS inhibitors through innovative strategies, such as the tethering approach. Structural analysis, performed through X-ray crystallography, has revealed that a cofactor derivative is entrapped inside the catalytic cavity of the hTS R175C variant. The peculiar binding mode of the cofactor analogue suggests new clues exploitable for the design of new hTS inhibitors.

Characterization of the AMP-binding protein gene family in Arabidopsis thaliana: will the real acyl-CoA synthetases please stand up?

2000 ◽  
Vol 28 (6) ◽  
pp. 955-957 ◽  
Author(s):  
J. Shockey ◽  
J. Schnurr ◽  
J. Browse
Keyword(s):  
Protein Gene ◽  
De Novo ◽  
Functional Characterization ◽  
De Novo Synthesis ◽  
Triacylglycerol Composition ◽  
Sequence Comparisons ◽  
Triacylglycerol Metabolism ◽  
Binding Protein Gene

One of the most prominent and important topics in modern agricultural biotechnology is the manipulation of oilseed triacylglycerol composition. Towards this goal, we have sought to identify and characterize acyl-CoA synthetases (ACSs), which play an important role in both de novo synthesis and modification of existing lipids. We have identified and cloned 20 different genes that bear strong sequence homology to known ACSs from other organisms. Through sequence comparisons and functional characterization, we have identified several members of this group that encode ACSs, while the other genes fall into the broader category of genes for AMP-binding proteins (AMPBPs). Distinguishing ACSs from AMPBPs will simplify our efforts to understand the role of ACS in triacylglycerol metabolism.

scholarly journals Structural and functional characterization of sugar epimerases in pathogenic bacteria

2017 ◽  
Vol 73 (a2) ◽  
pp. C265-C265
Author(s):  
M. Lavanyaa ◽  
Sai Rohit Guntupalli ◽  
Kirti Joshi ◽  
Vinod Nayak ◽  
S. Ramaswamy
Keyword(s):  
Pathogenic Bacteria ◽  
Functional Characterization

scholarly journals Clinical and Functional Characterization of the Recurrent TUBA1A p.(Arg2His) Mutation

2018 ◽  
Vol 8 (8) ◽  
pp. 145 ◽  
Author(s):  
Jennifer Gardner ◽  
Thomas Cushion ◽  
Georgios Niotakis ◽  
Heather Olson ◽  
P. Grant ◽  
...  
Keyword(s):  
De Novo ◽  
Functional Characterization ◽  
Clinical Manifestations ◽  
Fetal Brain ◽  
Detailed Comparison ◽  
Hek 293 ◽  
293 Cells ◽  
Brain Malformations ◽  
Computer Based

The TUBA1A gene encodes tubulin alpha-1A, a protein that is highly expressed in the fetal brain. Alpha- and beta-tubulin subunits form dimers, which then co-assemble into microtubule polymers: dynamic, scaffold-like structures that perform key functions during neurogenesis, neuronal migration, and cortical organisation. Mutations in TUBA1A have been reported to cause a range of brain malformations. We describe four unrelated patients with the same de novo missense mutation in TUBA1A, c.5G>A, p.(Arg2His), as found by next generation sequencing. Detailed comparison revealed similar brain phenotypes with mild variability. Shared features included developmental delay, microcephaly, hypoplasia of the cerebellar vermis, dysplasia or thinning of the corpus callosum, small pons, and dysmorphic basal ganglia. Two of the patients had bilateral perisylvian polymicrogyria. We examined the effects of the p.(Arg2His) mutation by computer-based protein structure modelling and heterologous expression in HEK-293 cells. The results suggest the mutation subtly impairs microtubule function, potentially by affecting inter-dimer interaction. Based on its sequence context, c.5G>A is likely to be a common recurrent mutation. We propose that the subtle functional effects of p.(Arg2His) may allow for other factors (such as genetic background or environmental conditions) to influence phenotypic outcome, thus explaining the mild variability in clinical manifestations.

Functional Characterization of NiTi Shape Memory Elements for Smart Micro-Actuation

2013 ◽  
Author(s):  
Carlo Alberto Biffi ◽  
Luca Bonacina ◽  
Adelaide Nespoli ◽  
Barbara Previtali ◽  
Ausonio Tuissi
Keyword(s):  
Shape Memory ◽  
Mechanical Response ◽  
Thermal Hysteresis ◽  
Functional Characterization ◽  
Functional Materials ◽  
Mechanical Tests ◽  
Calorimetric Analysis ◽  
Production Route ◽  
Definition Of

Shape Memory Alloys (SMAs) are smart and functional materials, which are considered good candidates for the activation of devices for the automotive, aerospace, biomedical and mechanical systems, thanks to the shape memory effect. In this work, a study on the mechanical response of NiTi SMA snake like elements has been proposed. The production route of these elements from thin sheets, was given by laser machining followed by chemical etching. The micro-elements were characterized by means of calorimetric analysis for the definition of the theoretical operating temperatures and by means of thermo-mechanical testing for the evaluation of their functional performances. Mechanical tests has been carried out to assess the tensile behavior of martensite and austenite separately, and to evaluate the thermal hysteresis under different constant loads. Moreover, Finite Element Modeling (FEM) has been also accomplished to study the numerical evaluation of the stress field that origins by the application of the different loads in both the martensitic and austenitic phases.

scholarly journals Structural and functional characterization of a putative de novo gene in Drosophila

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Andreas Lange ◽  
Prajal H. Patel ◽  
Brennen Heames ◽  
Adam M. Damry ◽  
Thorsten Saenger ◽  
...  
Keyword(s):  
De Novo ◽  
Functional Characterization ◽  
Comparative Genomic ◽  
Noncoding Dna ◽  
Protein Coding ◽  
Ancestral Sequences ◽  
De Novo Gene ◽  
Genomic Studies ◽  
Biochemical Genetic

AbstractComparative genomic studies have repeatedly shown that new protein-coding genes can emerge de novo from noncoding DNA. Still unknown is how and when the structures of encoded de novo proteins emerge and evolve. Combining biochemical, genetic and evolutionary analyses, we elucidate the function and structure of goddard, a gene which appears to have evolved de novo at least 50 million years ago within the Drosophila genus. Previous studies found that goddard is required for male fertility. Here, we show that Goddard protein localizes to elongating sperm axonemes and that in its absence, elongated spermatids fail to undergo individualization. Combining modelling, NMR and circular dichroism (CD) data, we show that Goddard protein contains a large central α-helix, but is otherwise partially disordered. We find similar results for Goddard’s orthologs from divergent fly species and their reconstructed ancestral sequences. Accordingly, Goddard’s structure appears to have been maintained with only minor changes over millions of years.

scholarly journals Structural and Functional Characterization of Sapovirus RNA-Dependent RNA Polymerase

2006 ◽  
Vol 81 (4) ◽  
pp. 1858-1871 ◽  
Author(s):  
Stephen W. B. Fullerton ◽  
Martina Blaschke ◽  
Bruno Coutard ◽  
Julia Gebhardt ◽  
Alexander Gorbalenya ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
De Novo ◽  
Functional Characterization ◽  
Transferase Activity ◽  
Dependent Manner ◽  
Rna Dependent Rna Polymerase ◽  
Key Enzyme

ABSTRACT Sapoviruses are one of the major agents of acute gastroenteritis in childhood. They form a tight genetic cluster (genus) in the Caliciviridae family that regroups both animal and human pathogenic strains. No permissive tissue culture has been developed for human sapovirus, limiting its characterization to surrogate systems. We report here on the first extensive characterization of the key enzyme of replication, the RNA-dependent RNA polymerase (RdRp) associated with the 3Dpol-like protein. Enzymatically active sapovirus 3Dpol and its defective mutant were expressed in Escherichia coli and purified. The overall structure of the sapovirus 3Dpol was determined by X-ray crystallography to 2.32-Å resolution. It revealed a right hand fold typical for template-dependent polynucleotide polymerases. The carboxyl terminus is located within the active site cleft, as observed in the RdRp of some (norovirus) but not other (lagovirus) caliciviruses. Sapovirus 3Dpol prefers Mn2+ over Mg2+ but may utilize either as a cofactor in vitro. In a synthetic RNA template-dependent reaction, sapovirus 3Dpol synthesizes a double-stranded RNA or labels the template 3′ terminus by terminal transferase activity. Initiation of RNA synthesis occurs de novo on heteropolymeric templates or in a primer-dependent manner on polyadenylated templates. Strikingly, this mode of initiation of RNA synthesis was also described for norovirus, but not for lagovirus, suggesting structural and functional homologies in the RNA-dependent RNA polymerase of human pathogenic caliciviruses. This first experimental evidence makes sapovirus 3Dpol an attractive target for developing drugs to control calicivirus infection in humans.

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