Structural and Functional Characterization of Camelus dromedarius Glutathione Transferase M1-1

Glutathione transferases (GSTs; EC. 2.5.1.18) are a large family of multifunctional enzymes that play crucial roles in the metabolism and inactivation of a broad range of xenobiotic compounds. In the present work, we report the kinetic and structural characterization of the isoenzyme GSTM1-1 from Camelus dromedarius (CdGSTM1-1). The CdGSΤM1-1 was expressed in E. coli BL21 (DE3) and was purified by affinity chromatography. Kinetics analysis showed that the enzyme displays a relative narrow substrate specificity and restricted ability to bind xenobiotic compounds. The crystal structures of CdGSΤM1-1 were determined by X-ray crystallography in complex with the substrate (GSH) or the reaction product (S-p-nitrobenzyl-GSH), providing snapshots of the induced-fit catalytic mechanism. The thermodynamic stability of CdGSTM1-1 was investigated using differential scanning fluorimetry (DSF) in the absence and in presence of GSH and S-p-nitrobenzyl-GSH and revealed that the enzyme’s structure is significantly stabilized by its ligands. The results of the present study advance the understanding of camelid GST detoxification mechanisms and their contribution to abiotic stress adaptation in harsh desert conditions.

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Structural and Functional Characterization of One Unclassified Glutathione S-Transferase in Xenobiotic Adaptation of Leptinotarsa decemlineata

International Journal of Molecular Sciences ◽

10.3390/ijms222111921 ◽

2021 ◽

Vol 22 (21) ◽

pp. 11921

Author(s):

Yanjun Liu ◽

Timothy Moural ◽

Sonu Koirala B K ◽

Jonathan Hernandez ◽

Zhongjian Shen ◽

...

Keyword(s):

Kinetic Parameters ◽

Functional Characterization ◽

Three Dimensional ◽

Large Family ◽

Dimensional Structure ◽

Glutathione S Transferase ◽

X Ray Crystallography ◽

Xenobiotic Exposure ◽

Catalytically Active ◽

Multifunctional Enzymes

Arthropod Glutathione S-transferases (GSTs) constitute a large family of multifunctional enzymes that are mainly associated with xenobiotic or stress adaptation. GST-mediated xenobiotic adaptation takes place through direct metabolism or sequestration of xenobiotics, and/or indirectly by providing protection against oxidative stress induced by xenobiotic exposure. To date, the roles of GSTs in xenobiotic adaptation in the Colorado potato beetle (CPB), a notorious agricultural pest of plants within Solanaceae, have not been well studied. Here, we functionally expressed and characterized an unclassified-class GST, LdGSTu1. The three-dimensional structure of the LdGSTu1 was solved with a resolution up to 1.8 Å by X-ray crystallography. The signature motif VSDGPPSL was identified in the “G-site”, and it contains the catalytically active residue Ser14. Recombinant LdGSTu1 was used to determine enzyme activity and kinetic parameters using 1-chloro-2, 4-dinitrobenzene (CDNB), GSH, p-nitrophenyl acetate (PNA) as substrates. The enzyme kinetic parameters and enzyme-substrate interaction studies demonstrated that LdGSTu1 could catalyze the conjugation of GSH to both CDNB and PNA, with a higher turnover number for CDNB than PNA. The LdGSTu1 enzyme inhibition assays demonstrated that the enzymatic conjugation of GSH to CDNB was inhibited by multiple pesticides, suggesting a potential function of LdGSTu1 in xenobiotic adaptation.

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Structural and functional characterization of proteins from the fire blight pathogen Erwinia amylovora. A review on the state of the art

Journal of Plant Pathology ◽

10.1007/s42161-020-00682-4 ◽

2020 ◽

Author(s):

Stefano Benini

Keyword(s):

Fire Blight ◽

Erwinia Amylovora ◽

Functional Characterization ◽

Data Bank ◽

Knock Out ◽

X Ray ◽

X Ray Crystallography ◽

Starting Point ◽

Good Starting Point

Abstract Together with genome analysis and knock-out mutants, structural and functional characterization of proteins provide valuable hints on the biology of the organism under investigation. Structural characterization can be achieved by techniques such as X-ray crystallography, NMR, Cryo-EM. The information derived from the structure are a good starting point to comprehend the details of the proteins molecular function for a better understanding of their biological role. This review aims at describing the progress in the structural and functional characterization of proteins from the plant pathogen Erwinia amylovora obtained by structural biology and currently deposited in the Protein Data Bank.

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

Molecules ◽

10.3390/molecules24071362 ◽

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.

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Characterization of βαβββ Resistance Proteins from Pseudomonas aeruginosa

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314092821 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C717-C717

Author(s):

Allegra Vit ◽

Monika Popp ◽

Eyad Kalawy-Fansa ◽

Shen Yu ◽

Wulf Blankenfeldt

Keyword(s):

Pseudomonas Aeruginosa ◽

Physiological Function ◽

Functional Characterization ◽

Conserved Residues ◽

X Ray ◽

Resistance Proteins ◽

X Ray Crystallography ◽

Phenazine Derivative ◽

Function Assignment

Pseudomonas aeruginosa is a multiresistant pathogen that can cause infection in immuno-compromized patients, for example in people suffering from cystic fibrosis. [1] It has complex patho-physiology and produces a large number of exoproducts, among which the phenazines are especially prominent. In P. aeruginosa, the blue phenazine derivative pyocyanin plays a crucial role in infection of the host. [2] This phenazine can generate reactive oxygen species and is thought to act as respiratory pigment and as a virulence factor at the same time. P. aeruginosa has to protect itself from its own phenazines because of the antibiotic action of these substances. Inspired by the fact that the phenazine biosynthesis operon of several bacteria contains a phenazine resistance factor of the βαβββ module protein family, we have searched the genome of P. aeruginosa for proteins of this fold. [3] In P. aeruginosa we could identify 22 of these genes, most without previous functional characterization. A structure-based sequence alignment made it possible to assign these proteins to two classes with two subgroups each, based on the conserved residues in the active site. Using X-ray crystallography and biophysical methods, we further demonstrate that several of these proteins indeed bind phenazines and possibly other antibiotics that contain aromatic moieties. Currently, we are working on the structural characterization and physiological function assignment of all of these βαβββ-module-containing proteins. Ultimately, these data may lead to novel anti-infective strategies.

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Mass spectrometry for fragment screening

Essays in Biochemistry ◽

10.1042/ebc20170071 ◽

2017 ◽

Vol 61 (5) ◽

pp. 465-473 ◽

Cited By ~ 11

Author(s):

Daniel Shiu-Hin Chan ◽

Andrew J. Whitehouse ◽

Anthony G. Coyne ◽

Chris Abell

Keyword(s):

Drug Discovery ◽

High Sensitivity ◽

Titration Calorimetry ◽

Label Free ◽

X Ray ◽

X Ray Crystallography ◽

Fragment Screening ◽

Differential Scanning Fluorimetry ◽

Biophysical Techniques

Fragment-based approaches in chemical biology and drug discovery have been widely adopted worldwide in both academia and industry. Fragment hits tend to interact weakly with their targets, necessitating the use of sensitive biophysical techniques to detect their binding. Common fragment screening techniques include differential scanning fluorimetry (DSF) and ligand-observed NMR. Validation and characterization of hits is usually performed using a combination of protein-observed NMR, isothermal titration calorimetry (ITC) and X-ray crystallography. In this context, MS is a relatively underutilized technique in fragment screening for drug discovery. MS-based techniques have the advantage of high sensitivity, low sample consumption and being label-free. This review highlights recent examples of the emerging use of MS-based techniques in fragment screening.

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Purification and functional characterization of pancreatic insulin from camel (Camelus dromedarius)

Saudi Journal of Biological Sciences ◽

10.1016/j.sjbs.2014.03.001 ◽

2014 ◽

Vol 21 (6) ◽

pp. 574-581 ◽

Cited By ~ 4

Author(s):

Babiker A. Elamin ◽

Abdulmajeed Al-Maleki ◽

Mohammad A. Ismael ◽

Mohammed Akli Ayoub

Keyword(s):

Functional Characterization ◽

Camelus Dromedarius ◽

Pancreatic Insulin

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Polyphosphate-dependent synthesis of ATP and ADP by the family-2 polyphosphate kinases in bacteria

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0807563105 ◽

2008 ◽

Vol 105 (46) ◽

pp. 17730-17735 ◽

Cited By ~ 66

Author(s):

Boguslaw Nocek ◽

Samvel Kochinyan ◽

Michael Proudfoot ◽

Greg Brown ◽

Elena Evdokimova ◽

...

Keyword(s):

Sinorhizobium Meliloti ◽

Molecular Mechanisms ◽

Catalytic Mechanism ◽

Functional Characterization ◽

High Energy ◽

Inorganic Polyphosphate ◽

The Family ◽

Adp Crystal ◽

Intracellular Energy

Inorganic polyphosphate (polyP) is a linear polymer of tens or hundreds of phosphate residues linked by high-energy bonds. It is found in all organisms and has been proposed to serve as an energy source in a pre-ATP world. This ubiquitous and abundant biopolymer plays numerous and vital roles in metabolism and regulation in prokaryotes and eukaryotes, but the underlying molecular mechanisms for most activities of polyP remain unknown. In prokaryotes, the synthesis and utilization of polyP are catalyzed by 2 families of polyP kinases, PPK1 and PPK2, and polyphosphatases. Here, we present structural and functional characterization of the PPK2 family. Proteins with a single PPK2 domain catalyze polyP-dependent phosphorylation of ADP to ATP, whereas proteins containing 2 fused PPK2 domains phosphorylate AMP to ADP. Crystal structures of 2 representative proteins, SMc02148 from Sinorhizobium meliloti and PA3455 from Pseudomonas aeruginosa, revealed a 3-layer α/β/α sandwich fold with an α-helical lid similar to the structures of microbial thymidylate kinases, suggesting that these proteins share a common evolutionary origin and catalytic mechanism. Alanine replacement mutagenesis identified 9 conserved residues, which are required for activity and include the residues from both Walker A and B motifs and the lid. Thus, the PPK2s represent a molecular mechanism, which potentially allow bacteria to use polyP as an intracellular energy reserve for the generation of ATP and survival.

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Structure and function of one unclassified-class glutathione S-transferase in Leptinotarsa decemlineata

10.1101/2021.09.29.462415 ◽

2021 ◽

Author(s):

Yanjun Liu ◽

Timothy W Moural ◽

Sonu BK Koirala ◽

Jonathan Hernandez ◽

Zhongjian Shen ◽

...

Keyword(s):

Kinetic Parameters ◽

Three Dimensional ◽

Large Family ◽

Dimensional Structure ◽

Glutathione S Transferase ◽

X Ray Crystallography ◽

Xenobiotic Exposure ◽

Multifunctional Enzymes ◽

Glutathione S Transferases ◽

And Function

Arthropod Glutathione S-transferases (GSTs) constitute a large family of multifunctional enzymes that are mainly associated with xenobiotic or stress adaptation. GST-mediated xenobiotic adaptation is through direct metabolism or sequestration of xenobiotics, and/or indirectly by providing protection against oxidative stress induced by xenobiotic exposure. To date, the roles of GSTs in xenobiotic adaptation in the Colorado potato beetle (CPB), a notorious agriculture pest of plants within Solanaceae have not been well studied. Here, we functionally expressed and characterized an unclassified-class GST, LdGSTu1. The three-dimensional structure of the LdGSTu1 was solved with a resolution up to 1.8 Å by x-ray crystallography. Recombinant LdGSTu1 was used to determine enzyme activity and kinetic parameters using 1-chloro-2,4-dinitrobenzene (CDNB), GSH, p-nitrophenyl acetate (PNA) as substrates. The enzyme kinetic parameters and enzyme-substrate interaction studies demonstrated that LdGSTu1 could catalyze the conjugation of GSH to both CDNB and PNA, with a higher turnover number for CDNB than PNA. The LdGSTu1 enzyme inhibition assays demonstrated that the enzymatic conjugation of GSH to CDNB could be inhibited by multiple pesticides, suggesting a potential function of LdGSTu1 in xenobiotic adaptation.

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Functional and Structural Investigation of Chalcone Synthases Based on Integrated Metabolomics and Transcriptome Analysis on Flavonoids and Anthocyanins Biosynthesis of the Fern Cyclosorus parasiticus

Frontiers in Plant Science ◽

10.3389/fpls.2021.757516 ◽

2021 ◽

Vol 12 ◽

Author(s):

Meng Niu ◽

Jie Fu ◽

Rong Ni ◽

Rui-Lin Xiong ◽

Ting-Ting Zhu ◽

...

Keyword(s):

Transcriptome Analysis ◽

Structural Investigation ◽

Catalytic Mechanism ◽

Functional Characterization ◽

Site Directed Mutagenesis ◽

Qrt Pcr ◽

Genes Encoding ◽

Similar Conformation ◽

Integrated Metabolomics

The biosynthesis of flavonoids and anthocyanidins has been exclusively investigated in angiosperms but largely unknown in ferns. This study integrated metabolomics and transcriptome to analyze the fronds from different development stages (S1 without spores and S2 with brown spores) of Cyclosorus parasiticus. About 221 flavonoid and anthocyanin metabolites were identified between S1 and S2. Transcriptome analysis revealed several genes encoding the key enzymes involved in the biosynthesis of flavonoids, and anthocyanins were upregulated in S2, which were validated by qRT-PCR. Functional characterization of two chalcone synthases (CpCHS1 and CpCHS2) indicated that CpCHS1 can catalyze the formation of pinocembrin, naringenin, and eriodictyol, respectively; however, CpCHS2 was inactive. The crystallization investigation of CpCHS1 indicated that it has a highly similar conformation and shares a similar general catalytic mechanism to other plants CHSs. And by site-directed mutagenesis, we found seven residues, especially Leu199 and Thr203 that are critical to the catalytic activity for CpCHS1.

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