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.

Current Status of the Use of Multifunctional Enzymes as Anti-Cancer Drug Targets

Fighting cancer is one of the major challenges of the 21st century. Among recently proposed treatments, molecular-targeted therapies are attracting particular attention. The potential targets of such therapies include a group of enzymes that possess the capability to catalyze at least two different reactions, so-called multifunctional enzymes. The features of such enzymes can be used to good advantage in the development of potent selective inhibitors. This review discusses the potential of multifunctional enzymes as anti-cancer drug targets along with the current status of research into four enzymes which by their inhibition have already demonstrated promising anti-cancer effects in vivo, in vitro, or both. These are PFK-2/FBPase-2 (involved in glucose homeostasis), ATIC (involved in purine biosynthesis), LTA4H (involved in the inflammation process) and Jmjd6 (involved in histone and non-histone posttranslational modifications). Currently, only LTA4H and PFK-2/FBPase-2 have inhibitors in active clinical development. However, there are several studies proposing potential inhibitors targeting these four enzymes that, when used alone or in association with other drugs, may provide new alternatives for preventing cancer cell growth and proliferation and increasing the life expectancy of patients.

Structural and Functional Studies of 3-Deoxy-D-arabino- Heptulosonate 7-Phosphate Synthase from Prevotella nigrescens

<p>Multifunctional enzymes, bearing two or more catalytic activities, provide exceptional contributions to the efficient and coherent function of metabolic pathways. Two main benefits of multifunctional enzymes have been clearly described. Firstly, linked catalytic modules can enhance the overall catalytic rate for consecutive reactions of a metabolic pathway due to substrate channelling. Secondly, the fusion of two protein domains can impart allosteric control, such that the catalytic function of one of the protein domains is altered by a ligand binding to the second, covalently linked domain. This study examines a bifunctional enzyme comprising a 3-deoxy-D-arabino heptulosonate 7-phosphate synthase (DAH7PS) domain covalently fused to a C-terminal chorismate mutase (CM) domain from Prevotella nigrescens (PniDAH7PS). DAH7PS catalyses the first reaction of the shikimate pathway leading to the biosynthesis of aromatic amino acids, whereas CM functions at a pathway branch point, leading to the biosynthesis of tyrosine and phenylalanine. Through the investigation of PniDAH7PS, a special functional interdependence between the two non-consecutive catalytic functionalities and the derived allosteric regulation was unravelled. Chapter 2 generally characterises the biochemical and structural features of PniDAH7PS. The two catalytic activities exhibit substantial hetero-interdependency and the separation of the two distinct catalytic domains results in a dramatic loss of both the DAH7PS and CM enzymatic activities. The structural investigation into this protein revealed a unique dimeric assembly and implicates a hetero-interaction between the DAH7PS and CM domains, providing a structural basis for the functional interdependence. Moreover, allosteric inhibition of DAH7PS by prephenate, the product of the CM-catalysed reaction, was observed. This allostery is accompanied by a striking conformational change, as observed by SAXS, implying that a manipulation of the hetero-domain interaction is the mechanism underpinning the allosteric inhibition. Chapter 3 looks into the mechanism underpinning the DAH7PS and CM functional interdependence. Rearrangements of the conformation of PniDAH7PS following the addition of substrate combinations were observed. This indicates that a dynamic interaction between the DAH7PS and CM domains is important for catalysis. Furthermore, perturbation of these conformational variations by either a truncation mutation in the CM domain or the presence of a high concentration of NaCl interrupted the both the DAH7PS and CM catalytic activities, implying that a dynamic hetero-domain interaction is essential for the delivering the normal DAH7PS and CM functions. This work also reveals a dual role for the DAH7PS domain, exerting catalysis and allosteric activation on the CM activity simultaneously. Chapter 4 investigates the mechanism of the allosteric inhibition of PniDAH7PS by prephenate. The structural effect of prephenate on PniDAH7PS, with the addition of substrate combinations, was inspected, and the results unravelled the same conformation of PniDAH7PS under different conditions, exhibiting high compactness and rigidity. This finding indicates that the probable inhibitory effect of prephenate on PniDAH7PS is realised by freezing the enzyme’s structure in order to deprive PniDAH7PS of the dynamic-dependent catalytic activity. Chapter 5 describes the development of a method for producing segmentally isotopically labelled PniDAH7PS using Expressed Protein Ligation (EPL). This chapter also details attempts to couple this method with small angle neutron scattering (SANS) and nuclear magnetic resonance spectroscopy (NMR) to gain more structural information regarding the catalytic and allosteric properties of PniDAH7PS.</p>

Structural and Functional Studies of 3-Deoxy-D-arabino- Heptulosonate 7-Phosphate Synthase from Prevotella nigrescens

<p>Multifunctional enzymes, bearing two or more catalytic activities, provide exceptional contributions to the efficient and coherent function of metabolic pathways. Two main benefits of multifunctional enzymes have been clearly described. Firstly, linked catalytic modules can enhance the overall catalytic rate for consecutive reactions of a metabolic pathway due to substrate channelling. Secondly, the fusion of two protein domains can impart allosteric control, such that the catalytic function of one of the protein domains is altered by a ligand binding to the second, covalently linked domain. This study examines a bifunctional enzyme comprising a 3-deoxy-D-arabino heptulosonate 7-phosphate synthase (DAH7PS) domain covalently fused to a C-terminal chorismate mutase (CM) domain from Prevotella nigrescens (PniDAH7PS). DAH7PS catalyses the first reaction of the shikimate pathway leading to the biosynthesis of aromatic amino acids, whereas CM functions at a pathway branch point, leading to the biosynthesis of tyrosine and phenylalanine. Through the investigation of PniDAH7PS, a special functional interdependence between the two non-consecutive catalytic functionalities and the derived allosteric regulation was unravelled. Chapter 2 generally characterises the biochemical and structural features of PniDAH7PS. The two catalytic activities exhibit substantial hetero-interdependency and the separation of the two distinct catalytic domains results in a dramatic loss of both the DAH7PS and CM enzymatic activities. The structural investigation into this protein revealed a unique dimeric assembly and implicates a hetero-interaction between the DAH7PS and CM domains, providing a structural basis for the functional interdependence. Moreover, allosteric inhibition of DAH7PS by prephenate, the product of the CM-catalysed reaction, was observed. This allostery is accompanied by a striking conformational change, as observed by SAXS, implying that a manipulation of the hetero-domain interaction is the mechanism underpinning the allosteric inhibition. Chapter 3 looks into the mechanism underpinning the DAH7PS and CM functional interdependence. Rearrangements of the conformation of PniDAH7PS following the addition of substrate combinations were observed. This indicates that a dynamic interaction between the DAH7PS and CM domains is important for catalysis. Furthermore, perturbation of these conformational variations by either a truncation mutation in the CM domain or the presence of a high concentration of NaCl interrupted the both the DAH7PS and CM catalytic activities, implying that a dynamic hetero-domain interaction is essential for the delivering the normal DAH7PS and CM functions. This work also reveals a dual role for the DAH7PS domain, exerting catalysis and allosteric activation on the CM activity simultaneously. Chapter 4 investigates the mechanism of the allosteric inhibition of PniDAH7PS by prephenate. The structural effect of prephenate on PniDAH7PS, with the addition of substrate combinations, was inspected, and the results unravelled the same conformation of PniDAH7PS under different conditions, exhibiting high compactness and rigidity. This finding indicates that the probable inhibitory effect of prephenate on PniDAH7PS is realised by freezing the enzyme’s structure in order to deprive PniDAH7PS of the dynamic-dependent catalytic activity. Chapter 5 describes the development of a method for producing segmentally isotopically labelled PniDAH7PS using Expressed Protein Ligation (EPL). This chapter also details attempts to couple this method with small angle neutron scattering (SANS) and nuclear magnetic resonance spectroscopy (NMR) to gain more structural information regarding the catalytic and allosteric properties of PniDAH7PS.</p>

The Complex Functions of the NME Family—A Matter of Location and Molecular Activity

The family of NME proteins represents a quite complex group of multifunctional enzymes [...]

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

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.

Genome-Wide Analysis of the Glutathione S-Transferase (GST) Genes and Functional Identification of MdGSTU12 Reveals the Involvement in the Regulation of Anthocyanin Accumulation in Apple

Anthocyanins have essential biological functions, affecting the development of horticultural production. They are synthesized in the cytoplasm through flavonoid metabolic pathways and finally transported into vacuoles for storage. Plant glutathione S-transferases (GSTs) are multifunctional enzymes involved in anthocyanin transportation. In this study, we identified 38 GSTs from the apple (Malus domestica) genome (HFTH1 Whole Genome v1.0) based on the sequence similarity with the GST family proteins of Arabidopsis. These MdGST genes could be grouped into nine chief subclasses: U, F, L, Z, T, GHR, EF1Bγ, TCHQD, and DHAR. The structures, motifs, three-dimensional models, and chromosomal distribution of MdGST genes were further analyzed. Elements which are responsive for some hormones and stress, and others that involve genes related to flavonoid biosynthesis were forecast in the promoter of MdGST. In addition, we identified 32 orthologous gene pairs between apple and Arabidopsis. These genes indicated that numerous apple and Arabidopsis counterparts appeared to be derived from a common ancestor. Amongst the 38 MdGST genes, MdGSTU12 was considerably correlated with anthocyanin variation in terms of extracting expression profiles from reported. Finally, further functional identification in apple transgenic calli and subcellular localization confirmed that MdGSTU12 was of great significance in anthocyanin accumulation in apple.

Internal Promoters and Their Effects on the Transcription of Operon Genes for Epothilone Production in Myxococcus xanthus

The biosynthetic genes for secondary metabolites are often clustered into giant operons with no transcription terminator before the end. The long transcripts are frangible and the transcription efficiency declines along with the process. Internal promoters might occur in operons to coordinate the transcription of individual genes, but their effects on the transcription of operon genes and the yield of metabolites have been less investigated. Epothilones are a kind of antitumor polyketides synthesized by seven multifunctional enzymes encoded by a 56-kb operon. In this study, we identified multiple internal promoters in the epothilone operon. We performed CRISPR-dCas9–mediated transcription activation of internal promoters, combined activation of different promoters, and activation in different epothilone-producing M. xanthus strains. We found that activation of internal promoters in the operon was able to promote the gene transcription, but the activation efficiency was distinct from the activation of separate promoters. The transcription of genes in the operon was influenced by not only the starting promoter but also internal promoters of the operon; internal promoters affected the transcription of the following and neighboring upstream/downstream genes. Multiple interferences between internal promoters thus changed the transcriptional profile of operon genes and the production of epothilones. Better activation efficiency for the gene transcription and the epothilone production was obtained in the low epothilone-producing strains. Our results highlight that interactions between promoters in the operon are critical for the gene transcription and the metabolite production efficiency.

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

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.