scholarly journals Functional Characterisation of Three Glycine N-Acyltransferase Variants and the Effect on Glycine Conjugation to Benzoyl–CoA

2021 ◽  
Vol 22 (6) ◽  
pp. 3129
Author(s):  
Johann M. Rohwer ◽  
Chantelle Schutte ◽  
Rencia van der Sluis
Keyword(s):  
Enzyme Activity ◽  
Energy Production ◽  
Catalytic Mechanism ◽  
Kinetic Mechanism ◽  
Coding Region ◽  
Functional Characterisation ◽  
Coa Ligase ◽  
Wide Range ◽  
Substrate Concentrations

The glycine conjugation pathway in humans is involved in the metabolism of natural substrates and the detoxification of xenobiotics. The interactions between the various substrates in this pathway and their competition for the pathway enzymes are currently unknown. The pathway consists of a mitochondrial xenobiotic/medium-chain fatty acid: coenzyme A (CoA) ligase (ACSM2B) and glycine N-acyltransferase (GLYAT). The catalytic mechanism and substrate specificity of both of these enzymes have not been thoroughly characterised. In this study, the level of evolutionary conservation of GLYAT missense variants and haplotypes were analysed. From these data, haplotype variants were selected (156Asn > Ser, [17Ser > Thr,156Asn > Ser] and [156Asn > Ser,199Arg > Cys]) in order to characterise the kinetic mechanism of the enzyme over a wide range of substrate concentrations. The 156Asn > Ser haplotype has the highest frequency and the highest relative enzyme activity in all populations studied, and hence was used as the reference in this study. Cooperative substrate binding was observed, and the kinetic data were fitted to a two-substrate Hill equation. The coding region of the GLYAT gene was found to be highly conserved and the rare 156Asn > Ser,199Arg > Cys variant negatively affected the relative enzyme activity. Even though the 156Asn > Ser,199Arg > Cys variant had a higher affinity for benzoyl-CoA (s0.5,benz = 61.2 µM), kcat was reduced to 9.8% of the most abundant haplotype 156Asn > Ser (s0.5,benz = 96.6 µM), while the activity of 17Ser > Thr,156Asn > Ser (s0.5,benz = 118 µM) was 73% of 156Asn > Ser. The in vitro kinetic analyses of the effect of the 156Asn > Ser,199Arg > Cys variant on human GLYAT enzyme activity indicated that individuals with this haplotype might have a decreased ability to metabolise benzoate when compared to individuals with the 156Asn > Ser variant. Furthermore, the accumulation of acyl-CoA intermediates can inhibit ACSM2B leading to a reduction in mitochondrial energy production.

scholarly journals The dependence on vitamin E and selenium of drug demethylation in rat liver microsomal fractions

1975 ◽  
Vol 146 (2) ◽  
pp. 339-350 ◽  
Author(s):  
A S M Giasuddin ◽  
C P J Caygill ◽  
A T Diplock ◽  
E H Jeffery
Keyword(s):  
Enzyme Activity ◽  
Vitamin E ◽  
Rat Liver ◽  
Selenium Deficiency ◽  
Wide Range ◽  
Km Value ◽  
Demethylase Activity ◽  
In Vitro Induction ◽  
Substrate Concentrations

1. The effects of vitamin E deficiency, and of vitamin E and selenium deficiency, on rat liver microsomal aminopyrine demethylase activity were investigated. It was found that, over a wide range of substrate concentrations, the enzyme activity in preparations from deficient animals was significantly lower than that in controls. 2. Addition of antioxidants in vitro, either to the homogenization or to the assay media, was without significant effect on the depressed enzyme activity. Castration and alteration in dietary protein concentration were also without effect. The rate of oxidation of NADPH was however, lower in preparations from deficient animals. 3. Lineweaver-Burk plots of the reciprocal of enzyme activity and substrate concentration showed a higher Km value in preparations from vitamin E-deficient animals, irrespective of whether selenium was present; the Vmax. was unaffected. These parameters were unchanged when antioxidants were added in vitro. Induction with phenobarbitone and 3-methylcholanthrene showed large changes in Km value which, for preparations from vitamin E-deficient animals, was higher than that for corresponding controls. 4. Examination of the synergism between NADH and NADPH as donors of reducing equivalents for aminopyrine demethylation showed that vitamin E and selenium were only minimally involved in the phenomenon. However, both the initial rate and the extent of demethylation were significantly lower in vitamin E- and selenium-deficient preparations and both nutrients were required for the restoration of full activity. 5. The significance of these results is discussed in the light of our working hypothesis.

scholarly journals Epigenetic control via allosteric regulation of mammalian protein arginine methyltransferases

2017 ◽  
Vol 114 (38) ◽  
pp. 10101-10106 ◽  
Author(s):  
Kanishk Jain ◽  
Cyrus Y. Jin ◽  
Steven G. Clarke
Keyword(s):  
Cancer Metastasis ◽  
Gene Activation ◽  
Kinetic Studies ◽  
Arginine Methylation ◽  
Histone H4 ◽  
Protein Arginine Methyltransferases ◽  
Wide Range ◽  
Substrate Concentrations

Arginine methylation on histones is a central player in epigenetics and in gene activation and repression. Protein arginine methyltransferase (PRMT) activity has been implicated in stem cell pluripotency, cancer metastasis, and tumorigenesis. The expression of one of the nine mammalian PRMTs, PRMT5, affects the levels of symmetric dimethylarginine (SDMA) at Arg-3 on histone H4, leading to the repression of genes which are related to disease progression in lymphoma and leukemia. Another PRMT, PRMT7, also affects SDMA levels at the same site despite its unique monomethylating activity and the lack of any evidence for PRMT7-catalyzed histone H4 Arg-3 methylation. We present evidence that PRMT7-mediated monomethylation of histone H4 Arg-17 regulates PRMT5 activity at Arg-3 in the same protein. We analyzed the kinetics of PRMT5 over a wide range of substrate concentrations. Significantly, we discovered that PRMT5 displays positive cooperativity in vitro, suggesting that this enzyme may be allosterically regulated in vivo as well. Most interestingly, monomethylation at Arg-17 in histone H4 not only raised the general activity of PRMT5 with this substrate, but also ameliorated the low activity of PRMT5 at low substrate concentrations. These kinetic studies suggest a biochemical explanation for the interplay between PRMT5- and PRMT7-mediated methylation of the same substrate at different residues and also suggest a general model for regulation of PRMTs. Elucidating the exact relationship between these two enzymes when they methylate two distinct sites of the same substrate may aid in developing therapeutics aimed at reducing PRMT5/7 activity in cancer and other diseases.

scholarly journals A hyperthermoactive-Cas9 editing tool reveals the role of a unique arsenite methyltransferase in the arsenic resistance system of Thermus thermophilus HB27

2021 ◽  
Author(s):  
Giovanni Gallo ◽  
Ioannis Mougiakos ◽  
Mauricio Bianco ◽  
Miriam Carbonaro ◽  
Andrea Carpentieri ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Fluorescent Protein ◽  
Efflux Pump ◽  
Thermus Thermophilus ◽  
Yellow Fluorescent Protein ◽  
Arsenic Resistance ◽  
Wide Range ◽  
Resistance System

Arsenic detoxification systems can be found in a wide range of organisms, from bacteria to man. In a previous study, we discovered an arsenic-responsive transcriptional regulator in the thermophilic bacterium Thermus thermophilus HB27 (TtSmtB). Here, we characterize the arsenic resistance system of T. thermophilus in more detail. We employed TtSmtB-based pull-down assays with protein extracts from cultures treated with arsenate and arsenite to obtain an S-adenosylmethionine (SAM)-dependent arsenite methyltransferase (TtArsM). In vivo and in vitro analyses were performed to shed light on this new component of the arsenic resistance network and its peculiar catalytic mechanism. Heterologous expression of TtarsM in Escherichia coli resulted in arsenite detoxification at mesophilic temperatures. Although TtArsM does not contain a canonical arsenite binding site, the purified protein does catalyse SAM-dependent arsenite methylation. In addition, in vitro analyses confirmed the unique interaction between TtArsM and TtSmtB. Next, a highly efficient ThermoCas9-based genome-editing tool was developed to delete the TtArsM-encoding gene on the T. thermophilus genome, and to confirm its involvement in the arsenite detoxification system. Finally, the TtarsX efflux pump gene in the T. thermophilus ΔTtarsM genome was substituted by a gene, encoding a stabilised yellow fluorescent protein (sYFP), to create a sensitive genome-based bioreporter system for the detection of arsenic ions.

scholarly journals Quantitative study of unsaturated transport of glycerol through aquaglyceroporin that has high affinity for glycerol

2020 ◽  
Author(s):  
Roberto A. Rodriguez ◽  
Ruth Chan ◽  
Huiyun Liang ◽  
Liao Y. Chen
Keyword(s):  
Free Energy ◽  
In Silico ◽  
Energy Profile ◽  
Free Energy Profile ◽  
High Affinity ◽  
Glycerol Transport ◽  
Wide Range ◽  
Unsaturated Transport ◽  
Substrate Concentrations

Graphical AbstractABSTRACTThe structures of several aquaglyceroporins have been resolved to atomic resolution showing two or more glycerols bound inside a channel and confirming a glycerol-facilitator’s affinity for its substrate glycerol. However, the kinetics data of glycerol transport experiments all point to unsaturated transport that is characteristic of low substrate affinity in terms of the Michaelis-Menten kinetics. In this article, we present an in silico-in vitro research focused on AQP3, one of the human aquaglyceroporins that is natively expressed in the abundantly available erythrocytes. We conducted 2.1 μs in silico simulations of AQP3 embedded in a model erythrocyte membrane with intracellular-extracellular asymmetries in leaflet lipid compositions and compartment salt ions. From the equilibrium molecular dynamics (MD), we elucidated the mechanism of glycerol transport at high substrate concentrations. From the steered MD simulations, we computed the Gibbs free-energy profile throughout the AQP3 channel. From the free-energy profile, we quantified the kinetics of glycerol transport that is unsaturated due to glycerol-glycerol interaction mediated by AQP3 resulting in the concerted movement of two glycerol molecules for the transport of one glycerol molecule across the cell membrane. We conducted in vitro experiments on glycerol uptake into human erythrocytes for a wide range of substrate concentrations and various temperatures. The experimental data quantitatively validated our theoretical-computational conclusions on the unsaturated glycerol transport through AQP3 that has high affinity for glycerol.

scholarly journals Octameric structure ofStaphylococcus aureusenolase in complex with phosphoenolpyruvate

2015 ◽  
Vol 71 (12) ◽  
pp. 2457-2470 ◽  
Author(s):  
Yunfei Wu ◽  
Chengliang Wang ◽  
Shenglong Lin ◽  
Minhao Wu ◽  
Lu Han ◽  
...  
Keyword(s):  
Energy Production ◽  
Biological Processes ◽  
Structural Studies ◽  
Positive Bacterium ◽  
Wide Range ◽  
Evolutionarily Conserved ◽  
Catalytically Active ◽  
Dimeric Form

Staphylococcus aureusis a Gram-positive bacterium with strong pathogenicity that causes a wide range of infections and diseases. Enolase is an evolutionarily conserved enzyme that plays a key role in energy production through glycolysis. Additionally, enolase is located on the surface ofS. aureusand is involved in processes leading to infection. Here, crystal structures ofSa_enolase with and without bound phosphoenolpyruvate (PEP) are presented at 1.6 and 2.45 Å resolution, respectively. The structure reveals an octameric arrangement; however, both dimeric and octameric conformations were observed in solution. Furthermore, enzyme-activity assays show that only the octameric variant is catalytically active. Biochemical and structural studies indicate that the octameric form ofSa_enolase is enzymatically activein vitroand likely alsoin vivo, while the dimeric form is catalytically inactive and may be involved in other biological processes.

scholarly journals Biochemical insight into redox regulation of plastidial 3-phosphoglycerate dehydrogenase from Arabidopsis thaliana

2020 ◽  
Vol 295 (44) ◽  
pp. 14906-14915
Author(s):  
Keisuke Yoshida ◽  
Kinuka Ohtaka ◽  
Masami Yokota Hirai ◽  
Toru Hisabori
Keyword(s):  
Arabidopsis Thaliana ◽  
Enzyme Activity ◽  
Disulfide Bond ◽  
Protein Modification ◽  
Redox Regulation ◽  
Site Directed Mutagenesis ◽  
Reductive Activation ◽  
Wide Range ◽  
Phosphoglycerate Dehydrogenase

Thiol-based redox regulation is a post-translational protein modification for controlling enzyme activity by switching oxidation/reduction states of Cys residues. In plant cells, numerous proteins involved in a wide range of biological systems have been suggested as the target of redox regulation; however, our knowledge on this issue is still incomplete. Here we report that 3-phosphoglycerate dehydrogenase (PGDH) is a novel redox-regulated protein. PGDH catalyzes the first committed step of Ser biosynthetic pathway in plastids. Using an affinity chromatography-based method, we found that PGDH physically interacts with thioredoxin (Trx), a key factor of redox regulation. The in vitro studies using recombinant proteins from Arabidopsis thaliana showed that a specific PGDH isoform, PGDH1, forms the intramolecular disulfide bond under nonreducing conditions, which lowers PGDH enzyme activity. MS and site-directed mutagenesis analyses allowed us to identify the redox-active Cys pair that is mainly involved in disulfide bond formation in PGDH1; this Cys pair is uniquely found in land plant PGDH. Furthermore, we revealed that some plastidial Trx subtypes support the reductive activation of PGDH1. The present data show previously uncharacterized regulatory mechanisms of PGDH and expand our understanding of the Trx-mediated redox-regulatory network in plants.

scholarly journals Singapore grouper iridovirus-encoded semaphorin homologue (SGIV-sema) contributes to viral replication, cytoskeleton reorganization and inhibition of cellular immune responses

2014 ◽  
Vol 95 (5) ◽  
pp. 1144-1155 ◽  
Author(s):  
Yang Yan ◽  
Huachun Cui ◽  
Chuanyu Guo ◽  
Jingguang Wei ◽  
Youhua Huang ◽  
...  
Keyword(s):  
Immune Responses ◽  
Viral Replication ◽  
Host Cells ◽  
Early Gene ◽  
Coding Region ◽  
Cytoskeletal Structure ◽  
Wide Range ◽  
Fish Cells ◽  
Cellular Immune

Semaphorins are a large, phylogenetically conserved family of proteins that are involved in a wide range of biological processes including axonal steering, organogenesis, neoplastic transformation, as well as immune responses. In this study, a novel semaphorin homologue gene belonging to the Singapore grouper iridovirus (SGIV), ORF155R (termed SGIV-sema), was cloned and characterized. The coding region of SGIV-sema is 1728 bp in length, encoding a predicted protein with 575 aa. SGIV-sema contains a ~370 aa N-terminal Sema domain, a conserved plexin-semaphorin-integrin (PSI) domain, and an immunoglobulin (Ig)-like domain near the C terminus. SGIV-sema is an early gene product during viral infection and predominantly distributed in the cytoplasm with a speckled and clubbed pattern of appearance. Functionally, SGIV-sema could promote viral replication during SGIV infection in vitro, with no effect on the proliferation of host cells. Intriguingly, ectopically expressed SGIV-sema could alter the cytoskeletal structure of fish cells, characterized by a circumferential ring of microtubules near the nucleus and a disrupted microfilament organization. Furthermore, SGIV-sema was able to attenuate the cellular immune response, as demonstrated by decreased expression of inflammation/immune-related genes such as IL-8, IL-15, TNF-α and mediator of IRF3 activation (MITA), in SGIV-sema-expressing cells before and after SGIV infection. Ultimately, our study identified a novel, functional SGIV gene that could regulate cytoskeletal structure, immune responses and facilitate viral replication.

scholarly journals Isolation and Identification of Inter-Species Enterovirus Recombinant Genomes

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2390
Author(s):  
Kirsten Bentley ◽  
Han Kang Tee ◽  
Ashley Pearson ◽  
Kym Lowry ◽  
Sheila Waugh ◽  
...  
Keyword(s):  
Rna Virus ◽  
Progeny Virus ◽  
Template Switching ◽  
Coding Region ◽  
Isolation And Identification ◽  
Positive Strand ◽  
Wide Range ◽  
Positive Strand Rna ◽  
Infectious Progeny Virus

Positive-strand RNA virus evolution is partly attributed to the process of recombination. Although common between closely genetically related viruses, such as within species of the Enterovirus genus of the Picornaviridae family, inter-species recombination is rarely observed in nature. Recent studies have shown recombination is a ubiquitous process, resulting in a wide range of recombinant genomes and progeny viruses. While not all recombinant genomes yield infectious progeny virus, their existence and continued evolution during replication have critical implications for the evolution of the virus population. In this study, we utilised an in vitro recombination assay to demonstrate inter-species recombination events between viruses from four enterovirus species, A-D. We show that inter-species recombinant genomes are generated in vitro with polymerase template-switching events occurring within the virus polyprotein coding region. However, these genomes did not yield infectious progeny virus. Analysis and attempted recovery of a constructed recombinant cDNA revealed a restriction in positive-strand but not negative-strand RNA synthesis, indicating a significant block in replication. This study demonstrates the propensity for inter-species recombination at the genome level but suggests that significant sequence plasticity would be required in order to overcome blocks in the virus life cycle and allow for the production of infectious viruses.

scholarly journals THE IDENTIFICATION, PRODUCTION AND CHARACTERIZATION OF NATTOKINASE, BACTERIOCIN FROM BACTERIAL SPECIES

2019 ◽  
Vol 2 (4) ◽  
pp. 91
Author(s):  
Lal Krishna
Keyword(s):  
Enzyme Activity ◽  
Bacillus Subtilis ◽  
Pathogenic Bacteria ◽  
Blood Clot ◽  
Bacterial Species ◽  
Antagonistic Activity ◽  
Bacterial Strains ◽  
Wide Range

The study was aimed at identification, production and characterization of nattokinase, bacteriocin from bacterial species. Nattokinase and bacteriocins finds a wide range of applications in Pharmaceutical industry, health care and medicine. Nattokinase is a highly active fibrinolytic enzyme secreted by Bacillus subtilis and bacteriocins are proteinaceous toxins produced by Lactobacillus to inhibit the growth of closely related bacterial strains. Bacillus subtilis and Lactobacillus isolates shown positive results to microscopic, biochemical analysis.  The nattokinase and bacteriocins were produced by optimizing the media. The enzymes were purified by ammonium sulfate precipitation and HPLC. The enzyme activity for nattokinase was found at 7 mg/ml, pH 8.0 and temperature 48 ºC and the enzyme activity for bacteriocin was found at 3.9 mg/ml, pH 6.5 and temperature 30 °C. Bacteriocins from Lactobacillus showed good antagonistic activity against pathogenic bacteria. Nattokinase from Bacillus subtilis played a significant role in thrombolytic and anti-coagulation at in vitro. The results indicated that the pure enzyme has a potential in dissolving blood clot.

scholarly journals Structural and Kinetic Analysis of Substrate Binding to the Sialyltransferase Cst-II from Campylobacter jejuni

2011 ◽  
Vol 286 (41) ◽  
pp. 35922-35932 ◽  
Author(s):  
Ho Jun Lee ◽  
Luke L. Lairson ◽  
Jamie R. Rich ◽  
Emilie Lameignere ◽  
Warren W. Wakarchuk ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Catalytic Mechanism ◽  
Kinetic Mechanism ◽  
Sialic Acids ◽  
Biological Processes ◽  
Human Pathogen ◽  
Cell Surfaces ◽  
Acceptor Specificity ◽  
Wide Range ◽  
Insight Into

Sialic acids play important roles in various biological processes and typically terminate the oligosaccharide chains on the cell surfaces of a wide range of organisms, including mammals and bacteria. Their attachment is catalyzed by a set of sialyltransferases with defined specificities both for their acceptor sugars and the position of attachment. However, little is known of how this specificity is encoded. The structure of the bifunctional sialyltransferase Cst-II of the human pathogen Campylobacter jejuni in complex with CMP and the terminal trisaccharide of its natural acceptor (Neu5Ac-α-2,3-Gal-β-1,3-GalNAc) has been solved at 1.95 Å resolution, and its kinetic mechanism was shown to be iso-ordered Bi Bi, consistent with its dual acceptor substrate specificity. The trisaccharide acceptor is seen to bind to the active site of Cst-II through interactions primarily mediated by Asn-51, Tyr-81, and Arg-129. Kinetic and structural analyses of mutants modified at these positions indicate that these residues are critical for acceptor binding and catalysis, thereby providing significant new insight into the kinetic and catalytic mechanism, and acceptor specificity of this pathogen-encoded bifunctional GT-42 sialyltransferase.

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