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Biomolecules Dec 2023For more than 60 years, glutathione transferases (GSTs) have attracted attention, but the research field of the GSTome [...].
For more than 60 years, glutathione transferases (GSTs) have attracted attention, but the research field of the GSTome [...].
Topics: Glutathione Transferase; Glutathione
PubMed: 38136620
DOI: 10.3390/biom13121749 -
FEBS Letters Dec 2001CoA-transferases are found in organisms from all lines of descent. Most of these enzymes belong to two well-known enzyme families, but recent work on unusual biochemical... (Review)
Review
CoA-transferases are found in organisms from all lines of descent. Most of these enzymes belong to two well-known enzyme families, but recent work on unusual biochemical pathways of anaerobic bacteria has revealed the existence of a third family of CoA-transferases. The members of this enzyme family differ in sequence and reaction mechanism from CoA-transferases of the other families. Currently known enzymes of the new family are a formyl-CoA: oxalate CoA-transferase, a succinyl-CoA: (R)-benzylsuccinate CoA-transferase, an (E)-cinnamoyl-CoA: (R)-phenyllactate CoA-transferase, and a butyrobetainyl-CoA: (R)-carnitine CoA-transferase. In addition, a large number of proteins of unknown or differently annotated function from Bacteria, Archaea and Eukarya apparently belong to this enzyme family. Properties and reaction mechanisms of the CoA-transferases of family III are described and compared to those of the previously known CoA-transferases.
Topics: Bacteria, Anaerobic; Bacterial Proteins; Binding, Competitive; Coenzyme A-Transferases; Kinetics; Substrate Specificity; Thiolester Hydrolases
PubMed: 11749953
DOI: 10.1016/s0014-5793(01)03178-7 -
Current Opinion in Biotechnology Feb 2019Produced as linear biopolymers from four major types of building blocks, DNA and RNA are further furnished with a range of covalent modifications. Despite the impressive... (Review)
Review
Produced as linear biopolymers from four major types of building blocks, DNA and RNA are further furnished with a range of covalent modifications. Despite the impressive specificity of natural enzymes, the transferred groups are often poor reporters and not amenable to further derivatization. Therefore, strategies based on repurposing some of these enzymatic reactions to accept derivatized versions of the transferrable groups have been exploited. By far the most widely used are S-adenosylmethionine-dependent methyltransferases, which along with several other nucleic acids modifying enzymes offer a broad selection of tagging chemistries and molecular features on DNA and RNA that can be targeted in vitro and in vivo. Engineered enzymatic reactions have been implemented in validated DNA sequencing-based protocols for epigenome analysis. The utility of chemo-enzymatic labeling is further enhanced with recent advances in physical detection of individual reporter groups on DNA using super resolution microscopy and nanopore sensing enabling single-molecule multiplex analysis of genetic and epigenetic marks in minute samples. Altogether, a number of new powerful techniques are currently in use or on the verge of real benchtop applications as research tools or next generation diagnostics.
Topics: DNA; Epigenesis, Genetic; Protein Engineering; RNA; Staining and Labeling; Transferases
PubMed: 30296696
DOI: 10.1016/j.copbio.2018.09.008 -
Plant Biotechnology Journal Nov 2019Natural rubber (NR) is a nonfungible and valuable biopolymer, used to manufacture ~50 000 rubber products, including tires and medical gloves. Current production of NR... (Review)
Review
Natural rubber (NR) is a nonfungible and valuable biopolymer, used to manufacture ~50 000 rubber products, including tires and medical gloves. Current production of NR is derived entirely from the para rubber tree (Hevea brasiliensis). The increasing demand for NR, coupled with limitations and vulnerability of H. brasiliensis production systems, has induced increasing interest among scientists and companies in potential alternative NR crops. Genetic/metabolic pathway engineering approaches, to generate NR-enriched genotypes of alternative NR plants, are of great importance. However, although our knowledge of rubber biochemistry has significantly advanced, our current understanding of NR biosynthesis, the biosynthetic machinery and the molecular mechanisms involved remains incomplete. Two spatially separated metabolic pathways provide precursors for NR biosynthesis in plants and their genes and enzymes/complexes are quite well understood. In contrast, understanding of the proteins and genes involved in the final step(s)-the synthesis of the high molecular weight rubber polymer itself-is only now beginning to emerge. In this review, we provide a critical evaluation of recent research developments in NR biosynthesis, in vitro reconstitution, and the genetic and metabolic pathway engineering advances intended to improve NR content in plants, including H. brasiliensis, two other prospective alternative rubber crops, namely the rubber dandelion and guayule, and model species, such as lettuce. We describe a new model of the rubber transferase complex, which integrates these developments. In addition, we highlight the current challenges in NR biosynthesis research and future perspectives on metabolic pathway engineering of NR to speed alternative rubber crop commercial development.
Topics: Hevea; Metabolic Engineering; Rubber; Transferases
PubMed: 31150158
DOI: 10.1111/pbi.13181 -
The Journal of Biological Chemistry Mar 1996
Review
Topics: Alkyl and Aryl Transferases; Amino Acid Sequence; Animals; Enzyme Inhibitors; Farnesyltranstransferase; Kinetics; Molecular Sequence Data; Protein Prenylation; Saccharomyces cerevisiae; Substrate Specificity; Transferases
PubMed: 8621375
DOI: 10.1074/jbc.271.10.5289 -
The Journal of Biological Chemistry 2021Brassinosteroids (BRs) are steroid hormones of plants that coordinate fundamental growth and development processes. Their homeostasis is controlled by diverse means,...
Brassinosteroids (BRs) are steroid hormones of plants that coordinate fundamental growth and development processes. Their homeostasis is controlled by diverse means, including glucosylation of the bioactive BR brassinolide (BL), which is catalyzed by the UDP-glycosyltransferases (UGTs) UGT73C5 and UGT73C6 and occurs mainly at the C-23 position. Additional evidence had suggested that the resultant BL-23-O-glucoside (BL-23-O-Glc) can be malonylated, but the physiological significance of and enzyme required for this reaction had remained unknown. Here, we show that in Arabidopsis thaliana malonylation of BL-23-O-Glc is catalyzed by the acyltransferase phenolic glucoside malonyl-transferase 1 (PMAT1), which is also known to malonylate phenolic glucosides and lipid amides. Loss of PMAT1 abolished BL-23-O-malonylglucoside formation and enriched BL-23-O-Glc, showing that the enzyme acts on the glucoside. An overexpression of PMAT1 in plants where UGT73C6 was also overexpressed, and thus, BL-23-O-Glc formation was promoted, enhanced the symptoms of BR-deficiency of UGT73C6oe plants, providing evidence that PMAT1 contributes to BL inactivation. Based on these results, a model is proposed in which PMAT1 acts in the conversion of both endogenous and xenobiotic glucosides to adjust metabolic homeostasis in spatial and temporal modes.
Topics: Acyltransferases; Arabidopsis; Arabidopsis Proteins; Brassinosteroids; Gene Expression Regulation, Plant; Glucosides; Glycosyltransferases; Plants, Genetically Modified; Steroids; Steroids, Heterocyclic; Transferases
PubMed: 33600798
DOI: 10.1016/j.jbc.2021.100424 -
Applied and Environmental Microbiology Apr 2019LeLoir glycosyltransferases are important biocatalysts for the production of glycosidic bonds in natural products, chiral building blocks, and pharmaceuticals. Trehalose...
LeLoir glycosyltransferases are important biocatalysts for the production of glycosidic bonds in natural products, chiral building blocks, and pharmaceuticals. Trehalose transferase (TreT) is of particular interest since it catalyzes the stereo- and enantioselective α,α-(1→1) coupling of a nucleotide sugar donor and monosaccharide acceptor for the synthesis of disaccharide derivatives. Heterologously expressed thermophilic trehalose transferases were found to be intrinsically aggregation prone and are mainly expressed as catalytically active inclusion bodies in To disfavor protein aggregation, the thermostable protein mCherry was explored as a fluorescent protein tag. The fusion of mCherry to trehalose transferase from (TreT) demonstrated increased protein solubility. Chaotropic agents like guanidine or the divalent cations Mn(II), Ca(II), and Mg(II) enhanced the enzyme activity of the fusion protein. The thermodynamic equilibrium constant, , for the reversible synthesis of trehalose from glucose and a nucleotide sugar was determined in both the synthesis and hydrolysis directions utilizing UDP-glucose and ADP-glucose, respectively. UDP-glucose was shown to achieve higher conversions than ADP-glucose, highlighting the importance of the choice of nucleotide sugars for LeLoir glycosyltransferases under thermodynamic control. The heterologous expression of proteins in is of great relevance for their functional and structural characterization and applications. However, the formation of insoluble inclusion bodies is observed in approximately 70% of all cases, and the subsequent effects can range from reduced soluble protein yields to a complete failure of the expression system. Here, we present an efficient methodology for the production and analysis of a thermostable, aggregation-prone trehalose transferase (TreT) from via its fusion with mCherry as a thermostable fluorescent protein tag. This fusion strategy allowed for increased enzyme stability and solubility and could be applied to other (thermostable) proteins, allowing rapid visualization and quantification of the mCherry-fused protein of interest. Finally, we have demonstrated that the enzymatic synthesis of trehalose from glucose and a nucleotide sugar is reversible by approaching the thermodynamic equilibrium in both the synthesis and hydrolysis directions. Our results show that uridine establishes an equilibrium constant which is more in favor of the product trehalose than when adenosine is employed as the nucleotide under identical conditions. The influence of different nucleotides on the reaction can be generalized for all LeLoir glycosyltransferases under thermodynamic control as the position of the equilibrium depends solely on the reaction conditions and is not affected by the nature of the catalyst.
Topics: Adenosine Diphosphate Glucose; Anions; Catalysis; Cations; Enzyme Activation; Enzyme Stability; Escherichia coli; Genetic Vectors; Glucose; Glycosyltransferases; Kinetics; Protein Aggregates; Pyrobaculum; Recombinant Fusion Proteins; Solubility; Transferases; Trehalose; Uridine Diphosphate Glucose
PubMed: 30737350
DOI: 10.1128/AEM.03084-18 -
Applied Microbiology and Biotechnology Mar 2023The ADP-ribosyl transferase activity of P. aeruginosa PE24 moiety expressed by E. coli BL21 (DE3) was assessed on nitrobenzylidene aminoguanidine (NBAG) and in vitro...
The ADP-ribosyl transferase activity of P. aeruginosa PE24 moiety expressed by E. coli BL21 (DE3) was assessed on nitrobenzylidene aminoguanidine (NBAG) and in vitro cultured cancer cell lines. Gene encoding PE24 was isolated from P. aeruginosa isolates, cloned into pET22b( +) plasmid, and expressed in E. coli BL21 (DE3) under IPTG induction. Genetic recombination was confirmed by colony PCR, the appearance of insert post digestion of engineered construct, and protein electrophoresis using sodium dodecyl-sulfate polyacrylamide gel (SDS-PAGE). The chemical compound NBAG has been used to confirm PE24 extract ADP-ribosyl transferase action through UV spectroscopy, FTIR, c-NMR, and HPLC before and after low-dose gamma irradiation (5, 10, 15, 24 Gy). The cytotoxicity of PE24 extract alone and in combination with paclitaxel and low-dose gamma radiation (both 5 Gy and one shot 24 Gy) was assessed on adherent cell lines HEPG2, MCF-7, A375, OEC, and Kasumi-1 cell suspension. Expressed PE24 moiety ADP-ribosylated NBAG as revealed by structural changes depicted by FTIR and NMR, and the surge of new peaks at different retention times from NBAG in HPLC chromatograms. Irradiating recombinant PE24 moiety was associated with a reduction in ADP-ribosylating activity. The PE24 extract IC50 values were < 10 μg/ml with an acceptable R value on cancer cell lines and acceptable cell viability at 10 μg/ml on normal OEC. Overall, the synergistic effects were observed upon combining PE24 extract with low-dose paclitaxel demonstrated by the reduction in IC50 whereas antagonistic effects and a rise in IC50 values were recorded after irradiation by low-dose gamma rays. KEY POINTS: • Recombinant PE24 moiety was successfully expressed and biochemically analyzed. • Low-dose gamma radiation and metal ions decreased the recombinant PE24 cytotoxic activity. • Synergism was observed upon combining recombinant PE24 with low-dose paclitaxel.
Topics: ADP Ribose Transferases; Pseudomonas aeruginosa; Gamma Rays; Escherichia coli
PubMed: 36808279
DOI: 10.1007/s00253-023-12401-x -
Scientific Reports Dec 2021Peptide bond formation on the ribosome requires that aminoacyl-tRNAs and peptidyl-tRNAs are properly positioned on the A site and the P site of the peptidyl transferase...
Peptide bond formation on the ribosome requires that aminoacyl-tRNAs and peptidyl-tRNAs are properly positioned on the A site and the P site of the peptidyl transferase center (PTC) so that nucleophilic attack can occur. Here we analyse some constraints associated with the induced-fit mechanism of the PTC, that promotes this positioning through a compaction around the aminoacyl ester orchestrated by U2506. The physical basis of PTC decompaction, that allows the elongated peptidyl-tRNA to free itself from that state and move to the P site of the PTC, is still unclear. From thermodynamics considerations and an analysis of published ribosome structures, the present work highlights the rational of this mechanism, in which the free-energy released by the new peptide bond is used to kick U2506 away from the reaction center. Furthermore, we show the evidence that decompaction is impaired when the nascent peptide is not yet anchored inside the exit tunnel, which may contribute to explain why the first rounds of elongation are inefficient, an issue that has attracted much interest for about two decades. Results in this field are examined in the light of the present analysis and a physico-chemical correlation in the genetic code, which suggest that elementary constraints associated with the size of the side-chain of the amino acids penalize early elongation events.
Topics: Amino Acids; Binding Sites; Models, Molecular; Peptide Chain Elongation, Translational; Peptides; Peptidyl Transferases; Protein Binding; Protein Biosynthesis; Protein Conformation; Ribosomes; Structure-Activity Relationship
PubMed: 34911999
DOI: 10.1038/s41598-021-02985-7 -
Biochemistry and Molecular Biology... 2011Argumentation and discourse are two integral parts of scientific investigation that are often overlooked in undergraduate science education. To address this limitation,... (Review)
Review
Argumentation and discourse are two integral parts of scientific investigation that are often overlooked in undergraduate science education. To address this limitation, the story of peptide bond formation by the ribosome can be used to illustrate the importance of evidence, claims, arguments, and counterarguments in scientific discourse. With the determination of the first structure of the large ribosomal subunit bound to a transition state inhibitor came an initial hypothesis about the role of the ribosome in peptide bond formation. This initial hypothesis was based on a few central assumptions about the transition state mimic and acid-base catalysis by serine proteases. The initial proposed mechanism started a flurry of scientific discourse in experimental articles and commentaries that tested the validity of the initial proposed mechanism. Using this civil argumentation as a guide, class discussions, assignments, and a debate were designed that allow students to analyze and question the claims and evidence about the mechanism of peptide bond synthesis. In the end, students develop a sense of critical skepticism, and an understanding of scientific discourse, while learning about the current consensus mechanism for peptide bond synthesis. Biochemistry and Molecular Biology Education Vol. 39, No. 3, pp. 185-190, 2011.
Topics: Catalysis; Comprehension; Consensus; Dissent and Disputes; Humans; Models, Biological; Models, Molecular; Peptidyl Transferases; Ribosomes; Teaching
PubMed: 21618381
DOI: 10.1002/bmb.20495