A revised mechanism for (p)ppGpp synthesis by Rel proteins: The critical role of the 2′-OH of GTP

Bacterial Rel proteins synthesize hyperphosphorylated guanosine nucleotides, denoted as (p)ppGpp, which by inhibiting energy requiring molecular pathways help bacteria to overcome the depletion of nutrients in its surroundings. (p)ppGpp synthesis by Rel involves transferring a pyrophosphate from ATP to the oxygen of 3′-OH of GTP/GDP. Initially, a conserved glutamate at the active site was believed to generate the nucleophile necessary to accomplish the reaction. Later this role was alluded to a Mg2+ ion. However, no study has unequivocally established a catalytic mechanism for (p)ppGpp synthesis. Here we present a revised mechanism, wherein for the first time we explore a role for 2′-OH of GTP and show how it is important in generating the nucleophile. Through a careful comparison of substrate-bound structures of Rel, we illustrate that the active site does not discriminate GTP from dGTP, for a substrate. Using biochemical studies, we demonstrate that both GTP and dGTP bind to Rel, but only GTP (but not dGTP) can form the product. Reactions performed using GTP analogs substituted with different chemical moieties at the 2′ position suggest a clear role for 2′-OH in catalysis by providing an indispensable hydrogen bond; preliminary computational analysis further supports this view. This study elucidating a catalytic role for 2′-OH of GTP in (p)ppGpp synthesis allows us to propose different mechanistic possibilities by which it generates the nucleophile for the synthesis reaction. This study underscores the selection of ribose nucleotides as second messengers and finds its roots in the old RNA world hypothesis.

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Randomness and Complexity

10.1093/oso/9780198814825.003.0007 ◽

2018 ◽

Author(s):

Steven E. Vigdor

Keyword(s):

Quantum Mechanics ◽

Natural Selection ◽

Rna World ◽

Biological Evolution ◽

Variable Theory ◽

Cosmological Natural Selection ◽

Einstein Podolsky Rosen ◽

World Hypothesis ◽

Rna World Hypothesis

Chapter 7 describes the fundamental role of randomness in quantum mechanics, in generating the first biomolecules, and in biological evolution. Experiments testing the Einstein–Podolsky–Rosen paradox have demonstrated, via Bell’s inequalities, that no local hidden variable theory can provide a viable alternative to quantum mechanics, with its fundamental randomness built in. Randomness presumably plays an equally important role in the chemical assembly of a wide array of polymer molecules to be sampled for their ability to store genetic information and self-replicate, fueling the sort of abiogenesis assumed in the RNA world hypothesis of life’s beginnings. Evidence for random mutations in biological evolution, microevolution of both bacteria and antibodies and macroevolution of the species, is briefly reviewed. The importance of natural selection in guiding the adaptation of species to changing environments is emphasized. A speculative role of cosmological natural selection for black-hole fecundity in the evolution of universes is discussed.

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Rapid kinetics of second messenger production in bitter taste

AJP Cell Physiology ◽

10.1152/ajpcell.1996.270.3.c926 ◽

1996 ◽

Vol 270 (3) ◽

pp. C926-C931 ◽

Cited By ~ 56

Author(s):

A. I. Spielman ◽

H. Nagai ◽

G. Sunavala ◽

M. Dasso ◽

H. Breer ◽

...

Keyword(s):

Bitter Taste ◽

Second Messengers ◽

Second Messenger ◽

Mouse Strains ◽

Protective Mechanism ◽

Transient Nature ◽

Sucrose Octaacetate ◽

Rapid Kinetics ◽

First Time

The tasting of bitter compounds may have evolved as a protective mechanism against ingestion of potentially harmful substances. We have identified second messengers involved in bitter taste and show here for the first time that they are rapid and transient. Using a quench-flow system, we have studied bitter taste signal transduction in a pair of mouse strains that differ in their ability to taste the bitter stimulus sucrose octaacetate (SOA); however, both strains taste the bitter agent denatonium. In both strains of mice, denatonium (10 mM) induced a transient and rapid increase in levels of the second messenger inositol 1,4,5-trisphosphate (IP3) with a maximal production near 75-100 ms after stimulation. In contrast, SOA (100 microM) brought about a similar increase in IP3 only in SOA-taster mice. The response to SOA was potentiated in the presence of GTP (1 microM). The GTP-enhanced SOA-response supports a G protein-mediated response for this bitter compound. The rapid kinetics, transient nature, and specificity of the bitter taste stimulus-induced IP3 formation are consistent with the role of IP3 as a second messenger in the chemoelectrical transduction of bitter taste.

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The role of the K-channel and the active-site tyrosine in the catalytic mechanism of cytochrome c oxidase

Biochimica et Biophysica Acta (BBA) - Bioenergetics ◽

10.1016/j.bbabio.2016.04.014 ◽

2016 ◽

Vol 1857 ◽

pp. e2

Author(s):

Vivek Sharma ◽

Mårten Wikström

Keyword(s):

Cytochrome C ◽

Cytochrome C Oxidase ◽

Active Site ◽

Catalytic Mechanism ◽

K Channel ◽

Active Site Tyrosine

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Investigating the role of noncoding regulatory DNA in plasmid development for Yarrowia lipolytica

10.22541/au.160691063.38058320/v1 ◽

2020 ◽

Author(s):

Carmen Lopez ◽

Mingfeng Cao ◽

Zhanyi Yao ◽

Zengyi Shao

Keyword(s):

Yarrowia Lipolytica ◽

Plasmid Stability ◽

Critical Role ◽

Fold Increase ◽

Microbial Cell Factories ◽

Cell Factories ◽

Expression Platform ◽

Regulatory Dna ◽

Selection Of

Production of industrially relevant compounds in microbial cell factories can employ either genomes or plasmids as an expression platform. Selection of plasmids as pathway carriers is advantageous for rapid demonstration but poses a challenge of stability. Yarrowia lipolytica has attracted great attention in the past decade for the biosynthesis of chemicals related to fatty acids at titers attractive to industry, and many genetic tools have been developed to explore its oleaginous potential. Our recent studies on the autonomously replicating sequences (ARSs) of nonconventional yeasts revealed that the ARSs from Y. lipolytica showcase a unique structure that includes a previously unannotated sequence (spacer) linking the origin of replication (ORI) and the centromeric (CEN) element and plays a critical role in modulating plasmid behavior. Maintaining a native 645-bp spacer yielded a 4.5-fold increase in gene expression and higher plasmid stability compared to a more universally employed minimized ARS. Testing the modularity of the ARS sub-elements indicated that plasmid stability exhibits a pronounced cargo dependency. Instability caused both plasmid loss and intramolecular rearrangements. Altogether, our work clarifies the appropriate application of various ARSs for the scientific community and sheds light on a previously unexplored DNA element as a potential target for engineering Y. lipolytica.

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Transcription and translation in an RNA world

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2006.1910 ◽

2006 ◽

Vol 361 (1474) ◽

pp. 1751-1760 ◽

Cited By ~ 8

Author(s):

William R Taylor

Keyword(s):

Amino Acid ◽

Active Site ◽

Rna World ◽

Large Subunit ◽

Trna Synthetase ◽

Nucleoside Triphosphate ◽

Amino Acid Binding ◽

World Hypothesis ◽

Protein Elongation ◽

Rna World Hypothesis

The RNA world hypothesis requires a ribozyme that was an RNA-directed RNA polymerase (ribopolymerase). If such a replicase makes a reverse complementary copy of any sequence (including itself), in a simple RNA world, there is no mechanism to prevent self-hybridization. It is proposed that this can be avoided through the synthesis of a parallel complementary copy. The logical consequences of this are pursued and developed in a computer simulation, where the behaviour of the parallel copy is compared to the conventional reverse complementary copy. It is found that the parallel copy is more efficient at higher temperatures (up to 90°C). A model for the ribopolymerase, based on the core of the large subunit (LSU) of the ribosome, is described. The geometry of a potential active site for this ribopolymerase suggests that it contained a cavity (now occupied by the aminoacyl-tRNA) and that an amino acid binding in this might have ‘poisoned’ the ribopolymerase by cross-reacting with the nucleoside-triphosphate before polymerization could occur. Based on a similarity to the active site components of the class-I tRNA synthetase enzymes, it is proposed that the amino acid could become attached to the nascent RNA transcript producing a variety of aminoacylated tRNA-like products. Using base-pairing interactions, some of these molecules might cross-link two ribopolymerases, giving rise to a precursor of the modern ribosome. A hybrid dimer, half polymerase and half proto-ribosome, could account for mRNA translocation before the advent of protein elongation factors.

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Unveiling the critical role of active site interaction in single atom catalyst towards hydrogen evolution catalysis

Nano Energy ◽

10.1016/j.nanoen.2021.106819 ◽

2022 ◽

Vol 93 ◽

pp. 106819

Author(s):

Feng Li ◽

Gao-Feng Han ◽

Yunfei Bu ◽

Shanshan Chen ◽

Ishfaq Ahmad ◽

...

Keyword(s):

Hydrogen Evolution ◽

Active Site ◽

Critical Role ◽

Single Atom

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A Few Experimental Suggestions Using Minerals to Obtain Peptides with a High Concentration of L-Amino Acids and Protein Amino Acids

Symmetry ◽

10.3390/sym12122046 ◽

2020 ◽

Vol 12 (12) ◽

pp. 2046

Author(s):

Dimas A. M. Zaia ◽

Cássia Thaïs B. V. Zaia

Keyword(s):

Amino Acids ◽

Rna World ◽

High Concentration ◽

Protein World ◽

Protein Amino Acids ◽

Hydrothermal Environment ◽

World Hypothesis ◽

Prebiotic Earth ◽

Hydrothermal Environments ◽

Selection Of

The peptides/proteins of all living beings on our planet are mostly made up of 19 L-amino acids and glycine, an achiral amino acid. Arising from endogenous and exogenous sources, the seas of the prebiotic Earth could have contained a huge diversity of biomolecules (including amino acids), and precursors of biomolecules. Thus, how were these amino acids selected from the huge number of available amino acids and other molecules? What were the peptides of prebiotic Earth made up of? How were these peptides synthesized? Minerals have been considered for this task, since they can preconcentrate amino acids from dilute solutions, catalyze their polymerization, and even make the chiral selection of them. However, until now, this problem has only been studied in compartmentalized experiments. There are separate experiments showing that minerals preconcentrate amino acids by adsorption or catalyze their polymerization, or separate L-amino acids from D-amino acids. Based on the [GADV]-protein world hypothesis, as well as the relative abundance of amino acids on prebiotic Earth obtained by Zaia, several experiments are suggested. The main goal of these experiments is to show that using minerals it is possible, at least, to obtain peptides whose composition includes a high quantity of L-amino acids and protein amino acids (PAAs). These experiments should be performed using hydrothermal environments and wet/dry cycles. In addition, for hydrothermal environment experiments, it is very important to use one of the suggested artificial seawaters, and for wet/dry environments, it is important to perform the experiments in distilled water and diluted salt solutions. Finally, from these experiments, we suggest that, without an RNA world or even a pre genetic world, a small peptide set could emerge that better resembles modern proteins.

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GlcNAcstatins are nanomolar inhibitors of human O-GlcNAcase inducing cellular hyper-O-GlcNAcylation

Biochemical Journal ◽

10.1042/bj20090110 ◽

2009 ◽

Vol 420 (2) ◽

pp. 221-227 ◽

Cited By ~ 56

Author(s):

Helge C. Dorfmueller ◽

Vladimir S. Borodkin ◽

Marianne Schimpl ◽

Daan M. F. van Aalten

Keyword(s):

Active Site ◽

Cell Biology ◽

Rational Design ◽

Catalytic Mechanism ◽

Conserved Residues ◽

Cellular Processes ◽

Acetyl Group ◽

Nanomolar Range ◽

Insight Into

O-GlcNAcylation is an essential, dynamic and inducible post-translational glycosylation of cytosolic proteins in metazoa and can show interplay with protein phosphorylation. Inhibition of OGA (O-GlcNAcase), the enzyme that removes O-GlcNAc from O-GlcNAcylated proteins, is a useful strategy to probe the role of this modification in a range of cellular processes. In the present study, we report the rational design and evaluation of GlcNAcstatins, a family of potent, competitive and selective inhibitors of human OGA. Kinetic experiments with recombinant human OGA reveal that the GlcNAcstatins are the most potent human OGA inhibitors reported to date, inhibiting the enzyme in the sub-nanomolar to nanomolar range. Modification of the GlcNAcstatin N-acetyl group leads to up to 160-fold selectivity against the human lysosomal hexosaminidases which employ a similar substrate-assisted catalytic mechanism. Mutagenesis studies in a bacterial OGA, guided by the structure of a GlcNAcstatin complex, provides insight into the role of conserved residues in the human OGA active site. GlcNAcstatins are cell-permeant and, at low nanomolar concentrations, effectively modulate intracellular O-GlcNAc levels through inhibition of OGA, in a range of human cell lines. Thus these compounds are potent selective tools to study the cell biology of O-GlcNAc.

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Albino mutants of Streptomyces glaucescens tyrosinase

Biochemical Journal ◽

10.1042/bj2740707 ◽

1991 ◽

Vol 274 (3) ◽

pp. 707-713 ◽

Cited By ~ 34

Author(s):

M P Jackman ◽

A Hajnal ◽

K Lerch

Keyword(s):

Active Site ◽

Critical Role ◽

Site Directed Mutagenesis ◽

Mutant Proteins ◽

Carbonyl Derivatives ◽

Hydrogen Bonding Interactions ◽

Albino Phenotype ◽

Derivatives Of ◽

Streptomyces Glaucescens

Site-directed mutagenesis was used to determine the functional role of several residues of Streptomyces glaucescens tyrosinase. Replacement of His-37, -53, -193 or -215 by glutamine yields albino phenotypes, as determined by expression on melanin-indicator plates. The purified mutant proteins display no detectable oxy-enzyme and increased Cu lability at the binuclear active site. The carbonyl derivatives of H189Q and H193Q luminesce, with lambda max. displaced more than 25 nm to a longer wavelength compared with native tyrosinase. The remaining histidine mutants display no detectable luminescence. The results are consistent with these histidine residues (together with His-62 and His-189 reported earlier) acting as Cu ligands in the Streptomyces glaucescens enzyme. Conservative substitution of the invariant Asn-190 by glutamine also gives an albino phenotype, no detectable oxy-enzyme and labilization of active-site Cu. The luminescence spectrum of carbonyl-N190Q, however, closely resembles that of the native enzyme under conditions promoting double Cu occupancy of the catalytic site. A critical role for Asn-190 in active-site hydrogen-bonding interactions is proposed.

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