Publications by authors named "Christopher S Francklyn"

35 Publications

De Novo and Bi-allelic Pathogenic Variants in NARS1 Cause Neurodevelopmental Delay Due to Toxic Gain-of-Function and Partial Loss-of-Function Effects.

Am J Hum Genet 2020 08 31;107(2):311-324. Epub 2020 Jul 31.

Bezmiâlem Vakıf Üniversitesi, Istanbul, 34093, Turkey.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitous, ancient enzymes that charge amino acids to cognate tRNA molecules, the essential first step of protein translation. Here, we describe 32 individuals from 21 families, presenting with microcephaly, neurodevelopmental delay, seizures, peripheral neuropathy, and ataxia, with de novo heterozygous and bi-allelic mutations in asparaginyl-tRNA synthetase (NARS1). We demonstrate a reduction in NARS1 mRNA expression as well as in NARS1 enzyme levels and activity in both individual fibroblasts and induced neural progenitor cells (iNPCs). Molecular modeling of the recessive c.1633C>T (p.Arg545Cys) variant shows weaker spatial positioning and tRNA selectivity. We conclude that de novo and bi-allelic mutations in NARS1 are a significant cause of neurodevelopmental disease, where the mechanism for de novo variants could be toxic gain-of-function and for recessive variants, partial loss-of-function.
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http://dx.doi.org/10.1016/j.ajhg.2020.06.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7413890PMC
August 2020

Neuropathy-associated histidyl-tRNA synthetase variants attenuate protein synthesis in vitro and disrupt axon outgrowth in developing zebrafish.

FEBS J 2021 01 6;288(1):142-159. Epub 2020 Jul 6.

Department of Biochemistry, University of Vermont, Burlington, VT, USA.

Charcot-Marie-Tooth disease (CMT) encompasses a set of genetically and clinically heterogeneous neuropathies characterized by length-dependent dysfunction of the peripheral nervous system. Mutations in over 80 diverse genes are associated with CMT, and aminoacyl-tRNA synthetases (ARS) constitute a large gene family implicated in the disease. Despite considerable efforts to elucidate the mechanistic link between ARS mutations and the CMT phenotype, the molecular basis of the pathology is unknown. In this work, we investigated the impact of three CMT-associated substitutions (V155G, Y330C, and R137Q) in the cytoplasmic histidyl-tRNA synthetase (HARS1) on neurite outgrowth and peripheral nervous system development. The model systems for this work included a nerve growth factor-stimulated neurite outgrowth model in rat pheochromocytoma cells (PC12), and a zebrafish line with GFP/red fluorescent protein reporters of sensory and motor neuron development. The expression of CMT-HARS1 mutations led to attenuation of protein synthesis and increased phosphorylation of eIF2α in PC12 cells and was accompanied by impaired neurite and axon outgrowth in both models. Notably, these effects were phenocopied by histidinol, a HARS1 inhibitor, and cycloheximide, a protein synthesis inhibitor. The mutant proteins also formed heterodimers with wild-type HARS1, raising the possibility that CMT-HARS1 mutations cause disease through a dominant-negative mechanism. Overall, these findings support the hypothesis that CMT-HARS1 alleles exert their toxic effect in a neuronal context, and lead to dysregulated protein synthesis. These studies demonstrate the value of zebrafish as a model for studying mutant alleles associated with CMT, and for characterizing the processes that lead to peripheral nervous system dysfunction.
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http://dx.doi.org/10.1111/febs.15449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7736457PMC
January 2021

Immunity-Guided Identification of Threonyl-tRNA Synthetase as the Molecular Target of Obafluorin, a β-Lactone Antibiotic.

ACS Chem Biol 2019 12 14;14(12):2663-2671. Epub 2019 Nov 14.

Department of Molecular Microbiology , John Innes Centre , Norwich Research Park , Norwich NR4 7UH , U.K.

To meet the ever-growing demands of antibiotic discovery, new chemical matter and antibiotic targets are urgently needed. Many potent natural product antibiotics which were previously discarded can also provide lead molecules and drug targets. One such example is the structurally unique β-lactone obafluorin, produced by ATCC 39502. Obafluorin is active against both Gram-positive and -negative pathogens; however, the biological target was unknown. We now report that obafluorin targets threonyl-tRNA synthetase, and we identify a homologue, ObaO, which confers immunity to the obafluorin producer. Disruption of in ATCC 39502 results in obafluorin sensitivity, whereas expression in sensitive strains confers resistance. Enzyme assays demonstrate that threonyl-tRNA synthetase is fully inhibited by obafluorin, whereas ObaO is only partly susceptible, exhibiting a very unusual partial inhibition mechanism. Altogether, our data highlight the utility of an immunity-guided approach for the identification of an antibiotic target and will ultimately enable the generation of improved obafluorin variants.
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http://dx.doi.org/10.1021/acschembio.9b00590DOI Listing
December 2019

Progress and challenges in aminoacyl-tRNA synthetase-based therapeutics.

J Biol Chem 2019 04 22;294(14):5365-5385. Epub 2019 Jan 22.

From the Department of Biochemistry, College of Medicine, University of Vermont, Burlington, Vermont 05405.

Aminoacyl-tRNA synthetases (ARSs) are universal enzymes that catalyze the attachment of amino acids to the 3' ends of their cognate tRNAs. The resulting aminoacylated tRNAs are escorted to the ribosome where they enter protein synthesis. By specifically matching amino acids to defined anticodon sequences in tRNAs, ARSs are essential to the physical interpretation of the genetic code. In addition to their canonical role in protein synthesis, ARSs are also involved in RNA splicing, transcriptional regulation, translation, and other aspects of cellular homeostasis. Likewise, aminoacylated tRNAs serve as amino acid donors for biosynthetic processes distinct from protein synthesis, including lipid modification and antibiotic biosynthesis. Thanks to the wealth of details on ARS structures and functions and the growing appreciation of their additional roles regulating cellular homeostasis, opportunities for the development of clinically useful ARS inhibitors are emerging to manage microbial and parasite infections. Exploitation of these opportunities has been stimulated by the discovery of new inhibitor frameworks, the use of semi-synthetic approaches combining chemistry and genome engineering, and more powerful techniques for identifying leads from the screening of large chemical libraries. Here, we review the inhibition of ARSs by small molecules, including the various families of natural products, as well as inhibitors developed by either rational design or high-throughput screening as antibiotics and anti-parasitic therapeutics.
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http://dx.doi.org/10.1074/jbc.REV118.002956DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6462538PMC
April 2019

A single Danio rerio hars gene encodes both cytoplasmic and mitochondrial histidyl-tRNA synthetases.

PLoS One 2017 21;12(9):e0185317. Epub 2017 Sep 21.

Department of Biology, University of Vermont, Burlington, VT, United States of America.

Histidyl tRNA Synthetase (HARS) is a member of the aminoacyl tRNA synthetase (ARS) family of enzymes. This family of 20 enzymes is responsible for attaching specific amino acids to their cognate tRNA molecules, a critical step in protein synthesis. However, recent work highlighting a growing number of associations between ARS genes and diverse human diseases raises the possibility of new and unexpected functions in this ancient enzyme family. For example, mutations in HARS have been linked to two different neurological disorders, Usher Syndrome Type IIIB and Charcot Marie Tooth peripheral neuropathy. These connections raise the possibility of previously undiscovered roles for HARS in metazoan development, with alterations in these functions leading to complex diseases. In an attempt to establish Danio rerio as a model for studying HARS functions in human disease, we characterized the Danio rerio hars gene and compared it to that of human HARS. Using a combination of bioinformatics, molecular biology, and cellular approaches, we found that while the human genome encodes separate genes for cytoplasmic and mitochondrial HARS protein, the Danio rerio genome encodes a single hars gene which undergoes alternative splicing to produce the respective cytoplasmic and mitochondrial versions of Hars. Nevertheless, while the HARS genes of humans and Danio differ significantly at the genomic level, we found that they are still highly conserved at the amino acid level, underscoring the potential utility of Danio rerio as a model organism for investigating HARS function and its link to human diseases in vivo.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0185317PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608375PMC
October 2017

The Usher Syndrome Type IIIB Histidyl-tRNA Synthetase Mutation Confers Temperature Sensitivity.

Biochemistry 2017 07 7;56(28):3619-3631. Epub 2017 Jul 7.

Department of Biochemistry, University of Vermont , Burlington, Vermont 05405, United States.

Histidyl-tRNA synthetase (HARS) is a highly conserved translation factor that plays an essential role in protein synthesis. HARS has been implicated in the human syndromes Charcot-Marie-Tooth (CMT) Type 2W and Type IIIB Usher (USH3B). The USH3B mutation, which encodes a Y454S substitution in HARS, is inherited in an autosomal recessive fashion and associated with childhood deafness, blindness, and episodic hallucinations during acute illness. The biochemical basis of the pathophysiologies linked to USH3B is currently unknown. Here, we present a detailed functional comparison of wild-type (WT) and Y454S HARS enzymes. Kinetic parameters for enzymes and canonical substrates were determined using both steady state and rapid kinetics. Enzyme stability was examined using differential scanning fluorimetry. Finally, enzyme functionality in a primary cell culture was assessed. Our results demonstrate that the Y454S substitution leaves HARS amino acid activation, aminoacylation, and tRNA binding functions largely intact compared with those of WT HARS, and the mutant enzyme dimerizes like the wild type does. Interestingly, during our investigation, it was revealed that the kinetics of amino acid activation differs from that of the previously characterized bacterial HisRS. Despite the similar kinetics, differential scanning fluorimetry revealed that Y454S is less thermally stable than WT HARS, and cells from Y454S patients grown at elevated temperatures demonstrate diminished levels of protein synthesis compared to those of WT cells. The thermal sensitivity associated with the Y454S mutation represents a biochemical basis for understanding USH3B.
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http://dx.doi.org/10.1021/acs.biochem.7b00114DOI Listing
July 2017

Characterization of aminoacyl-tRNA synthetase stability and substrate interaction by differential scanning fluorimetry.

Methods 2017 01 26;113:64-71. Epub 2016 Oct 26.

Department of Biochemistry, University of Vermont, Burlington, VT 05405, USA. Electronic address:

Differential scanning fluorimetry (DSF) is a fluorescence-based assay to evaluate protein stability by determining protein melting temperatures. Here, we describe the application of DSF to investigate aminoacyl-tRNA synthetase (AARS) stability and interaction with ligands. Employing three bacterial AARS enzymes as model systems, methods are presented here for the use of DSF to measure the apparent temperatures at which AARSs undergo melting transitions, and the effect of AARS substrates and inhibitors. One important observation is that the extent of temperature stability realized by an AARS in response to a particular bound ligand cannot be predicted a priori. The DSF method thus serves as a rapid and highly quantitative approach to measure AARS stability, and the ability of ligands to influence the temperature at which unfolding transitions occur.
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http://dx.doi.org/10.1016/j.ymeth.2016.10.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5493479PMC
January 2017

Aminoacyl-Transfer RNA Synthetases: Connecting Nutrient Status to Angiogenesis Through the Unfolded Protein Response.

Arterioscler Thromb Vasc Biol 2016 Apr;36(4):582-3

From the Departments of Pharmacology (K.M.L.) and Biochemistry (C.S.F.), University of Vermont, College of Medicine, Burlington.

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http://dx.doi.org/10.1161/ATVBAHA.116.307193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4807867PMC
April 2016

Aminoacyl-tRNA synthetase dependent angiogenesis revealed by a bioengineered macrolide inhibitor.

Sci Rep 2015 Aug 14;5:13160. Epub 2015 Aug 14.

Department of Biochemistry, University of Vermont.

Aminoacyl-tRNA synthetases (AARSs) catalyze an early step in protein synthesis, but also regulate diverse physiological processes in animal cells. These include angiogenesis, and human threonyl-tRNA synthetase (TARS) represents a potent pro-angiogenic AARS. Angiogenesis stimulation can be blocked by the macrolide antibiotic borrelidin (BN), which exhibits a broad spectrum toxicity that has discouraged deeper investigation. Recently, a less toxic variant (BC194) was identified that potently inhibits angiogenesis. Employing biochemical, cell biological, and biophysical approaches, we demonstrate that the toxicity of BN and its derivatives is linked to its competition with the threonine substrate at the molecular level, which stimulates amino acid starvation and apoptosis. By separating toxicity from the inhibition of angiogenesis, a direct role for TARS in vascular development in the zebrafish could be demonstrated. Bioengineered natural products are thus useful tools in unmasking the cryptic functions of conventional enzymes in the regulation of complex processes in higher metazoans.
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http://dx.doi.org/10.1038/srep13160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4536658PMC
August 2015

Structural basis for full-spectrum inhibition of translational functions on a tRNA synthetase.

Nat Commun 2015 Mar 31;6:6402. Epub 2015 Mar 31.

Department of Cancer Biology, Scripps Research Institute, Scripps Florida, 130 Scripps Way, Jupiter, Florida 33458, USA.

The polyketide natural product borrelidin displays antibacterial, antifungal, antimalarial, anticancer, insecticidal and herbicidal activities through the selective inhibition of threonyl-tRNA synthetase (ThrRS). How borrelidin simultaneously attenuates bacterial growth and suppresses a variety of infections in plants and animals is not known. Here we show, using X-ray crystal structures and functional analyses, that a single molecule of borrelidin simultaneously occupies four distinct subsites within the catalytic domain of bacterial and human ThrRSs. These include the three substrate-binding sites for amino acid, ATP and tRNA associated with aminoacylation, and a fourth 'orthogonal' subsite created as a consequence of binding. Thus, borrelidin competes with all three aminoacylation substrates, providing a potent and redundant mechanism to inhibit ThrRS during protein synthesis. These results highlight a surprising natural design to achieve the quadrivalent inhibition of translation through a highly conserved family of enzymes.
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http://dx.doi.org/10.1038/ncomms7402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4389257PMC
March 2015

Regulation of angiogenesis by aminoacyl-tRNA synthetases.

Int J Mol Sci 2014 Dec 19;15(12):23725-48. Epub 2014 Dec 19.

Department of Pharmacology, College of Medicine, University of Vermont, Burlington, VT 05405, USA.

In addition to their canonical roles in translation the aminoacyl-tRNA synthetases (ARSs) have developed secondary functions over the course of evolution. Many of these activities are associated with cellular survival and nutritional stress responses essential for homeostatic processes in higher eukaryotes. In particular, six ARSs and one associated factor have documented functions in angiogenesis. However, despite their connection to this process, the ARSs are mechanistically distinct and exhibit a range of positive or negative effects on aspects of endothelial cell migration, proliferation, and survival. This variability is achieved through the appearance of appended domains and interplay with inflammatory pathways not found in prokaryotic systems. Complete knowledge of the non-canonical functions of ARSs is necessary to understand the mechanisms underlying the physiological regulation of angiogenesis.
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http://dx.doi.org/10.3390/ijms151223725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4284789PMC
December 2014

Analogs of natural aminoacyl-tRNA synthetase inhibitors clear malaria in vivo.

Proc Natl Acad Sci U S A 2014 Dec 8;111(51):E5508-17. Epub 2014 Dec 8.

Institute for Research in Biomedicine, 08028 Barcelona, Catalonia, Spain; Catalan Institution for Research and Advanced Studies, 08010 Barcelona, Catalonia, Spain

Malaria remains a major global health problem. Emerging resistance to existing antimalarial drugs drives the search for new antimalarials, and protein translation is a promising pathway to target. Here we explore the potential of the aminoacyl-tRNA synthetase (ARS) family as a source of antimalarial drug targets. First, a battery of known and novel ARS inhibitors was tested against Plasmodium falciparum cultures, and their activities were compared. Borrelidin, a natural inhibitor of threonyl-tRNA synthetase (ThrRS), stands out for its potent antimalarial effect. However, it also inhibits human ThrRS and is highly toxic to human cells. To circumvent this problem, we tested a library of bioengineered and semisynthetic borrelidin analogs for their antimalarial activity and toxicity. We found that some analogs effectively lose their toxicity against human cells while retaining a potent antiparasitic activity both in vitro and in vivo and cleared malaria from Plasmodium yoelii-infected mice, resulting in 100% mice survival rates. Our work identifies borrelidin analogs as potent, selective, and unexplored scaffolds that efficiently clear malaria both in vitro and in vivo.
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http://dx.doi.org/10.1073/pnas.1405994111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4280603PMC
December 2014

Threonyl-tRNA synthetase overexpression correlates with angiogenic markers and progression of human ovarian cancer.

BMC Cancer 2014 Aug 27;14:620. Epub 2014 Aug 27.

Departments of Pharmacology, University of Vermont, College of Medicine, Burlington, Vermont 05405, USA.

Background: Ovarian tumors create a dynamic microenvironment that promotes angiogenesis and reduces immune responses. Our research has revealed that threonyl-tRNA synthetase (TARS) has an extracellular angiogenic activity separate from its function in protein synthesis. The objective of this study was to test the hypothesis that TARS expression in clinical samples correlates with angiogenic markers and ovarian cancer progression.

Methods: Protein and mRNA databases were explored to correlate TARS expression with ovarian cancer. Serial sections of paraffin embedded ovarian tissues from 70 patients diagnosed with epithelial ovarian cancer and 12 control patients were assessed for expression of TARS, vascular endothelial growth factor (VEGF) and PECAM using immunohistochemistry. TARS secretion from SK-OV-3 human ovarian cancer cells was measured. Serum samples from 31 tissue-matched patients were analyzed by ELISA for TARS, CA-125, and tumor necrosis factor-α (TNF-α).

Results: There was a strong association between the tumor expression of TARS and advancing stage of epithelial ovarian cancer (p < 0.001). TARS expression and localization were also correlated with VEGF (p < 0.001). A significant proportion of samples included heavy TARS staining of infiltrating leukocytes which also correlated with stage (p = 0.017). TARS was secreted by ovarian cancer cells, and patient serum TARS was related to tumor TARS and angiogenic markers, but did not achieve significance with respect to stage. Multivariate Cox proportional hazard models revealed a surprising inverse relationship between TARS expression and mortality risk in late stage disease (p = 0.062).

Conclusions: TARS expression is increased in epithelial ovarian cancer and correlates with markers of angiogenic progression. These findings and the association of TARS with disease survival provide clinical validation that TARS is associated with angiogenesis in ovarian cancer. These results encourage further study of TARS as a regulator of the tumor microenvironment and possible target for diagnosis and/or treatment in ovarian cancer.
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http://dx.doi.org/10.1186/1471-2407-14-620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4155084PMC
August 2014

Transfer RNA and human disease.

Front Genet 2014 3;5:158. Epub 2014 Jun 3.

Department of Biochemistry, College of Medicine, University of Vermont Burlington, VT, USA.

Pathological mutations in tRNA genes and tRNA processing enzymes are numerous and result in very complicated clinical phenotypes. Mitochondrial tRNA (mt-tRNA) genes are "hotspots" for pathological mutations and over 200 mt-tRNA mutations have been linked to various disease states. Often these mutations prevent tRNA aminoacylation. Disrupting this primary function affects protein synthesis and the expression, folding, and function of oxidative phosphorylation enzymes. Mitochondrial tRNA mutations manifest in a wide panoply of diseases related to cellular energetics, including COX deficiency (cytochrome C oxidase), mitochondrial myopathy, MERRF (Myoclonic Epilepsy with Ragged Red Fibers), and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). Diseases caused by mt-tRNA mutations can also affect very specific tissue types, as in the case of neurosensory non-syndromic hearing loss and pigmentary retinopathy, diabetes mellitus, and hypertrophic cardiomyopathy. Importantly, mitochondrial heteroplasmy plays a role in disease severity and age of onset as well. Not surprisingly, mutations in enzymes that modify cytoplasmic and mitochondrial tRNAs are also linked to a diverse range of clinical phenotypes. In addition to compromised aminoacylation of the tRNAs, mutated modifying enzymes can also impact tRNA expression and abundance, tRNA modifications, tRNA folding, and even tRNA maturation (e.g., splicing). Some of these pathological mutations in tRNAs and processing enzymes are likely to affect non-canonical tRNA functions, and contribute to the diseases without significantly impacting on translation. This chapter will review recent literature on the relation of mitochondrial and cytoplasmic tRNA, and enzymes that process tRNAs, to human disease. We explore the mechanisms involved in the clinical presentation of these various diseases with an emphasis on neurological disease.
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http://dx.doi.org/10.3389/fgene.2014.00158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4042891PMC
June 2014

Standardizing analysis of circulating microRNA: clinical and biological relevance.

J Cell Biochem 2014 May;115(5):805-11

Vermont Cancer Center for Basic and Translational Research, University of Vermont College of Medicine, Burlington, Vermont, 05405; Department of Biochemistry, University of Vermont College of Medicine, Burlington, Vermont, 05405.

Circulating microRNAs (c-miRNAs) provide a new dimension as clinical biomarkers for disease diagnosis, progression, and response to treatment. However, the discovery of individual miRNAs from biofluids that reliably reflect disease states is in its infancy. The highly variable nature of published studies exemplifies a need to standardize the analysis of miRNA in circulation. Here, we show that differential sample handling of serum leads to inconsistent and incomparable results. We present a standardized method of RNA isolation from serum that eliminates multiple freeze/thaw cycles, provides at least three normalization mechanisms, and can be utilized in studies that compare both archived and prospectively collected samples. It is anticipated that serum processed as described here can be profiled, either globally or on a gene by gene basis, for c-miRNAs and other non-coding RNA in the circulation to reveal novel, clinically relevant epigenetic signatures for a wide range of diseases.
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http://dx.doi.org/10.1002/jcb.24745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3992702PMC
May 2014

Secreted Threonyl-tRNA synthetase stimulates endothelial cell migration and angiogenesis.

Sci Rep 2013 ;3:1317

Department of Pharmacology, University of Vermont, College of Medicine, Burlington, VT 05405, USA.

Aminoacyl-tRNA synthetases classically regulate protein synthesis but some also engage in alternative signaling functions related to immune responses and angiogenesis. Threonyl-tRNA synthetase (TARS) is an autoantigen in the autoimmune disorder myositis, and borrelidin, a potent inhibitor of TARS, inhibits angiogenesis. We explored a mechanistic link between these findings by testing whether TARS directly affects angiogenesis through inflammatory mediators. When human vascular endothelial cells were exposed to tumor necrosis factor-α (TNF-α) or vascular endothelial growth factor (VEGF), TARS was secreted into the cell media. Furthermore, exogenous TARS stimulated endothelial cell migration and angiogenesis in both in vitro and in vivo assays. The borrelidin derivative BC194 reduced the angiogenic effect of both VEGF and TARS, but not a borrelidin-resistant TARS mutant. Our findings reveal a previously undiscovered function for TARS as an angiogenic, pro-migratory extracellular signaling molecule. TARS thus provides a potential target for detecting or interdicting disease-related inflammatory or angiogenic responses.
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http://dx.doi.org/10.1038/srep01317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3578223PMC
August 2013

The α-amino group of the threonine substrate as the general base during tRNA aminoacylation: a new version of substrate-assisted catalysis predicted by hybrid DFT.

J Phys Chem A 2011 Nov 26;115(45):13050-60. Epub 2011 Sep 26.

Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada.

Density functional theory-based methods in combination with large chemical models have been used to investigate the mechanism of the second half-reaction catalyzed by Thr-tRNA synthetase: aminoacyl transfer from Thr-AMP onto the (A76)3'OH of the cognate tRNA. In particular, we have examined pathways in which an active site His309 residue is either protonated or neutral (i.e., potentially able to act as a base). In the protonated His309-assisted mechanism, the rate-limiting step is formation of the tetrahedral intermediate. The barrier for this step is 155.0 kJ mol(-1), and thus, such a pathway is concluded to not be enzymatically feasible. For the neutral His309-assisted mechanism, two models were used with the difference being whether Lys465 was included. For either model, the barrier of the rate-limiting step is below the upper thermodynamic enzymatic limit of ~125 kJ mol(-1). Specifically, without Lys465, the rate-limiting barrier is 122.1 kJ mol(-1) and corresponds to a rotation about the tetrahedral intermediate C(carb)-OH bond. For the model with Lys465, the rate-limiting barrier is slightly lower and corresponds to the formation of the tetrahedral intermediate. Importantly, for both "neutral His309" models, the neutral amino group of the threonyl substrate directly acts as the proton acceptor; in the formation of the tetrahedral intermediate, the (A76)3'OH proton is directly transferred onto the Thr-NH(2). Therefore, the overall mechanism follows a general substrate-assisted catalytic mechanism.
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http://dx.doi.org/10.1021/jp205037aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3773706PMC
November 2011

Substrate specificity and catalysis by the editing active site of Alanyl-tRNA synthetase from Escherichia coli.

Biochemistry 2011 Mar 31;50(9):1474-82. Epub 2011 Jan 31.

Department of Biochemistry, College of Medicine, Health Sciences Complex, 89 Beaumont Avenue, University of Vermont, Burlington, Vermont 05405, United States.

Aminoacyl-tRNA synthetases (ARSs) enhance the fidelity of protein synthesis through multiple mechanisms, including hydrolysis of the adenylate and cleavage of misacylated tRNA. Alanyl-tRNA synthetase (AlaRS) limits misacylation with glycine and serine by use of a dedicated editing domain, and a mutation in this activity has been genetically linked to a mouse model of a progressive neurodegenerative disease. Using the free-standing Pyrococcus horikoshii AlaX editing domain complexed with serine as a model and both Ser-tRNA(Ala) and Ala-tRNA(Ala) as substrates, the deacylation activities of the wild type and five different Escherichia coli AlaRS editing site substitution mutants were characterized. The wild-type AlaRS editing domain deacylated Ser-tRNA(Ala) with a k(cat)/K(M) of 6.6 × 10(5) M(-1) s(-1), equivalent to a rate enhancement of 6000 over the rate of enzyme-independent deacylation but only 12.2-fold greater than the rate with Ala-tRNA(Ala). While the E664A and T567G substitutions only minimally decreased k(cat)/K(M,) Q584H, I667E, and C666A AlaRS were more compromised in activity, with decreases in k(cat)/K(M) in the range of 6-, 6.6-, and 15-fold. C666A AlaRS was 1.7-fold more active on Ala-tRNA(Ala) relative to Ser-tRNA(Ala), providing the only example of a true reversal of substrate specificity and highlighting a potential role of the coordinated zinc in editing substrate specificity. Along with the potentially serious physiological consequences of serine misincorporation, the relatively modest specificity of the AlaRS editing domain may provide a rationale for the widespread phylogenetic distribution of AlaX free-standing editing domains, thereby contributing a further mechanism to lower concentrations of misacylated tRNA(Ala).
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http://dx.doi.org/10.1021/bi1013535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3069921PMC
March 2011

Fidelity escape by the unnatural amino acid β-hydroxynorvaline: an efficient substrate for Escherichia coli threonyl-tRNA synthetase with toxic effects on growth.

Biochemistry 2011 Feb 24;50(6):1101-9. Epub 2011 Jan 24.

Cell and Molecular Biology Program, College of Medicine, Health Sciences Complex, University of Vermont, Burlington, Vermont 05405-0068, United States.

In all living systems, the fidelity of translation is maintained in part by the editing mechanisms of aminoacyl-tRNA synthetases (ARSs). Some nonproteogenic amino acids, including β-hydroxynorvaline (HNV) are nevertheless efficiently aminoacylated and become incorporated into proteins. To investigate the basis of HNV's ability to function in protein synthesis, the utilization of HNV by Escherichia coli threonyl-tRNA synthetase (ThrRS) was investigated through both in vitro functional experiments and bacterial growth studies. The measured specificity constant (k(cat)/K(M)) for HNV was found to be only 20-30-fold less than that of cognate threonine. The rate of aminoacyl transfer (10.4 s(-1)) was 10-fold higher than the multiple turnover k(cat) value (1 s(-1)), indicating that, as for cognate threonine, amino acid activation is likely to be the rate-limiting step. Like noncognate serine, HNV enhances the ATPase function of the synthetic site, at a rate not increased by nonaminoacylatable (3'-dA76) tRNA. ThrRS also failed to exhibit posttransfer editing activity against HNV. In growing bacteria, the addition of HNV dramatically suppressed growth rates, which indicates either negative phenotypic consequences associated with its incorporation into protein or inhibition of an unidentified metabolic reaction. The inability of wild ThrRS to prevent utilization of HNV as a substrate illustrates that, for at least one ARS, the naturally occurring enzyme lacks the capability to effectively discriminate against nonproteogenic amino acids that are not encountered under normal physiological conditions. Other examples of "fidelity escape" in the ARSs may serve as useful starting points in the design of ARSs with specificity for unnatural amino acids.
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http://dx.doi.org/10.1021/bi101360aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3063515PMC
February 2011

Aminoacyl transfer rate dictates choice of editing pathway in threonyl-tRNA synthetase.

J Biol Chem 2010 Jul 26;285(31):23810-7. Epub 2010 May 26.

Cell and Molecular Biology Program, University of Vermont, Burlington, VT 05405, USA.

Aminoacyl-tRNA synthetases hydrolyze aminoacyl adenylates and aminoacyl-tRNAs formed from near-cognate amino acids, thereby increasing translational fidelity. The contributions of pre- and post-transfer editing pathways to the fidelity of Escherichia coli threonyl-tRNA synthetase (ThrRS) were investigated by rapid kinetics. In the pre-steady state, asymmetric activation of cognate threonine and noncognate serine was observed in the active sites of dimeric ThrRS, with similar rates of activation. In the absence of tRNA, seryl-adenylate was hydrolyzed 29-fold faster by the ThrRS catalytic domain than threonyl-adenylate. The rate of seryl transfer to cognate tRNA was only 2-fold slower than threonine. Experiments comparing the rate of ATP consumption to the rate of aminoacyl-tRNA(AA) formation demonstrated that pre-transfer hydrolysis contributes to proofreading only when the rate of transfer is slowed significantly. Thus, the relative contributions of pre- and post-transfer editing in ThrRS are subject to modulation by the rate of aminoacyl transfer.
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http://dx.doi.org/10.1074/jbc.M110.105320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2911285PMC
July 2010

tRNA as an active chemical scaffold for diverse chemical transformations.

FEBS Lett 2010 Jan;584(2):366-75

Cell and Molecular Biology Program, University of Vermont, Burlington, VT 05405, United States.

During protein synthesis, tRNA serves as the intermediary between cognate amino acids and their corresponding RNA trinucleotide codons. Aminoacyl-tRNA is also a biosynthetic precursor and amino acid donor for other macromolecules. AA-tRNAs allow transformations of acidic amino acids into their amide-containing counterparts, and seryl-tRNA(Ser) donates serine for antibiotic synthesis. Aminoacyl-tRNA is also used to cross-link peptidoglycan, to lysinylate the lipid bilayer, and to allow proteolytic turnover via the N-end rule. These alternative functions may signal the use of RNA in early evolution as both a biological scaffold and a catalyst to achieve a wide variety of chemical transformations.
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http://dx.doi.org/10.1016/j.febslet.2009.11.045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3241936PMC
January 2010

Asymmetric amino acid activation by class II histidyl-tRNA synthetase from Escherichia coli.

J Biol Chem 2009 Jul 1;284(31):20753-62. Epub 2009 Jun 1.

Department of Biochemistry, College of Medicine, Health Sciences Complex, University of Vermont, Burlington, Vermont 05405, USA.

Aminoacyl-tRNA synthetases (ARSs) join amino acids to their cognate tRNAs to initiate protein synthesis. Class II ARS possess a unique catalytic domain fold, possess active site signature sequences, and are dimers or tetramers. The dimeric class I enzymes, notably TyrRS, exhibit half-of-sites reactivity, but its mechanistic basis is unclear. In class II histidyl-tRNA synthetase (HisRS), amino acid activation occurs at different rates in the two active sites when tRNA is absent, but half-of-sites reactivity has not been observed. To investigate the mechanistic basis of the asymmetry, and explore the relationship between adenylate formation and conformational events in HisRS, a fluorescently labeled version of the enzyme was developed by conjugating 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC) to a cysteine introduced at residue 212, located in the insertion domain. The binding of the substrates histidine, ATP, and 5'-O-[N-(l-histidyl)sulfamoyl]adenosine to MDCC-HisRS produced fluorescence quenches on the order of 6-15%, allowing equilibrium dissociation constants to be measured. The rates of adenylate formation measured by rapid quench and domain closure as measured by stopped-flow fluorescence were similar and asymmetric with respect to the two active sites of the dimer, indicating that conformational change may be rate-limiting for product formation. Fluorescence resonance energy transfer experiments employing differential labeling of the two monomers in the dimer suggested that rigid body rotation of the insertion domain accompanies adenylate formation. The results support an alternating site model for catalysis in HisRS that may prove to be common to other class II aminoacyl-tRNA synthetases.
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http://dx.doi.org/10.1074/jbc.M109.021311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2743188PMC
July 2009

RNA-assisted catalysis in a protein enzyme: The 2'-hydroxyl of tRNA(Thr) A76 promotes aminoacylation by threonyl-tRNA synthetase.

Proc Natl Acad Sci U S A 2008 Nov 7;105(46):17748-53. Epub 2008 Nov 7.

Cell and Molecular Biology Program, College of Medicine, Health Sciences Complex, 89 Beaumont Avenue, University of Vermont, Burlington, VT 05405, USA.

Aminoacyl-tRNA synthetases (aaRSs) join amino acids to 1 of 2 terminal hydroxyl groups of their cognate tRNAs, thereby contributing to the overall fidelity of protein synthesis. In class II histidyl-tRNA synthetase (HisRS) the nonbridging S(p)-oxygen of the adenylate is a potential general base for aminoacyl transfer. To test for conservation of this mechanism in other aaRSs and the role of terminal hydroxyls of tRNA in aminoacyl transfer, we investigated the class II Escherichia coli threonyl-tRNA synthetase (ThrRS). As with other class II aaRSs, the rate-determining step for ThrRS is amino acid activation. In ThrRS, however, the 2'-OH of A76 of tRNA(Thr) and a conserved active-site histidine (His-309) collaborate to catalyze aminoacyl transfer by a mechanism distinct from HisRS. Conserved residues in the ThrRS active site were replaced with alanine, and then the resulting mutant proteins were analyzed by steady-state and rapid kinetics. Nearly all mutants preferentially affected the amino acid activation step, with only a modest effect on aminoacyl transfer. By contrast, H309A ThrRS decreased transfer 242-fold and imposed a kinetic block to CCA accommodation. His-309 hydrogen bonds to the 2'-OH of A76, and substitution of the latter by hydrogen or fluorine decreased aminoacyl transfer by 763- and 94-fold, respectively. The proton relay mechanism suggested by these data to promote aminoacylation is reminiscent of the NAD(+)-dependent mechanisms of alcohol dehydrogenases and sirtuins and the RNA-mediated catalysis of the ribosomal peptidyl transferase center.
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http://dx.doi.org/10.1073/pnas.0804247105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2584683PMC
November 2008

DNA polymerases and aminoacyl-tRNA synthetases: shared mechanisms for ensuring the fidelity of gene expression.

Biochemistry 2008 Nov 14;47(45):11695-703. Epub 2008 Oct 14.

Department of Biochemistry, Department of Microbiology, College of Medicine, University of Vermont, Burlington, Vermont 05405, USA.

DNA polymerases and aminoacyl-tRNA synthetases (ARSs) represent large enzyme families with critical roles in the transformation of genetic information from DNA to RNA to protein. DNA polymerases carry out replication and collaborate in the repair of the genome, while ARSs provide aminoacylated tRNA precursors for protein synthesis. Enzymes of both families face the common challenge of selecting their cognate small molecule substrates from a pool of chemically related molecules, achieving high levels of discrimination with the assistance of proofreading mechanisms. Here, the fidelity preservation mechanisms in these two important systems are reviewed and similar features highlighted. Among the noteworthy features common to both DNA polymerases and ARSs are the use of multidomain architectures that segregate synthetic and proofreading functions into discrete domains; the use of induced fit to enhance binding selectivity; the imposition of fidelity at the level of chemistry; and the use of postchemistry error correction mechanisms to hydrolyze incorrect products in a discrete editing domain. These latter mechanisms further share the common property that error correction involves the translocation of misincorporated products from the synthetic to the editing site and that the accuracy of the process may be influenced by the rates of translocation in either direction. Fidelity control in both families can thus be said to rely on multiple elementary steps, each with its contribution to overall fidelity. The summed contribution of these kinetic checkpoints provides the high observed overall accuracy of DNA replication and aminoacylation.
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http://dx.doi.org/10.1021/bi801500zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2638074PMC
November 2008

Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases.

Methods 2008 Feb;44(2):100-18

Department of Biochemistry, University of Vermont, Health Sciences Complex, 89 Beaumont Avenue, Burlington, VT 05405, USA.

The accuracy of protein synthesis relies on the ability of aminoacyl-tRNA synthetases (aaRSs) to discriminate among true and near cognate substrates. To date, analysis of aaRSs function, including identification of residues of aaRS participating in amino acid and tRNA discrimination, has largely relied on the steady state kinetic pyrophosphate exchange and aminoacylation assays. Pre-steady state kinetic studies investigating a more limited set of aaRS systems have also been undertaken to assess the energetic contributions of individual enzyme-substrate interactions, particularly in the adenylation half reaction. More recently, a renewed interest in the use of rapid kinetics approaches for aaRSs has led to their application to several new aaRS systems, resulting in the identification of mechanistic differences that distinguish the two structurally distinct aaRS classes. Here, we review the techniques for thermodynamic and kinetic analysis of aaRS function. Following a brief survey of methods for the preparation of materials and for steady state kinetic analysis, this review will describe pre-steady state kinetic methods employing rapid quench and stopped-flow fluorescence for analysis of the activation and aminoacyl transfer reactions. Application of these methods to any aaRS system allows the investigator to derive detailed kinetic mechanisms for the activation and aminoacyl transfer reactions, permitting issues of substrate specificity, stereochemical mechanism, and inhibitor interaction to be addressed in a rigorous and quantitative fashion.
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http://dx.doi.org/10.1016/j.ymeth.2007.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2288706PMC
February 2008

Kinetic discrimination of tRNA identity by the conserved motif 2 loop of a class II aminoacyl-tRNA synthetase.

Mol Cell 2007 Feb;25(4):531-42

Department of Biochemistry, University of Vermont, Health Sciences Complex, Burlington, VT 05405, USA.

The selection of tRNAs by their cognate aminoacyl-tRNA synthetases is critical for ensuring the fidelity of protein synthesis. While nucleotides that comprise tRNA identity sets have been readily identified, their specific role in the elementary steps of aminoacylation is poorly understood. By use of a rapid kinetics analysis employing mutants in tRNA(His) and its cognate aminoacyl-tRNA synthetase, the role of tRNA identity in aminoacylation was investigated. While mutations in the tRNA anticodon preferentially affected the thermodynamics of initial complex formation, mutations in the acceptor stem or the conserved motif 2 loop of the tRNA synthetase imposed a specific kinetic block on aminoacyl transfer and decreased tRNA-mediated kinetic control of amino acid activation. The mechanistic basis of tRNA identity is analogous to fidelity control by DNA polymerases and the ribosome, whose reactions also demand high accuracy.
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http://dx.doi.org/10.1016/j.molcel.2007.01.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2020812PMC
February 2007

Substrate recognition by the hetero-octameric ATP phosphoribosyltransferase from Lactococcus lactis.

Biochemistry 2006 Dec;45(50):14933-43

Department of Microbiology and Molecular Genetics, University of Vermont, B403 Given Building, 89 Beaumont Avenue, Burlington, Vermont 05405, USA.

Two families of ATP phosphoribosyl transferases (ATP-PRT) join ATP and 5-phosphoribosyl-1 pyrophosphate (PRPP) in the first reaction of histidine biosynthesis. These consist of a homohexameric form found in all three kingdoms and a hetero-octameric form largely restricted to bacteria. Hetero-octameric ATP-PRTs consist of four HisGS catalytic subunits related to periplasmic binding proteins and four HisZ regulatory subunits that resemble histidyl-tRNA synthetases. To clarify the relationship between the two families of ATP-PRTs and among phosphoribosyltransferases in general, we determined the steady state kinetics for the hetero-octameric form and characterized the active site by mutagenesis. The KmPRPP (18.4 +/- 3.5 microM) and kcat (2.7 +/- 0.3 s-1) values for the PRPP substrate are similar to those of hexameric ATP-PRTs, but the Km for ATP (2.7 +/- 0.3 mM) is 4-fold higher, suggestive of tighter regulation by energy charge. Histidine and AMP were determined to be noncompetitive (Ki = 81.1 microM) and competitive (Ki = 1.44 mM) inhibitors, respectively, with values that approximate their intracellular concentrations. Mutagenesis experiments aimed at investigating the side chains recognizing PRPP showed that 5'-phosphate contacts (T159A and T162A) had the largest (25- and 155-fold, respectively) decreases in kcat/Km, while smaller decreases were seen with mutants making cross subunit contacts (K50A and K8A) to the pyrophosphate moiety or contacts to the 2'-OH group. Despite their markedly different quaternary structures, hexameric and hetero-octameric ATRP-PRTs exhibit similar functional parameters and employ mechanistic strategies reminiscent of the broader PRT superfamily.
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http://dx.doi.org/10.1021/bi061802vDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2567060PMC
December 2006

Evolutionary conservation of a functionally important backbone phosphate group critical for aminoacylation of histidine tRNAs.

RNA 2006 Jul 1;12(7):1315-22. Epub 2006 Jun 1.

Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.

All histidine tRNA molecules have an extra nucleotide, G-1, at the 5' end of the acceptor stem. In bacteria, archaea, and eukaryotic organelles, G-1 base pairs with C73, while in eukaryotic cytoplasmic tRNAHis, G-1 is opposite A73. Previous studies of Escherichia coli histidyl-tRNA synthetase (HisRS) have demonstrated the importance of the G-1:C73 base pair to tRNAHis identity. Specifically, the 5'-monophosphate of G-1 and the major groove amine of C73 are recognized by E. coli HisRS; these individual atomic groups each contribute approximately 4 kcal/mol to transition state stabilization. In this study, two chemically synthesized 24-nucleotide RNA microhelices, each of which recapitulates the acceptor stem of either E. coli or Saccharomyces cervisiae tRNAHis, were used to facilitate an atomic group "mutagenesis" study of the -1:73 base pair recognition by S. cerevisiae HisRS. Compared with E. coli HisRS, microhelixHis is a much poorer substrate relative to full-length tRNAHis for the yeast enzyme. However, the data presented here suggest that, similar to the E. coli system, the 5' monophosphate of yeast tRNA(His) is critical for aminoacylation by yeast HisRS and contributes approximately 3 kcal/mol to transition state stability. The primary role of the unique -1:73 base pair of yeast tRNAHis appears to be to properly position the critical 5' monophosphate for interaction with the yeast enzyme. Our data also suggest that the eukaryotic HisRS/tRNAHis interaction has coevolved to rely less on specific major groove interactions with base atomic groups than the bacterial system.
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http://dx.doi.org/10.1261/rna.78606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1484442PMC
July 2006

Activation of the hetero-octameric ATP phosphoribosyl transferase through subunit interface rearrangement by a tRNA synthetase paralog.

J Biol Chem 2005 Oct 28;280(40):34096-104. Epub 2005 Jul 28.

Department of Microbiology and Molecular Genetics and Biochemistry, University of Vermont, Burlington, Vermont 05405, USA.

ATP phosphoribosyl transferase (ATP-PRT) joins ATP and 5-phosphoribosyl-1-pyrophosphate (PRPP) in a highly regulated reaction that initiates histidine biosynthesis. The unusual hetero-octameric version of ATP-PRT includes four HisG(S) catalytic subunits based on the periplasmic binding protein fold and four HisZ regulatory subunits that resemble histidyl-tRNA synthetases. Here, we present the first structure of a PRPP-bound ATP-PRT at 2.9 A and provide a structural model for allosteric activation based on comparisons with other inhibited and activated ATP-PRTs from both the hetero-octameric and hexameric families. The activated state of the octameric enzyme is characterized by an interstitial phosphate ion in the HisZ-HisG interface and new contacts between the HisZ motif 2 loop and the HisG(S) dimer interface. These contacts restructure the interface to recruit conserved residues to the active site, where they activate pyrophosphate to promote catalysis. Additionally, mutational analysis identifies the histidine binding sites within a region highly conserved between HisZ and the functional HisRS. Through the oligomerization and functional re-assignment of protein domains associated with aminoacylation and phosphate binding, the HisZ-HisG octameric ATP-PRT acquired the ability to initiate the synthesis of a key metabolic intermediate in an allosterically regulated fashion.
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http://dx.doi.org/10.1074/jbc.M505041200DOI Listing
October 2005
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