The heterotrimeric Thermus thermophilus Asp‐tRNAAsn amidotransferase can also generate Gln‐tRNAGln

Becker, Hubert Dominique; Min, Bokkee; Jacobi, Carsten; Raczniak, Gregory; Pelaschier, Joanne; Roy, Hervé; Klein, Sylvain; Kern, Daniel; Söll, Dieter

doi:10.1016/s0014-5793(00)01697-5

Cited by 42 publications

(49 citation statements)

References 9 publications

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“…Based on our functional studies, it is unlikely that this At5g64440 gene product functions as a glutamyl-tRNA amidotransferase, and this activity has not been attributed to membrane-bound mammalian FAAH enzymes. Additionally, in plants these glutamyltRNA amidotransferases are localized in the stroma of chloroplasts as soluble, oligomeric complexes of multiple subunits (45,46). In fact, nuclear-encoded, chloroplast-localized orthologues of the glutamyl-tRNA amidotransferase subunits have been cloned from Arabidopsis and expressed/characterized by the Soll group (GenBank TM accession numbers, AF241841, AF240465, AF239836, and AF224745), and these proteins share less than 24% amino acid sequence identity with the At5g64440 NAE amidohydrolase.…”

Section: Discussionmentioning

confidence: 99%

Molecular Identification of a Functional Homologue of the Mammalian Fatty Acid Amide Hydrolase in Arabidopsis thaliana

Shrestha

Dixon

Chapman

2003

Journal of Biological Chemistry

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N-Acylethanolamines (NAEs) are endogenous constituents of plant and animal tissues, and in vertebrates their hydrolysis terminates their participation as lipid mediators in the endocannabinoid signaling system. The membrane-bound enzyme responsible for NAE hydrolysis in mammals has been identified at the molecular level (designated fatty acid amide hydrolase, FAAH), and although an analogous enzyme activity was identified in microsomes of cotton seedlings, no molecular information is available for this enzyme in plants. Here we report the identification, the heterologous expression (in Escherichia coli), and the biochemical characterization of an Arabidopsis thaliana FAAH homologue. Candidate Arabidopsis DNA sequences containing a characteristic amidase signature sequence (PS00571) were identified in plant genome data bases, and a cDNA was isolated by reverse transcriptase-PCR using Arabidopsis genome sequences to develop appropriate oligonucleotide primers. The cDNA was sequenced and predicted to encode a protein of 607 amino acids with 37% identity to rat FAAH within the amidase signature domain (18% over the entire length). Residues determined to be important for FAAH catalysis were conserved between the Arabidopsis and rat protein sequences. In addition, a single transmembrane domain near the N terminus was predicted in the Arabidopsis protein sequence, similar to that of the rat FAAH protein. The putative plant FAAH cDNA was expressed as an epitope/ His-tagged fusion protein in E. coli and solubilized from cell lysates in the nonionic detergent, dodecyl maltoside. Affinity-purified recombinant protein was indeed active in hydrolyzing a variety of naturally occurring N-acylethanolamine types. Kinetic parameters and inhibition data for the recombinant Arabidopsis protein were consistent with these properties of the enzyme activity characterized previously in plant and animal systems. Collectively these data now provide support at the molecular level for a conserved mechanism between plants and animals for the metabolism of NAEs.N-Acylethanolamines (NAEs) 1 are lipid mediators that are produced from the phospholipase D-mediated hydrolysis of Nacylphosphatidylethanolamines, a minor membrane lipid constituent of cellular membranes (1). In animal systems, anandamide (NAE 20:4) acts as an endogenous ligand for cannabinoid receptors and has varied physiological roles, such as the modulation of neurotransmission in the central nervous system (2). Anandamide also activates vanilloid receptors, functions as an endogenous analgesic (3), and appears to be involved in neuroprotection (4,5). In other tissues, NAEs have been implicated in immunomodulation (6), synchronization of embryo development (7), and induction of apoptosis (8). These endogenous bioactive molecules lose their signaling activity upon hydrolysis by fatty acid amide hydrolase (FAAH; see Ref. 9).In plants NAEs are present in substantial amounts in desiccated seeds (ϳ1 g g Ϫ1 fresh weight), and their levels decline after a few hours of imbibition (10). Ind...

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Section: Discussionmentioning

confidence: 99%

Molecular Identification of a Functional Homologue of the Mammalian Fatty Acid Amide Hydrolase in Arabidopsis thaliana

Shrestha

Dixon

Chapman

2003

Journal of Biological Chemistry

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“…In other bacteria, archaea, and some organelles however, AspRS is a non-canonical, non-discriminating enzyme (ND-AspRS) that generates both AsptRNA Asp and the misacylated Asp-tRNA Asn (5)(6)(7)12,3,14). This Asp-tRNA Asn is converted to Asn-tRNA Asn by a glutamine-dependent Asp-tRNA Asn /Glu-tRNA Gln amidotransferase (Asp/ Glu-Adt) (26,27,9,28,12). ND-AspRS is most commonly found in organisms lacking asparaginyl-tRNA synthetase (AsnRS) and in these cases, the combination of ND-AspRS and Asp/Glu-Adt provides the sole route for Asn-tRNA Asn biosynthesis and, in some cases, for asparagine biosynthesis as well (7,29).…”

Section: Introductionmentioning

confidence: 99%

The Nondiscriminating Aspartyl-tRNA Synthetase from Helicobacter pylori: Anticodon-Binding Domain Mutations That Impact tRNA Specificity and Heterologous Toxicity

Chuawong

Hendrickson

2006

Biochemistry

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Divergent tRNA substrate recognition patterns distinguish the two distinct forms of aspartyl-tRNA synthetase (AspRS) that exist in different bacteria. In some cases, a canonical, discriminating AspRS (D-AspRS) specifically generates Asp-tRNA Asp and usually co-exists with asparaginyl-tRNA synthetase (AsnRS). In other bacteria, particularly those that lack AsnRS, AspRS is nondiscriminating (ND-AspRS) and generates both Asp-tRNA Asp and the non-canonical, misacylated Asp-tRNA Asn ; this misacylated tRNA is subsequently repaired by the glutamine-dependent AsptRNA Asn /Glu-tRNA Gln amidotransferase (Asp/Glu-Adt). The molecular features that distinguish the closely related bacterial D-AspRS and ND-AspRS are not well understood. Here we report the first characterization of the ND-AspRS from the human pathogen Helicobacter pylori. This enzyme is toxic when heterologously overexpressed in E. coli. This toxicity is rescued upon co-expression of the H. pylori Asp/Glu-Adt, indicating that Hp Asp/Glu-Adt can utilize E. coli Asp-tRNA Asn as a substrate. Finally, mutations in the anticodon-binding domain of Hp ND-AspRS reduce this enzyme's ability to misacylate tRNA Asn , in a manner that correlates with the toxicity of the enzyme in E. coli.

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“…Genome analyses of bacteria and archaea have revealed that the presence of the ND-AspRS is always accompanied by the occurrence of the heterotrimeric GatCAB amidotransferase, an enzyme capable of converting the misacylated Asp-tRNA Asn to Asn-tRNA Asn (2,5,19). Presumably, this is to avoid introducing the misacylated Asp-tRNA Asn into an organism's translational apparatus and potentially endangering protein synthesis.…”

mentioning

confidence: 99%

Protein Synthesis in Escherichia coli with Mischarged tRNA

Min¹,

Kitabatake²,

Polycarpo³

et al. 2003

J Bacteriol

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Two types of aspartyl-tRNA synthetase exist: the discriminating enzyme (D-AspRS) forms only Asp-tRNA Asp , while the nondiscriminating one (ND-AspRS) also synthesizes Asp-tRNA Asn , a required intermediate in protein synthesis in many organisms (but not in Escherichia coli). On the basis of the E. coli trpA34 missense mutant transformed with heterologous ND-aspS genes, we developed a system with which to measure the in vivo formation of Asp-tRNA Asn and its acceptance by elongation factor EF-Tu. While large amounts of Asp-tRNA Asn are detrimental to E. coli, smaller amounts support protein synthesis and allow the formation of up to 38% of the wild-type level of missense-suppressed tryptophan synthetase.Aspartyl-tRNA synthetase (AspRS) exists in two different forms with respect to tRNA recognition (7). The discriminating enzyme (D-AspRS) recognizes only tRNA Asp , while the nondiscriminating one (ND-AspRS) also recognizes tRNA Asn and therefore forms both Asp-tRNA Asn and Asp-tRNA Asp . Most bacteria and archaea lack asparaginyl-tRNA synthetase and are unable to synthesize Asn-tRNA Asn by direct acylation of tRNA. These organisms rely on the ND-AspRS to produce the misacylated Asp-tRNA Asn , which is then converted by a tRNA-dependent amidotransferase to the correctly acylated Asn-tRNA Asn (1,4,5,19). Thus, the ND-AspRS is essential in organisms that form Asn-tRNA by transamidation.The primary sequence distinguishes two general groups of AspRS. There is a predominantly bacterial type of AspRS that is about 580 amino acids, in addition to a shorter archaealeukaryotic type of about 430 amino acids. In vitro data have made clear that discriminating and nondiscriminating enzymes exist in both groups (16,20). The determinants in the protein sequence responsible for tRNA discrimination are not known.The two AspRS types are usually separated in nature. Genome analyses of bacteria and archaea have revealed that the presence of the ND-AspRS is always accompanied by the occurrence of the heterotrimeric GatCAB amidotransferase, an enzyme capable of converting the misacylated Asp-tRNA Asn to Asn-tRNA Asn (2,5,19). Presumably, this is to avoid introducing the misacylated Asp-tRNA Asn into an organism's translational apparatus and potentially endangering protein synthesis. This reasoning is supported by the fact that the heterologous expression of ND-AspRS or ND-GluRS in Escherichia coli, which lacks GatCAB, is highly toxic to the cell, especially when the synthetase genes are overexpressed (15). However, some organisms (e.g., Deinococcus radiodurans and Thermus thermophilus) contain a D-AspRS in addition to an ND-AspRS and a GatCAB amidotransferase (1,3,5,9).We wanted to observe how E. coli copes with in vivo mischarging effected by the ND-AspRS, as this organism is unable to eliminate the toxic Asp-tRNA Asn . Therefore, we developed an approach that would, in fact, require E. coli to be dependent on the presence of mischarged Asp-tRNA Asn for growth. To this aim, we used missense suppression of a specific mutation in the trp...

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The heterotrimeric Thermus thermophilus Asp‐tRNA^Asn amidotransferase can also generate Gln‐tRNA^Gln

Cited by 42 publications

References 9 publications

Molecular Identification of a Functional Homologue of the Mammalian Fatty Acid Amide Hydrolase in Arabidopsis thaliana

Molecular Identification of a Functional Homologue of the Mammalian Fatty Acid Amide Hydrolase in Arabidopsis thaliana

The Nondiscriminating Aspartyl-tRNA Synthetase from Helicobacter pylori: Anticodon-Binding Domain Mutations That Impact tRNA Specificity and Heterologous Toxicity

Protein Synthesis in Escherichia coli with Mischarged tRNA

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