Functional Divergence of a Unique C-terminal Domain of Leucyl-tRNA Synthetase to Accommodate Its Splicing and Aminoacylation Roles

Hsu, Jennifer L.; Rho, Seung Bae; Vannella, Kevin M.; Martinis, Susan A.

doi:10.1074/jbc.m601606200

Cited by 40 publications

(70 citation statements)

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“…Interestingly, the R vec L, and E vec K single mutations had K m values of 1.6 and 1.3 μM respectively, which were comparable to the wild type enzyme (12). However, a large decrease in the apparent k cat of about 25-fold results in substantially diminished activity.…”

Section: Chimeric Leurs and Valrs Mutants Restore Limited Aminoacylatmentioning

confidence: 92%

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A Unique Insert of Leucyl-tRNA Synthetase Is Required for Aminoacylation and Not Amino Acid Editing

Martinis

2007

Biochemistry

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Leucyl-tRNA synthetase (LeuRS) is a class I enzyme, which houses its aminoacylation active site in a canonical core that is defined by a Rossmann nucleotide binding fold. In addition, many LeuRSs bear a unique polypeptide insert comprised of about 50 amino acids located just upstream of the conserved KMSKS sequence. The role of this leucine-specific domain (LS-domain) remains undefined. We hypothesized that this domain may be important for substrate recognition in aminoacylation and/or amino acid editing. We carried out a series of deletion mutations and chimeric swaps within the leucine-specific domain of Escherichia coli. Our results support that the leucinespecific domain is critical for aminoacylation, but not required for editing activity. Kinetic analysis determined that deletion of the LS-domain primarily impacts k cat . Because of its proximity to the aminoacylation active site, we propose that this domain interacts with the tRNA during amino acid activation and/or tRNA aminoacylation. Although the leucine-specific domain does not appear to be important to the editing complex, it remains possible that it aids the dynamic translocation process that moves tRNA from the aminoacylation to the editing complex.Aminoacylation is catalyzed by an ancient family of enzymes called the aminoacyl-tRNA synthetases (aaRS) (1,2). It is important to translation that the aaRS correctly link amino acid to the corresponding tRNA acceptors. Each of the aaRS is responsible for covalently attaching one of twenty standard amino acids to a set of cognate tRNA isoacceptors. The esterification of amino acid to tRNA occurs via a two-step reaction:The first reversible step involves the activation of an amino acid concomitant with ATP hydrolysis to form an aminoacyl-adenylate intermediate. The amino acid is then transferred from the adenylate intermediate to the 3′ terminal adenosine moiety of the tRNA. The charged aminoacyl-tRNA binds to EF-Tu and is transported to the ribosome to extend the polypeptide chain.Leucyl-tRNA synthetase (LeuRS) is a class I aaRS, and is responsible for accurately aminoacylating leucine to its cognate tRNA Leu isoacceptors (3). As is characteristic of all class I synthetases, the catalytic core is defined by a Rossmann nucleotide binding fold (4) that comprises the main body of the enzyme (5). This canonical class I core of LeuRS contains inserts and appendages (5-7). For example, the enzyme has an amino acid editing function that † This work was supported by the National Institutes of Health (GM63789). resides within a large insert called the connective polypeptide 1 (CP1) (8). In addition, a novel C-terminal domain has been proposed to be important for tRNA interactions (9-12).One unique small insert called the leucine-specific domain (LS-domain) is found in many bacterial LeuRSs (5). Its function remains undefined. This compact domain that is comprised of five β-strands and two short α-helices flanks the entrance of the aminoacylation active site (5). The LS-domain is inserted after the last β-...

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Section: Chimeric Leurs and Valrs Mutants Restore Limited Aminoacylatmentioning

confidence: 92%

“…In addition, a novel C-terminal domain has been proposed to be important for tRNA interactions (9)(10)(11)(12).…”

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confidence: 99%

A Unique Insert of Leucyl-tRNA Synthetase Is Required for Aminoacylation and Not Amino Acid Editing

Martinis

2007

Biochemistry

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“…The structure model for the C-terminal domain is based on weak, but unambiguous, electron density present in difference Fourier maps. The C-terminal domain is linked to the core of the enzyme by a flexible tether (11) and interacts with the corner of the L-shaped tRNA. Thus, stabilization of the position of this mobile domain in either the editing or aminoacylation complex requires tRNA binding (29).…”

Section: X-ray Crystal Structure Of M Mobile Leurs-leuams Is Similar Tomentioning

confidence: 99%

“…In the posttransfer editing pathway (Eq. 3), mischarged tRNA is hydrolyzed in an aaRS domain that is distinct from the synthetic aminoacylation domain (9)(10)(11)(12)(13)(14)(15)(16). In addition, editing of mischarged tRNA can occur in trans by independent proteins that function as tRNA-specific deacylases (17,18):…”

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confidence: 99%

Leucyl-tRNA synthetase editing domain functions as a molecular rheostat to control codon ambiguity in Mycoplasma pathogens

Palencia

Lukk³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Mycoplasma leucyl-tRNA synthetases (LeuRSs) have been identified in which the connective polypeptide 1 (CP1) amino acid editing domain that clears mischarged tRNAs are missing (Mycoplasma mobile) or highly degenerate (Mycoplasma synoviae). Thus, these enzymes rely on a clearance pathway called pretransfer editing, which hydrolyzes misactivated aminoacyl-adenylate intermediate via a nebulous mechanism that has been controversial for decades. Even as the sole fidelity pathway for clearing amino acid selection errors in the pathogenic M. mobile, pretransfer editing is not robust enough to completely block mischarging of tRNA Leu , resulting in codon ambiguity and statistical proteins. A high-resolution X-ray crystal structure shows that M. mobile LeuRS structurally overlaps with other LeuRS cores. However, when CP1 domains from different aminoacyl-tRNA synthetases and origins were fused to this common LeuRS core, surprisingly, pretransfer editing was enhanced. It is hypothesized that the CP1 domain evolved as a molecular rheostat to balance multiple functions. These include distal control of specificity and enzyme activity in the ancient canonical core, as well as providing a separate hydrolytic active site for clearing mischarged tRNA.protein evolution | quality control | statistical proteins | aminoacylation | translation

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“…3). LeuRSs are also composed of a number of additional domains involved in binding tRNA Leu or in proofreading activities [4][5][6][7] . X-ray crystal structures have been obtained for LeuRSs from both archaeal and bacterial origins in complex with a number of substrates and substrate analogues, including Leu 5 and tRNA Leu (refs 6,7), a reaction-intermediate analogue of , and also substrate mimics of both postand pre-transfer editing reactions 8 .…”

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confidence: 99%

Plant tumour biocontrol agent employs a tRNA-dependent mechanism to inhibit leucyl-tRNA synthetase

et al. 2013

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Leucyl-tRNA synthetases (LeuRSs) have an essential role in translation and are promising targets for antibiotic development. Agrocin 84 is a LeuRS inhibitor produced by the biocontrol agent Agrobacterium radiobacter K84 that targets pathogenic strains of A. tumefaciens, the causative agent of plant tumours. Agrocin 84 acts as a molecular Trojan horse and is processed inside the pathogen into a toxic moiety (TM84). Here we show using crystal structure, thermodynamic and kinetic analyses, that this natural antibiotic employs a unique and previously undescribed mechanism to inhibit LeuRS. TM84 requires tRNA Leu for tight binding to the LeuRS synthetic active site, unlike any previously reported inhibitors. TM84 traps the enzyme-tRNA complex in a novel 'aminoacylation-like' conformation, forming novel interactions with the KMSKS loop and the tRNA 3 0 -end. Our findings reveal an intriguing tRNAdependent inhibition mechanism that may confer a distinct evolutionary advantage in vivo and inform future rational antibiotic design.

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Functional Divergence of a Unique C-terminal Domain of Leucyl-tRNA Synthetase to Accommodate Its Splicing and Aminoacylation Roles

Cited by 40 publications

References 32 publications

A Unique Insert of Leucyl-tRNA Synthetase Is Required for Aminoacylation and Not Amino Acid Editing

A Unique Insert of Leucyl-tRNA Synthetase Is Required for Aminoacylation and Not Amino Acid Editing

Leucyl-tRNA synthetase editing domain functions as a molecular rheostat to control codon ambiguity in Mycoplasma pathogens

Plant tumour biocontrol agent employs a tRNA-dependent mechanism to inhibit leucyl-tRNA synthetase

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