Substrate Specificity and Catalysis by the Editing Active Site of Alanyl-tRNA Synthetase from <i>Escherichia coli</i>

Pasman, Zvi; Robey-Bond, Susan M.; Mirando, Adam C.; Smith, Gregory J.; Lague, Astrid; Francklyn, Christopher S.

doi:10.1021/bi1013535

Cited by 25 publications

(19 citation statements)

References 51 publications

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“…Furthermore, larger amino acids are also accepted by the editing site, e.g., Ser by AlaRS84 or Tyr by PheRS 85. Editing AARSs have been known to catalyze deacylation of cognate AA‐tRNA at significant rates,10 confirmed recently for AlaRS,25 which deacylates Ala‐tRNA Ala only 12.2‐fold less efficiently than Ser‐tRNA Ala . The ability of AARS to deacylate cognate AA‐tRNA suggests that cognate AA‐tRNA enters editing site,33 as directly demonstrated by crystallographic studies of ThrRS 57…”

Section: Posttransfer Editing At a Dedicated Sitementioning

confidence: 96%

“…AARSs have major selectivity problems with homocysteine (Hcy), misactivated by MetRS,30 IleRS,30 LeuRS,31 ValRS,10 and LysRS,24 and with Ser, misactivated by AlaRS32 and ThrRS 5. Chemical bases for the limited discrimination against Hcy3,33–35 and Ser5,32,36,25 have been elucidated. Other selectivity problems faced by AARSs include10: Val, Leu, Cys versus Ile by IleRS37; Met, Ile versus Leu by LeuRS26; Cys, Thr, Ile versus Val by ValRS37; ornithine (Orn) versus Lys by LysRS24; Tyr versus Phe by PheRS; and Ala,27 Cys versus Pro by ProRS 23.…”

Section: Selectivity Of Aarssmentioning

confidence: 99%

“…For example, the N2 domain of ThRS5 deacylates Ser‐tRNA Thr . Editing domain of AlaRS25,78 decylates Ser‐tRNA Ala and Gly‐tRNA Ala . A unique domain present in ProRS79 deacylates Ala‐tRNA Pro but not Cys‐tRNA Pro .…”

Section: Posttransfer Editing At a Dedicated Sitementioning

confidence: 99%

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Quality control in tRNA charging

Jakubowski

2011

WIREs RNA

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Faithful translation of the genetic code during protein synthesis is fundamental to the growth, development, and function of living organisms. Aminoacyl-tRNA synthetases (AARSs), which define the genetic code by correctly pairing amino acids with their cognate tRNAs, are responsible for 'quality control' in the flow of information from a gene to a protein. When differences in binding energies of amino acids to an AARS are inadequate, editing is used to achieve high selectivity. Editing occurs at the synthetic active site by hydrolysis of noncognate aminoacyl-adenylates (pretransfer editing) and at a dedicated editing site located in a separate domain by deacylation of mischarged aminoacyl-tRNA (posttransfer editing). Access of nonprotein amino acids, such as homocysteine or ornithine, to the genetic code is prevented by the editing function of AARSs, which functionally partitions amino acids present in living cells into protein and nonprotein amino acids. Continuous editing is part of the tRNA aminoacylation process in living organisms from bacteria to human beings. Preventing mistranslation by the clearance of misactivated amino acids is crucial to cellular homeostasis and has a role in etiology of disease. Although there is a strong selective pressure to minimize mistranslation, some organisms possess error-prone AARSs that cause mistranslation. Elevated levels of mistranslation and the synthesis of statistical proteins can be beneficial for pathogens by increasing phenotypic variation essential for the evasion of host defenses.

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Section: Posttransfer Editing At a Dedicated Sitementioning

confidence: 96%

Section: Selectivity Of Aarssmentioning

confidence: 99%

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Quality control in tRNA charging

Jakubowski

2011

WIREs RNA

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“…2. exception is the lower misactivation frequency of Ser by ThrRS (Thr/Ser discrimination factor = 1067; Table 1) [89], where modulation of the interaction with the active site zinc ion may possibly contribute to higher selectivity. In all these cases, the low initial selectivity is compensated by editing, which proceeds, predominately, through the post-transfer editing [26,39,41,42,45,46,48,58,86,[98][99][100], with the exception of MetRS which edits Hcy prior to its transfer through cyclization of HCys-AMP [61,62].…”

Section: The Hydroxyl Paradox: Discrimination Against Substrates Largmentioning

confidence: 99%

How evolution shapes enzyme selectivity – lessons from aminoacyl‐tRNA synthetases and other amino acid utilizing enzymes

Tawfik

Gruić-Sovulj

2020

The FEBS Journal

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Aminoacyl‐tRNA synthetases (AARSs) charge tRNA with their cognate amino acids. Many other enzymes use amino acids as substrates, yet discrimination against noncognate amino acids that threaten the accuracy of protein translation is a hallmark of AARSs. Comparing AARSs to these other enzymes allowed us to recognize patterns in molecular recognition and strategies used by evolution for exercising selectivity. Overall, AARSs are 2–3 orders of magnitude more selective than most other amino acid utilizing enzymes. AARSs also reveal the physicochemical limits of molecular discrimination. For example, amino acids smaller by a single methyl moiety present a discrimination ceiling of ~200, while larger ones can be discriminated by up to 105‐fold. In contrast, substrates larger by a hydroxyl group challenge AARS selectivity, due to promiscuous H‐bonding with polar active site groups. This ‘hydroxyl paradox’ is resolved by editing. Indeed, when the physicochemical discrimination limits are reached, post‐transfer editing – hydrolysis of tRNAs charged with noncognate amino acids, evolved. The editing site often selectively recognizes the edited noncognate substrate using the very same feature that the synthetic site could not efficiently discriminate against. Finally, the comparison to other enzymes also reveals that the selectivity of AARSs is an explicitly evolved trait, showing some clear examples of how selection acted not only to optimize catalytic efficiency with the target substrate, but also to abolish activity with noncognate threat substrates (‘negative selection’).

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“…Purified E. coli enzymes Alanyl-tRNA synthetase (AlaRS), Histidyl-tRNA synthetase (HisRS) and Threonyl-tRNA synthetase (ThrRS) were isolated by Ni-column chromatography as previously described [25–27]. This represents the minimum degree of recommended purification; some enzymes may require additional steps for best results.…”

Section: Differential Scanning Fluorimetry Methodsmentioning

confidence: 99%

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

Abbott

Livingston

Egri

et al. 2017

Methods

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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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Substrate Specificity and Catalysis by the Editing Active Site of Alanyl-tRNA Synthetase from Escherichia coli

Cited by 25 publications

References 51 publications

Quality control in tRNA charging

Quality control in tRNA charging

How evolution shapes enzyme selectivity – lessons from aminoacyl‐tRNA synthetases and other amino acid utilizing enzymes

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

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