The tRNA A76 Hydroxyl Groups Control Partitioning of the tRNA-dependent Pre- and Post-transfer Editing Pathways in Class I tRNA Synthetase

Cvetešić, Nevena; Biluš, Mirna; Gruić-Sovulj, Ita

doi:10.1074/jbc.m115.648568

Cited by 31 publications

(45 citation statements)

References 74 publications

(89 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To measure the chemical step, one hydrolytic turnover has to be followed because under multiturnover conditions product dissociation is rate limiting [14]. Single-turnover analysis reported a rate constant of 44 s À1 (Table 4), which is similar to the previously published rates for Val [13] and Nva [16]. That Abu is rapidly edited is surprising because IleRS efficiently rejects Abu in the synthetic reaction with the discrimination factor of 7100 (Table 1).…”

Section: Abu Is Efficiently Edited By the Ilers Editing Domainsupporting

confidence: 72%

“…Abu was transferred more rapidly than Ile (Table ) confirming that, as is the case of Nva and Val , the transfer step is nondiscriminative. Apparently, given that the K M , but not k cat , changes in the case of Nva and Abu activation, interactions with the side chain are not an inherent part of the transition state for either the transfer or the activation step.…”

Section: Resultsmentioning

confidence: 65%

“…To stimulate release of pyrophosphate, the additional wash was performed using 15 CV of the same buffer without Purification and labeling of tRNA Ile tRNA Ile GAU (with G1-C72 instead of WT A1-U72 sequence) was overexpressed in E. coli. Substitution of the first base pair enhances transcription and does not affect tRNA Ile participation in the IleRS synthetic and editing reactions [13,46]. tRNA Ile was purified using reverse phase chromatography as described [11].…”

Section: Purification Of Ile/leu/valrs Enzymesmentioning

confidence: 99%

See 2 more Smart Citations

Mechanism of discrimination of isoleucyl‐tRNA synthetase against nonproteinogenic α‐aminobutyrate and its fluorinated analogues

et al. 2019

View full text Add to dashboard Cite

Isoleucyl‐tRNA synthetase (IleRS) is a paradigm for understanding how specificity against smaller hydrophobic substrates evolved in both the synthetic and editing reactions. IleRS misactivates nonproteinogenic norvaline (Nva) and proteinogenic valine (Val), with a 200‐fold lower efficiency than the cognate isoleucine (Ile). Translational errors are, however, prevented by IleRS hydrolytic editing. Nva and Val are both smaller than Ile by a single methylene group. How does the removal of one additional methylene group affects IleRS specificity? We found that the nonproteinogenic α‐aminobutyrate (Abu) is activated 30‐fold less efficiently than Nva and Val, indicating that the removal of the second methylene group comes with a lower penalty. As with Nva and Val, discrimination against Abu predominantly originated from a higher KM. To examine whether increased hydrophobicity could compensate for the loss of van der Waals interactions, we tested fluorinated Abu analogues. We found that fluorination further hampered activation by IleRS, and even more so by the evolutionary‐related ValRS. This suggests that hydrophobicity is not a main driving force of substrate binding in these enzymes. Finally, a discrimination factor of 7100 suggests that IleRS is not expected to edit Abu. However, we found that the IleRS editing domain hydrolyzes Abu‐tRNAIle with a rate of 40 s−1 and the introduction of fluorine did not slow down the hydrolysis. This raises interesting questions regarding the mechanism of specificity of the editing domain and its evolution. Understanding what shapes IleRS specificity is also of importance for reengineering translation to accommodate artificial substrates including fluorinated amino acids. Enzymes Isoleucyl‐tRNA synthetase (EC 6.1.1.5), leucyl‐tRNA synthetase (EC 6.1.1.4), valyl‐tRNA synthetase (EC 6.1.1.9).

show abstract

Section: Abu Is Efficiently Edited By the Ilers Editing Domainsupporting

confidence: 72%

Section: Resultsmentioning

confidence: 65%

Section: Purification Of Ile/leu/valrs Enzymesmentioning

confidence: 99%

See 1 more Smart Citation

Mechanism of discrimination of isoleucyl‐tRNA synthetase against nonproteinogenic α‐aminobutyrate and its fluorinated analogues

et al. 2019

View full text Add to dashboard Cite

show abstract

“…For GlnRS and GluRS that activate amino acids in the strictly tRNA‐dependent manner, interaction with the cognate tRNA substantially promotes amino acid selectivity . In contrast, the interaction with tRNA does not influence discrimination against the noncognate Val and norvaline (Nva) in the activation step catalyzed by isoleucyl‐tRNA synthetase (IleRS), which may activate amino acids in the absence of tRNA . To what extent this can be applied to other AARSs awaits further experimental work.…”

Section: Aarss Mechanism Of Actionmentioning

confidence: 99%

“…For the majority of AARSs, however, pretransfer editing is weak and is not stimulated by tRNA ( tRNA‐independent pretransfer editing , or tRNA independent ). Only in few cases, IleRS being the best documented one , the rate of pretransfer editing increases when the cognate tRNA is bound to the enzyme ( tRNA‐dependent pretransfer editing or tRNA dependent ) and pretransfer editing may therefore contribute to higher accuracy in vivo . A physiologically relevant case of pretransfer editing is the exclusion of the nonproteinogenic amino acid homocysteine (Hcy) by the synthetic site of methionyl‐tRNA synthetase (MetRS), which occurs via an intramolecular cyclization of Hyc‐AMP rather than via a hydrolytic reaction .…”

Section: Aarss Mechanism Of Actionmentioning

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

View full text Add to dashboard Cite

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’).

show abstract

Gly56 in the synthetic site of isoleucyl‐tRNA synthetase confers specificity and maintains communication with the editing site

Dulic,

Krpan,

Gruic‐Sovulj

2023

FEBS Letters

View full text Add to dashboard Cite

Isoleucyl‐tRNA synthetase (IleRS) links isoleucine to cognate tRNA via the Ile‐AMP intermediate. Non‐cognate valine is often mistakenly recognized as the IleRS substrate; therefore, to maintain the accuracy of translation, IleRS hydrolyzes Val‐AMP within the synthetic site (pre‐transfer editing). As this activity is not efficient enough, Val‐tRNAIle is formed and hydrolyzed in the distant post‐transfer editing site. A strictly conserved synthetic site residue Gly56 was previously shown to safeguard Ile‐to‐Val discrimination during aminoacyl (aa)‐AMP formation. Here, we show that the Gly56Ala variant lost its specificity in pre‐transfer editing, confirming that this residue ensures the selectivity of all synthetic site reactions. Moreover, we found that the Gly56Ala mutation affects IleRS interaction with aa‐tRNA likely by disturbing tRNA‐dependent communication between the two active sites.

show abstract

The tRNA A76 Hydroxyl Groups Control Partitioning of the tRNA-dependent Pre- and Post-transfer Editing Pathways in Class I tRNA Synthetase

Cited by 31 publications

References 74 publications

Mechanism of discrimination of isoleucyl‐tRNA synthetase against nonproteinogenic α‐aminobutyrate and its fluorinated analogues

Mechanism of discrimination of isoleucyl‐tRNA synthetase against nonproteinogenic α‐aminobutyrate and its fluorinated analogues

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

Gly56 in the synthetic site of isoleucyl‐tRNA synthetase confers specificity and maintains communication with the editing site

Contact Info

Product

Resources

About