Fidelity escape by the unnatural amino acid β-hydroxynorvaline: an efficient substrate for Escherichia coli threonyl-tRNA synthetase with toxic effects on growth.
Fidelity escape by the unnatural amino acid β-hydroxynorvaline: an efficient substrate for Escherichia coli threonyl-tRNA synthetase with toxic effects on growth.
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DOI：
10.1021/bi101360a
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发表时间：
2011-02-15
期刊：
影响因子：
2.9
通讯作者：
Francklyn, Christopher S.
中科院分区：
文献类型：
--
作者：
Minajigi, Anand;Deng, Bin;Francklyn, Christopher S.
In all living systems, the fidelity of translation is maintained in part by the editing mechanisms of aminoacyl-tRNA synthetases (ARSs). Some non-proteogenic 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 E. coli threonyl-tRNA synthetase (ThrRS) was investigated through both in vitro functional experiments and bacterial growth studies. The measured specificity constant (kcat/KM) 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 kcat value (1 s−1), indicating that, as for cognate threonine, amino acid activation is likely to be the rate-limiting step. Like non-cognate serine, HNV enhances the ATPase function of the synthetic site, at a rate not increased by non-aminoacylatable (3′-dA76) tRNA. ThrRS also failed to exhibit post-transfer 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 non-proteogenic 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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影响因子：
2.9
作者：
Francklyn, Christopher S.
通讯作者：
Francklyn, Christopher S.
影响因子：
4.8
作者：
Ledoux, Sarah;Uhlenbeck, Olke C.
通讯作者：
Uhlenbeck, Olke C.
DOI：
10.1073/pnas.0509219103
发表时间：
2006-03-21
影响因子：
11.1
作者：
Hartman, MCT;Josephson, K;Szostak, JW
通讯作者：
Szostak, JW
影响因子：
5.1
作者：
HILL, RE;WHITE, RL;SMITH, KC
通讯作者：
SMITH, KC
影响因子：
4.8
作者：
Dulic, Morana;Cvetesic, Nevena;Gruic-Sovulj, Ita
通讯作者：
Gruic-Sovulj, Ita