Fidelity Escape by the Unnatural Amino Acid β-Hydroxynorvaline: An Efficient Substrate forEscherichia coliThreonyl-tRNA Synthetase with Toxic Effects on Growth

Abstract

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 (k cat /K M ) 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 k cat 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.Aminoacyl-tRNA synthetases establish the fidelity of the genetic code in a two-step aminoacylation reaction, covalently linking specific amino acids to their corresponding tRNAs. In the first step, amino acid is condensed with ATP to form the aminoacyl adenylate (adenylation), while in the second step, the amino acid moiety of adenylate is transferred to the A76 of tRNA (aminoacyl transfer), along with the release of pyrophosphate (PPi) and AMP, respectively.

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

“…The second step of aminoacylation, transfer of the aminoacyl moiety to tRNA, generally does not significantly contribute to amino acid selectivity as measured for AARSs with limited initial selectivity . In contrast, selectivity in the already selective GlnRS is further increased by 10 3 ‐fold when the second step is included .…”

“…A conspicuous exception among the noncognate substrates presented in Table is β‐hydroxynorvaline (β‐HNV). Although its side chain is larger than that of Thr by one methylene group, the synthetic site of ThrRS discriminates against this unnatural amino acid by 29‐fold only . However, ThrRS has never been challenged with β‐HNV; while in the aforementioned AARSs, the presence of larger noncognate amino acids had explicitly shaped the synthetic sites to discriminate against these physiologically relevant threat substrates (discussed further in ‘The explicit evolution of selectivity – negative selection’ below).…”

Fluorothreonyl-tRNA deacylase prevents mistranslation in the organofluorine producer Streptomyces cattleya

“…To avoid toxicity due to tricarboxylic acid cycle shutdown, S. cattleya employs both regulatory and enzymatic detoxification strategies (12)(13)(14). In comparison, the fate of FThr remains relatively unknown, although it provides a possible substrate for both amino acid metabolism and translation based on its structural similarity to threonine (15,16).…”

Progress and challenges in aminoacyl-tRNA synthetase-based therapeutics

“…For example, an inbred mouse strain containing an editing-deficient AlaRS allele (the sticky (Sti) allele) exhibits Purkinje cell degeneration and associated deficits in cerebellar function (30). Some nonproteogenic amino acids are able to evade natural editing systems, with potentially toxic consequences (31,32). Interestingly, editing functions may be diminished in certain obligate parasitic species, such as Mycoplasma (33,34).…”