YqfB protein from Escherichia coli: an atypical amidohydrolase active towards N4-acylcytosine derivatives

Abstract

Human activating signal cointegrator homology (ASCH) domain-containing proteins are widespread and diverse but, at present, the vast majority of those proteins have no function assigned to them. This study demonstrates that the 103-amino acid Escherichia coli protein YqfB, previously identified as hypothetical, is a unique ASCH domain-containing amidohydrolase responsible for the catabolism of N 4-acetylcytidine (ac4C). YqfB has several interesting and unique features: i) it is the smallest monomeric amidohydrolase described to date, ii) it is active towards structurally different N 4-acylated cytosines/cytidines, and iii) it has a high specificity for these substrates (k cat /K m up to 2.8 × 10 6 M −1 s −1). Moreover, our results suggest that YqfB contains a unique Thr-Lys-Glu catalytic triad, and Arg acting as an oxyanion hole. The mutant lacking the yqfB gene retains the ability to grow, albeit poorly, on N 4-acetylcytosine as a source of uracil, suggesting that an alternative route for the utilization of this compound exists in E. coli. Overall, YqfB ability to hydrolyse various N 4-acylated cytosines and cytidines not only sheds light on the long-standing mystery of how ac4C is catabolized in bacteria, but also expands our knowledge of the structural diversity within the active sites of amidohydrolases. Human activating signal cointegrator 1 (ASC-1), otherwise known as the thyroid hormone receptor interactor protein 4 (ASC-1/TRIP4), interacts with a wide range of unrelated transcription factors to facilitate nuclear receptors-mediated transcription. It also plays a pivotal role in the transactivation of serum response factor (SRF), activating protein 1 (AP-1), and nuclear factor κB (NF-κB) 1,2. In 2006 it was shown that the C-terminal domain of ASC-1 defines a large ASC-1 homology (ASCH) domain superfamily 2. To date, the ASCH-containing proteins have been reported for a wide range of organisms representing all three kingdoms of life, and have also been found in viruses 3. Although it has long been suggested that this domain of ~110 residues may be responsible for RNA binding during transcription coactivation, RNA processing, and regulation of translation, the vast majority of ASCH proteins are small (~140 residues) hypothetical proteins, which at present have no function assigned to them 2. One such protein is the product of the yqfB gene in Escherichia coli. Herein we demonstrate that the 103-amino acid YqfB is a unique monomeric amidohydrolase responsible for the catabolism of the modified nucleoside, N 4-acetylcytidine (ac4C), in E. coli. More than 160 of differently modified nucleotides play a crucial role in various biological processes 1,2,4,5. The biosynthetic pathways of many modified bases, nucleosides and nucleotides are well understood 5,6 , but the catabolism or salvage of those compounds are scarcely studied. Just like the ASCH proteins, the modified nucleoside ac4C is found in organisms within all three domains of life 4-9. It prevents misreading of AUA isoleucine codons during protein synthes...

A Rapid Method for the Selection of Amidohydrolases from Metagenomic Libraries by Applying Synthetic Nucleosides and a Uridine Auxotrophic Host

“…and N 4 -(4-benzoyl-benzoyl)-2'-deoxycytidine were synthesised as described previously [58]. N 2 -acetylisocytosine and N 4 -acetyl-5-fluorocytosine were prepared as published previously [50].…”

“…The amino acid sequence analysis using SMART [46] revealed that D8_RL contains the human activating signal cointegrator homology (ASCH) [47] and growth-arrest-specific protein 2 (GAS2) domains [48]. ASCH domain-containing proteins are widespread and diverse but, at present, the vast majority of those proteins have no function assigned to them [47], except for an ASCH domain-containing ribonuclease from Zymomonas mobilis [49] and a recently characterized unique ASCH domain-containing amidohydrolase YqfB from Escherichia coli, which is active towards N 4 -acylcytidines [50]. The comparison of YqfB and D8_RL amino acid sequences showed merely 17 identical residues between these two proteins, and only 2 of the 4 YqfB catalytic amino acids were among them, although the aspartic acid residue in position 72 of the D8_RL and the catalytic glutamic acid residue in position 74 of the YqfB enzyme can also be considered conservative ones (Figure 3 and Figure S2).…”

“…A more detailed analysis of the D8_RL hydrolase was carried out. Its kinetic parameters and substrate specificity was compared to those of a recently characterized amidohydrolase YqfB [50]. The kinetic parameters of both D8_RL and YqfB are shown in Table 2.…”

“…The absorption of the reaction mixture at 405 nm was measured against enzyme-free blank to compensate for the substrate auto-hydrolysis [62]. The hydrolytic activity using acylcytidines/cytosines was assayed in reaction mixture, which consisted of 50 mM potassium phosphate buffer, pH 7.5, with 0.1-5 mM substrate and 5-300 ng of protein as described earlier [50]. A kinetic study of D8_RL hydrolase was performed as described previously [50].…”

“…The hydrolytic activity using acylcytidines/cytosines was assayed in reaction mixture, which consisted of 50 mM potassium phosphate buffer, pH 7.5, with 0.1-5 mM substrate and 5-300 ng of protein as described earlier [50]. A kinetic study of D8_RL hydrolase was performed as described previously [50]. The reactions were started by adding an appropriate amount of protein to 50 mM potassium phosphate buffer, pH 8.0, supplemented with 0.1-0.5 mM substrate.…”