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...
SummaryA high‐throughput method (≥ 106 of clones can be analysed on a single agar plate) for the selection of ester‐hydrolysing enzymes was developed based on the uridine auxotrophy of Escherichia coli strain DH10B ΔpyrFEC and the acylated derivatives 2′,3′,5′‐O‐tri‐acetyluridine and 2′,3′,5′‐O‐tri‐hexanoyluridine as the sole source of uridine. The proposed approach permits the selection of hydrolases belonging to different families and active towards different substrates. Moreover, the ester group of the substrate used for the selection, at least partly, determined the specificity of the selected enzymes.
In this study, the development of a rapid, high-throughput method for the selection of amide-hydrolysing enzymes from the metagenome is described. This method is based on uridine auxotrophic Escherichia coli strain DH10B ∆pyrFEC and the use of N4-benzoyl-2’-deoxycytidine as a sole source of uridine in the minimal microbial M9 medium. The approach described here permits the selection of unique biocatalysts, e.g., a novel amidohydrolase from the activating signal cointegrator homology (ASCH) family and a polyethylene terephthalate hydrolase (PETase)-related enzyme.
Cytidine deaminases (CDAs) catalyze the hydrolytic deamination of cytidine and 2′-deoxycytidine to uridine and 2′-deoxyuridine. Here, we report that prokaryotic homo-tetrameric CDAs catalyze the nucleophilic substitution at the fourth position of N 4 -acyl-cytidines, N 4 -alkyl-cytidines, and N 4 -alkyloxycarbonyl-cytidines, and S 4 -alkylthio-uridines and O 4 -alkyl-uridines, converting them to uridine and corresponding amide, amine, carbamate, thiol, or alcohol as leaving groups. The x-ray structure of a metagenomic CDA_F14 and the molecular modeling of the CDAs used in this study show a relationship between the bulkiness of a leaving group and the volume of the binding pocket, which is partly determined by the flexible β3α3 loop of CDAs. We propose that CDAs that are active toward a wide range of substrates participate in salvage and/or catabolism of variously modified pyrimidine nucleosides. This identified promiscuity of CDAs expands the knowledge about the cellular turnover of cytidine derivatives, including the pharmacokinetics of pyrimidine-based prodrugs.
There is an extensive list of applications for nucleosides, nucleotides, and their analogues that spans from substrates and inhibitors in enzymatic research to anticancer and antiviral drugs. Nucleoside phosphates are often obtained by chemical phosphorylation reactions, although enzymatic nucleoside phosphorylation is a promising green alternative. In this work two nucleoside kinases, D. melanogaster deoxynucleoside kinase and B. subtilis deoxycytidine kinase, have been employed for the phosphorylation of various canonical and modified nucleosides, and the results between the two enzymes have been compared. It was determined that both kinases are suitable candidates for enzymatic nucleoside 5′-monophosphate synthesis, as the reaction yields are often in the 40–90% range. Deoxynucleoside kinase, however, often outperforms deoxycytidine kinase and accepts a wider range of nucleoside analogues as substrates. Hence, deoxynucleoside kinase and deoxycytidine kinase were active towards 43 and 34 of 57 tested compounds, respectively. Both nucleoside kinases have been also tested for a larger-scale synthesis of nucleoside monophosphates in the presence of a GTP regeneration system using acetate kinase from E. coli.
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