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 acidEscherichia coliprotein YqfB, previously identified as hypothetical, is a unique ASCH domain-containing amidohydrolase responsible for the catabolism ofN⁴-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 differentN⁴-acylated cytosines/cytidines, and iii) it has a high specificity for these substrates (k_cat/K_mup to 2.8 × 10⁶ 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 theyqfBgene retains the ability to grow, albeit poorly, onN⁴-acetylcytosine as a source of uracil, suggesting that an alternative route for the utilization of this compound exists inE. coli. Overall, YqfB ability to hydrolyse variousN⁴-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.

Summary: A high‐throughput method (≥ 10 ⁶ 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 DH 10B Δpyr FEC 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.

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.

Enzymatic degradation of plastic pollution offers a promising environmentally friendly waste management strategy, however, suitable biocatalysts must be screened and developed. Traditional screening methods using soluble or solubilised polymers do not necessarily identify enzymes that are effective against solid or crystalline polymers. This study presents a simple, time-saving and cost-effective method for identifying microorganisms and enzymes capable of degrading polymeric films. The method was tested on polycaprolactone (PCL), polyethylene terephthalate (PET), polylactate (PLA) and polyhydroxybutyrate/polyhydroxyvalerate (PHB/PHV) films. It involves two steps: first, screening for PCL diol (PCLD)-degrading microorganisms on agar plates, and second, testing these microorganisms on polyester films. Using this screening method, over 100 PCLD-degrading microorganisms and 27 E. coli clones carrying genomic or metagenomic DNA fragments have been isolated. In addition, recombinant cutinases from Streptomyces scabiei and Thermobifida fusca have been tested. Approximately 66 % of the microorganisms forming halos on PCLD agar plates hydrolysed PCL and 6 % - the biaxially oriented PET film. In addition, five PLA- and four PHB/PHV-degrading esterases have been identified. The proposed method is effective for detecting both wild-type and recombinant microorganisms, as well as recombinant enzymes from in vitro transcription-translation reactions. Screening for thermostable and thermophilic enzymes, including those resistant to organic solvents or environmental inhibitors, is also easily implemented.

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.