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...

No abstract

A cryptic plasmid from Arthrobacter rhombi PRH1, designated as pPRH, was sequenced and characterized. It was 5000 bp in length with a G+C content of 66 mol%. The plasmid pPRH was predicted to encode six putative open reading frames (ORFs), in which ORF2 and ORF3 formed the minimal replicon of plasmid pPRH and shared 55-61% and 60-69% homology, respectively, with the RepA and RepB proteins of reported rhodococcal plasmids. Sequence analysis revealed a typical ColE2-type ori located 45 bp upstream of the gene repA. Sequence and phylogenetic analysis led to the conclusion that pPRH is a representative of a novel group of pAL5000 subfamily of ColE2 family plasmids. Three shuttle vectors pRMU824, pRMU824Km and pRMU824Tc, encoding chloramphenicol resistance, were constructed. The latter two harboured additional antibiotic resistance genes kan and tet, respectively. All vectors successfully replicated in Escherichia coli, Arthrobacter and Rhodococcus spp. The vector pRMU824Km was employed for functional screening of 2-hydroxypyridine catabolism encoding genes from Arthrobacter sp. PY22. Sequence analysis of the cloned 6-kb DNA fragment revealed eight putative ORFs, among which hpyB gene encoded a putative monooxygenase.

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.

Bacterial strain 68b was isolated from contaminated soil. According to 16S rDNA analysis it belongs to genus Arthrobacter. This strain is capable to utilise phthalic acid as a sole carbon source. This ability was proved by physiological and biochemical tests. By using resting cells, it was found out that Arthrobacter sp. 68b cells could use phthalic acid or convert quinolinic acid if they were pre-grown in the presence of phthalic acid. While analysing the results of a partially sequenced genome, the putative phthalate degradation operon (pht) was detected. It consisted of eight genes; seven genes could code the conversion of phthalate to protocatechuate. It was determined that the gene (pehA) of putative phthalate ester hydrolase is located upstream of pht operon. Genes of putative phthalate degradation operon were re-sequenced and their sequences fully corresponded to the de novo sequencing data. The homology search of genes revealed that all gene products are most similar to phthalate degradation proteins from other Arthrobacter spp. strains and confirmed that the strain 68b converts phthalate to protocatechuate by 3,4-dioxygenase pathway.

Pyridine and its derivatives constitute the majority of heterocyclic aromatic compounds that occur largely as a result of human activities and contribute to environmental pollution. It is known that they can be degraded by various bacteria in the environment; however, the degradation of unsubstituted pyridine has not yet been completely resolved. In this study, we present data on the pyridine catabolic pathway in Arthrobacter sp. strain 68b at the level of genes, enzymes, and metabolites. The pyr gene cluster, responsible for the degradation of pyridine, was identified in a catabolic plasmid, p2MP. The pathway of pyridine metabolism consisted of four enzymatic steps and ended by the formation of succinic acid. The first step in the degradation of pyridine proceeds through a direct ring cleavage catalyzed by a two-component flavin-dependent monooxygenase system, encoded by pyrA (pyridine monooxygenase) and pyrE genes. The genes pyrB, pyrC, and pyrD were found to encode (Z)-N-(4-oxobut-1-enyl)formamide dehydrogenase, amidohydrolase, and succinate semialdehyde dehydrogenase, respectively. These enzymes participate in the subsequent steps of pyridine degradation. The metabolites of these enzymatic reactions were identified, and this allowed us to reconstruct the entire pyridine catabolism pathway in Arthrobacter sp. 68b. IMPORTANCE The biodegradation pathway of pyridine, a notorious toxicant, is relatively unexplored, as no genetic data related to this process have ever been presented. In this paper, we describe the plasmid-borne pyr gene cluster, which includes the complete set of genes responsible for the degradation of pyridine. A key enzyme, the monooxygenase PyrA, which is responsible for the first step of the catabolic pathway, performs an oxidative cleavage of the pyridine ring without typical activation steps such as reduction or hydroxylation of the heterocycle. This work provides new insights into the metabolism of N-heterocyclic compounds in nature.

An inducible promoter region, PTTMP (tetramethylpyrazine [TTMP]), has been identified upstream of the tpdABC operon, which contains the genes required for the initial degradation of 2,3,5,6-tetramethylpyrazine in Rhodococcus jostii TMP1 bacteria. In this work, the promoter region was fused with the gene for the enhanced green fluorescent protein (EGFP) to investigate the activity of PTTMP by measuring the fluorescence of bacteria. The highest promoter activity was observed when bacteria were grown in a nutrient broth (NB) medium supplemented with 5 mM 2,3,5,6-tetramethylpyrazine for 48 h. Using a primer extension reaction, two transcriptional start sites for tpdA were identified, and the putative −35 and −10 promoter motifs were determined. The minimal promoter along with two 15 bp long direct repeats and two 7 bp inverted sequences were identified. Also, the influence of the promoter elements on the activity of PTTMP were determined using site-directed mutagenesis. Furthermore, PTTMP was shown to be induced by pyrazine derivatives containing methyl groups in the 2- and 5-positions of the heterocyclic ring, in the presence of the LuxR family transcriptional activator TpdR.

Aminotransferases (ATs) are pyridoxal 5′-phosphate-dependent enzymes that catalyze the reversible transfer of an amino group from an amino donor to a keto substrate. ATs are promising biocatalysts that are replacing traditional chemical routes for the production of chiral amines. In this study, an in silico-screening of a metagenomic library isolated from the Curonian Lagoon identified 11 full-length fold type IV aminotransferases that were successfully expressed and used for substrate profiling. Three of them (AT-872, AT-1132, and AT-4421) were active toward (R)-methylbenzylamine. Purified proteins showed activity with L- and D-amino acids and various aromatic compounds such as (R)-1-aminotetraline. AT-872 and AT-1132 exhibited thermostability and retained about 55% and 80% of their activities, respectively, even after 24 h of incubation at 50 °C. Active site modeling revealed that AT-872 and AT-4421 have an unusual active site environment similar to the AT of Haliscomenobacter hydrossis, while AT-1132 appeared to be structurally related to the AT from thermophilic archaea Geoglobus acetivorans. Thus, we have identified and characterized PLP fold type IV ATs that were active toward both amino acids and a variety of (R)-amines.