Cytidine deaminases catalyze the conversion of N(S,O)4-substituted pyrimidine nucleosides.

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 N4-acyl-cytidines, N4-alkyl-cytidines, and N4-alkyloxycarbonyl-cytidines, and S4-alkylthio-uridines and O4-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 ß3α3 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.

An efficient and regioselective biocatalytic synthesis of aromatic N-oxides by using a soluble di-iron monooxygenase PmlABCDEF produced in the Pseudomonas species.

Here, we present an improved whole-cell biocatalysis system for the synthesis of heteroaromatic N-oxides based on the production of a soluble di-iron monooxygenase PmlABCDEF in Pseudomonas sp. MIL9 and Pseudomonas putida KT2440. The presented biocatalysis system performs under environmentally benign conditions, features a straightforward and inexpensive procedure and possesses a high substrate conversion and product yield. The capacity of gram-scale production was reached in the simple shake-flask cultivation. The template substrates (pyridine, pyrazine, 2-aminopyrimidine) have been converted into pyridine-1-oxide, pyrazine-1-oxide and 2-aminopyrimidine-1-oxide in product titres of 18.0, 19.1 and 18.3 g l-1 , respectively. To our knowledge, this is the highest reported productivity of aromatic N-oxides using biocatalysis methods. Moreover, comparing to the chemical method of aromatic N-oxides synthesis based on meta-chloroperoxybenzoic acid, the developed approach is applicable for a regioselective oxidation that is an additional advantageous option in the preparation of the anticipated N-oxides.

Biochemical Pathways Leading to the Formation of Wyosine Derivatives in tRNA of Archaea.

Tricyclic wyosine derivatives are present at position 37 in tRNAPhe of both eukaryotes and archaea. In eukaryotes, five different enzymes are needed to form a final product, wybutosine (yW). In archaea, 4-demethylwyosine (imG-14) is an intermediate for the formation of three different wyosine derivatives, yW-72, imG, and mimG. In this review, current knowledge regarding the archaeal enzymes involved in this process and their reaction mechanisms are summarized. The experiments aimed to elucidate missing steps in biosynthesis pathways leading to the formation of wyosine derivatives are suggested. In addition, the chemical synthesis pathways of archaeal wyosine nucleosides are discussed, and the scheme for the formation of yW-86 and yW-72 is proposed. Recent data demonstrating that wyosine derivatives are present in the other tRNA species than those specific for phenylalanine are discussed.

Microbial Degradation of Pyridine: a Complete Pathway in Arthrobacter sp. Strain 68b Deciphered.

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.

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

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 N4-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 N4-acylated cytosines/cytidines, and iii) it has a high specificity for these substrates (kcat/Km up 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 the yqfB gene retains the ability to grow, albeit poorly, on N4-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 N4-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.

Transient N4 -Acyl-DNA Protection against Cleavage by Restriction Endonucleases.

A set of five N4 -acyl-modified 2'-deoxycytidine 5'-triphosphates were incorporated into modified DNA by using phi29 DNA polymerase, and cleavage by selected restriction endonucleases was studied. Modified DNA containing N4 -acyl functional groups in either one or both strands of a DNA molecule was resistant to the majority of restriction enzymes tested, whereas modifications outside of the recognition sequences were well tolerated. The N4 -acylated cytidine derivatives were subjected to competitive nucleotide incorporation by using phi29 DNA polymerase, showing that a high-fidelity phi29 DNA polymerase efficiently used the modified analogues in the presence of its natural counterpart. These N4 modifications were also demonstrated to be easily removed in an aqueous ethanolamine solution, in which all steps, including primer extension, demodification, and cleavage by restriction endonuclease, could be performed in a one-pot procedure that eliminated additional purification stages. It is suggested that N4 -modified nucleotides are promising building blocks for a programmable; transient; and, most importantly, straightforward DNA protection against specific endonucleases.

Engineering of a chromogenic enzyme screening system based on an auxiliary indole-3-carboxylic acid monooxygenase.

Here, we present a proof-of-principle for a new high-throughput functional screening of metagenomic libraries for the selection of enzymes with different activities, predetermined by the substrate being used. By this approach, a total of 21 enzyme-coding genes were selected, including members of xanthine dehydrogenase, aldehyde dehydrogenase (ALDH), and amidohydrolase families. The screening system is based on a pro-chromogenic substrate, which is transformed by the target enzyme to indole-3-carboxylic acid. The later compound is converted to indoxyl by a newly identified indole-3-carboxylate monooxygenase (Icm). Due to the spontaneous oxidation of indoxyl to indigo, the target enzyme-producing colonies turn blue. Two types of pro-chromogenic substrates have been tested. Indole-3-carboxaldehydes and the amides of indole-3-carboxylic acid have been applied as substrates for screening of the ALDHs and amidohydrolases, respectively. Both plate assays described here are rapid, convenient, easy to perform, and adaptable for the screening of a large number of samples both in Escherichia coli and Rhodococcus sp. In addition, the fine-tuning of the pro-chromogenic substrate allows screening enzymes with the desired substrate specificity.

Application of the uridine auxotrophic host and synthetic nucleosides for a rapid selection of hydrolases from metagenomic libraries.

A 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.

Identification of a 2'-O-Methyluridine Nucleoside Hydrolase Using the Metagenomic Libraries.

Ribose methylation is among the most ubiquitous modifications found in RNA. 2'-O-methyluridine is found in rRNA, snRNA, snoRNA and tRNA of Archaea, Bacteria, and Eukaryota. Moreover, 2'-O-methylribonucleosides are promising starting materials for the production of nucleic acid-based drugs. Despite the countless possibilities of practical use for the metabolic enzymes associated with methylated nucleosides, there are very few reports regarding the metabolic fate and enzymes involved in the metabolism of 2'-O-alkyl nucleosides. The presented work focuses on the cellular degradation of 2'-O-methyluridine. A novel enzyme was found using a screening strategy that employs Escherichia coli uracil auxotroph and the metagenomic libraries. A 2'-O-methyluridine hydrolase (RK9NH) has been identified together with an aldolase (RK9DPA)-forming a part of a probable gene cluster that is involved in the degradation of 2'-O-methylated nucleosides. The RK9NH is functional in E. coli uracil auxotroph and in vitro. The RK9NH nucleoside hydrolase could be engineered to enzymatically produce 2'-O-methylated nucleosides that are of great demand as raw materials for production of nucleic acid-based drugs. Moreover, RK9NH nucleoside hydrolase converts 5-fluorouridine, 5-fluoro-2'-deoxyuridine and 5-fluoro-2'-O-methyluridine into 5-fluorouracil, which suggests it could be employed in cancer therapy.

A versatile method for the UVA-induced cross-linking of acetophenone- or benzophenone-functionalized DNA.

Bioconjugation, biosensing, bioimaging, bionanomaterials, etc., are only a few examples of application of functionalized DNA. Since base-modified nucleic acids contribute not only to a broad range of biotechnological fields but also to the understanding of various cellular processes, it is crucial to design novel modifications with unique properties. Here, we demonstrate the utilization of N4-cytidine modified oligonucleotides, which contain reactive acetophenone (AP) or benzophenone (BP) groups, for the UV-induced cross-linking. We find that terminal deoxynucleotidyl transferase-mediated 3'-tailing using AP/BP-containing modified nucleotides generates photoactive DNA, suitable for a straightforward covalent cross-linking with both interacting proteins and a variety of well-known solid polymeric supports. Moreover, we show that AP/BP-functionalization of nucleic acid molecules induces an efficient cross-linking upon exposure to UVA light. Our findings reveal that 3'-tailed single-stranded DNA bearing AP/BP-moieties is easily photoimmobilized onto untreated polystyrene, polypropylene, polylactate, polydimethylsiloxane, sol-gel and borosilicate glass substrates. Furthermore, we demonstrate that such immobilized DNA probes can be further used for successful hybridization of complementary DNA targets. Our results establish novel N4-cytosine nucleobase modifications as photoreactive labels and suggest an effortless approach for photoimmobilization of nucleic acids.

Discovery of Bacterial Deaminases That Convert 5-Fluoroisocytosine Into 5-Fluorouracil.

Cytosine is one of the four letters of a standard genetic code, found both in DNA and in RNA. This heterocyclic base can be converted into uracil upon the action of the well-known cytosine deaminase. Isocytosine (2-aminouracil) is an isomer of cytosine, yet the enzymes that could convert it into uracil were previously mainly overlooked. In order to search for the isocytosine deaminases we used a selection strategy that is based on uracil auxotrophy and the metagenomic libraries, which provide a random pool of genes from uncultivated soil bacteria. Several genes that encode isocytosine deaminases were found and two respective recombinant proteins were purified. It was established that both novel deaminases do not use cytosine as a substrate. Instead, these enzymes are able to convert not only isocytosine into uracil, but also 5-fluoroisocytosine into 5-fluorouracil. Our findings suggest that novel isocytosine deaminases have a potential to be efficiently used in targeted cancer therapy instead of the classical cytosine deaminases. Use of isocytosine instead of cytosine would produce fewer side effects since deaminases produced by the commensal E. coli gut flora are ten times less efficient in degrading isocytosine than cytosine. In addition, there are no known homologs of isocytosine deaminases in human cells that would induce the toxicity when 5-fluoroisocytosine would be used as a prodrug.

N4-acyl-2'-deoxycytidine-5'-triphosphates for the enzymatic synthesis of modified DNA.

A huge diversity of modified nucleobases is used as a tool for studying DNA and RNA. Due to practical reasons, the most suitable positions for modifications are C5 of pyrimidines and C7 of purines. Unfortunately, by using these two positions only, one cannot expand a repertoire of modified nucleotides to a maximum. Here, we demonstrate the synthesis and enzymatic incorporation of novel N4-acylated 2'-deoxycytidine nucleotides (dCAcyl). We find that a variety of family A and B DNA polymerases efficiently use dCAcylTPs as substrates. In addition to the formation of complementary CAcyl•G pair, a strong base-pairing between N4-acyl-cytosine and adenine takes place when Taq, Klenow fragment (exo-), Bsm and KOD XL DNA polymerases are used for the primer extension reactions. In contrast, a proofreading phi29 DNA polymerase successfully utilizes dCAcylTPs but is prone to form CAcyl•A base pair under the same conditions. Moreover, we show that terminal deoxynucleotidyl transferase is able to incorporate as many as several hundred N4-acylated-deoxycytidine nucleotides. These data reveal novel N4-acylated deoxycytidine nucleotides as beneficial substrates for the enzymatic synthesis of modified DNA, which can be further applied for specific labelling of DNA fragments, selection of aptamers or photoimmobilization.

Conjugation of phosphonoacetic acid to nucleobase promotes a mechanism-based inhibition.

Small molecule inhibitors have a powerful blocking action on viral polymerases. The bioavailability of the inhibitor, nevertheless, often raise a significant selectivity constraint and may substantially limit the efficacy of therapy. Phosphonoacetic acid has long been known to possess a restricted potential to block DNA biosynthesis. In order to achieve a better affinity, this compound has been linked with natural nucleotide at different positions. The structural context of the resulted conjugates has been found to be crucial for the acquisition by DNA polymerases. We show that nucleobase-conjugated phosphonoacetic acid is being accepted, but this alters the processivity of DNA polymerases. The data presented here not only provide a mechanistic rationale for a switch in the mode of DNA synthesis, but also highlight the nucleobase-targeted nucleotide functionalization as a route for enhancing the specificity of small molecule inhibitors.

Modified Nucleotides as Substrates of Terminal Deoxynucleotidyl Transferase.

The synthesis of novel modified nucleotides and their incorporation into DNA sequences opens many possibilities to change the chemical properties of oligonucleotides (ONs), and, therefore, broaden the field of practical applications of modified DNA. The chemical synthesis of nucleotide derivatives, including ones bearing thio-, hydrazino-, cyano- and carboxy groups as well as 2-pyridone nucleobase-containing nucleotides was carried out. The prepared compounds were tested as substrates of terminal deoxynucleotidyl transferase (TdT). The nucleotides containing N4-aminocytosine, 4-thiouracil as well as 2-pyridone, 4-chloro- and 4-bromo-2-pyridone as a nucleobase were accepted by TdT, thus allowing enzymatic synthesis of 3'-terminally modified ONs. The successful UV-induced cross-linking of 4-thiouracil-containing ONs to TdT was carried out. Enzymatic post-synthetic 3'-modification of ONs with various photo- and chemically-reactive groups opens novel possibilities for future applications, especially in analysis of the mechanisms of polymerases and the development of photo-labels, sensors, and self-assembling structures.

Oxyfunctionalization of pyridine derivatives using whole cells of Burkholderia sp. MAK1.

Pyridinols and pyridinamines are important intermediates with many applications in chemical industry. The pyridine derivatives are in great demand as synthons for pharmaceutical products. Moreover, pyridines are used either as biologically active substances or as building blocks for polymers with unique physical properties. Application of enzymes or whole cells is an attractive strategy for preparation of hydroxylated pyridines since the methods for chemical synthesis of pyridinols, particularly aminopyridinols, are usually limited or inefficient. Burkholderia sp. MAK1 (DSM102049), capable of using pyridin-2-ol as the sole carbon and energy source, was isolated from soil. Whole cells of Burkholderia sp. MAK1 were confirmed to possess a good ability to convert different pyridin-2-amines and pyridin-2-ones into their 5-hydroxy derivatives. Moreover, several methylpyridines as well as methylated pyrazines were converted to appropriate N-oxides. In conclusion, regioselective oxyfunctionalization of pyridine derivatives using whole cells of Burkholderia sp. MAK1 is a promising method for the preparation of various pyridin-5-ols and pyridin-N-oxides.

Evolution of tRNAPhe:imG2 methyltransferases involved in the biosynthesis of wyosine derivatives in Archaea.

Tricyclic wyosine derivatives are found at position 37 of eukaryotic and archaeal tRNAPhe In Archaea, the intermediate imG-14 is targeted by three different enzymes that catalyze the formation of yW-86, imG, and imG2. We have suggested previously that a peculiar methyltransferase (aTrm5a/Taw22) likely catalyzes two distinct reactions: N1-methylation of guanosine to yield m1G; and C7-methylation of imG-14 to yield imG2. Here we show that the recombinant aTrm5a/Taw22-like enzymes from both Pyrococcus abyssi and Nanoarchaeum equitans indeed possess such dual specificity. We also show that substitutions of individual conservative amino acids of P. abyssi Taw22 (P260N, E173A, and R174A) have a differential effect on the formation of m1G/imG2, while replacement of R134, F165, E213, and P262 with alanine abolishes the formation of both derivatives of G37. We further demonstrate that aTrm5a-type enzyme SSO2439 from Sulfolobus solfataricus, which has no N1-methyltransferase activity, exhibits C7-methyltransferase activity, thereby producing imG2 from imG-14. We thus suggest renaming such aTrm5a methyltransferases as Taw21 to distinguish between monofunctional and bifunctional aTrm5a enzymes.

Synthesis of Pyridone-based Nucleoside Analogues as Substrates or Inhibitors of DNA Polymerases.

The synthesis and characterization of novel acyclic and cyclic pyridone-based nucleosides and nucleotides is described. In total, seven nucleosides and four nucleotides were synthesized. None of the tested nucleosides showed inhibitory properties against Klenow exo- polymerase and M.MuLV and HIV-1 reverse transcriptases. The nucleotides containing 4-chloro- and 4-bromo-2-pyridone as a nucleobase were accepted by the Klenow fragment, but at the expense of fidelity and extension efficiency.

A 2-Hydroxypyridine Catabolism Pathway in Rhodococcus rhodochrous Strain PY11.

Rhodococcus rhodochrous PY11 (DSM 101666) is able to use 2-hydroxypyridine as a sole source of carbon and energy. By investigating a gene cluster (hpo) from this bacterium, we were able to reconstruct the catabolic pathway of 2-hydroxypyridine degradation. Here, we report that in Rhodococcus rhodochrous PY11, the initial hydroxylation of 2-hydroxypyridine is catalyzed by a four-component dioxygenase (HpoBCDF). A product of the dioxygenase reaction (3,6-dihydroxy-1,2,3,6-tetrahydropyridin-2-one) is further oxidized by HpoE to 2,3,6-trihydroxypyridine, which spontaneously forms a blue pigment. In addition, we show that the subsequent 2,3,6-trihydroxypyridine ring opening is catalyzed by the hypothetical cyclase HpoH. The final products of 2-hydroxypyridine degradation in Rhodococcus rhodochrous PY11 are ammonium ion and α-ketoglutarate.

Ketoreductase TpdE from Rhodococcus jostii TMP1: characterization and application in the synthesis of chiral alcohols.

Background. Production of highly pure enantiomers of vicinal diols is desirable, but difficult to achieve. Enantiomerically pure diols and acyloins are valuable bulk chemicals, promising synthones and potential building blocks for chiral polymers. Enzymatic reduction of ketones is a useful technique for the synthesis of the desired enantiomeric alcohols. Here, we report on the characterization of a ketoreductase TpdE from Rhodococcus jostii TMP1 that is a prospective tool for the synthesis of such compounds. Results. In this study, NADPH-dependent short-chain dehydrogenase/reductase TpdE from Rhodococcus jostii TMP1 was characterized. The enzyme exhibited broad substrate specificity towards aliphatic 2,3-diketones, butan-3-one-2-yl alkanoates, as well as acetoin and its acylated derivatives. TpdE stereospecifically reduced α-diketones to the corresponding diols. The GC-MS analysis of the reduction products of 2,3- and 3,4-diketones indicated that TpdE is capable of reducing both keto groups in its substrate leading to the formation of two new chiral atoms in the product molecule. Bioconversions of diketones to corresponding diols occurred using either purified enzyme or a whole-cell Escherichia coli BL21 (DE3) biocatalyst harbouring recombinant TpdE. The optimum temperature and pH were determined to be 30-35 °C and 7.5, respectively. Conclusions. The broad substrate specificity and stereoselectivity of TpdE from Rhodococcus jostii TMP1 make it a promising biocatalyst for the production of enantiomerically pure diols that are difficult to obtain by chemical routes.

Identification and characterization of a tetramethylpyrazine catabolic pathway in Rhodococcus jostii TMP1.

At present, there are no published data on catabolic pathways of N-heterocyclic compounds, in which all carbon atoms carry a substituent. We identified the genetic locus and characterized key reactions in the aerobic degradation of tetramethylpyrazine in Rhodococcus jostii strain TMP1. By comparing protein expression profiles, we identified a tetramethylpyrazine-inducible protein of 40 kDa and determined its identity by tandem mass spectrometry (MS-MS) de novo sequencing. Searches against an R. jostii TMP1 genome database allowed the identification of the tetramethylpyrazine-inducible protein-coding gene. The tetramethylpyrazine-inducible gene was located within a 13-kb genome cluster, denominated the tetramethylpyrazine degradation (tpd) locus, that encoded eight proteins involved in tetramethylpyrazine catabolism. The genes from this cluster were cloned and transferred into tetramethylpyrazine-nondegrading Rhodococcus erythropolis strain SQ1. This allowed us to verify the function of the tpd locus, to isolate intermediate metabolites, and to reconstruct the catabolic pathway of tetramethylpyrazine. We report that the degradation of tetramethylpyrazine is a multistep process that includes initial oxidative aromatic-ring cleavage by tetramethylpyrazine oxygenase, TpdAB; subsequent hydrolysis by (Z)-N,N'-(but-2-ene-2,3-diyl)diacetamide hydrolase, TpdC; and further intermediate metabolite reduction by aminoalcohol dehydrogenase, TpdE. Thus, the genes responsible for bacterial degradation of pyrazines have been identified, and intermediate metabolites of tetramethylpyrazine degradation have been isolated for the first time.