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Aim Characterization of biosurfactant produced by a carbazole degrading bacterium Roseomonas cervicalis and proteomic analysis of intracellular proteins of bacterium while growing on glucose and carbazole medium. Methods and results The bacterium R. cervicalis was isolated from a soil sample contaminated with crude petroleum oil. PCR amplification ascertained the existence of some hydrocarbon-degrading catabolic genes (alkB and PAH-RHDα, C12O, and C23O) in the bacterial genome. GC-MS and RP-HPLC analyses demonstrated 62% and 60% carbazole degradation, respectively, by R. cervicalis 144 h post-incubation at 37°C and pH 6.5. Due to the paucity of protein databases, expressions of only 29 and 14 intracellular proteins were explicitly recognized and quantitated by mass spectrometry analysis when R. cervicalis was grown in carbazole and glucose medium, respectively. FTIR, NMR, and HR-MS/MS analyses demonstrated the lipopeptide nature of the purified biosurfactant produced by R. cervicalis. The biosurfactant is also presumed to assist in the solubilization of carbazole. Conclusion The isolated R. cervicalis strain is a potential candidate for the bioremediation of carbazole in petroleum-oil contaminated sites. Significance and impact of the study This is the first report of the promising R. cervicalis strain proficient in carbazole biodegradation.

This work assessed the catabolic versatility of functional genes in hydrocarbon-utilizing bacteria obtained from the rhizosphere of plants harvested in aged polluted soil sites in Ogoni and their attenuation efficacy in a bioremediation study. Rhizosphere soil was enumerated for its hydrocarbon-utilizing bacteria. The bacteria were in-vitro screened and selected through the quantification of their total protein and specific intermediate pathway enzyme (catechol 2,3-dioxygenase) activity in the metabolism of hydrocarbon. Thereafter, agarose gel electrophoresis technique was deployed to profile the genome of the selected strains for catechol 2,3-dioxygenase (C23O), 1,2-alkane monooxygenase (alkB), and naphthalene dioxygenase (nahR). Four rhizobacterial isolates namely Pseudomonas fluorescens (A3), Achromobacter agilis (A4), Bacillus thuringiensis (D2), and Staphylococcus lentus (L1) were selected based on the presence of C23O, alkB, and nahR genes. The gel electrophoresis results showed an approximate molecular weight of 200 bp for alkB, 300 bp for C23O, and 400 bp for nahR. The gas chromatogram for residual total petroleum hydrocarbon (TPH) revealed mineralization of fractions C8-C17, phytane, C18-C30. TPH for in-vitro bioremediation of crude oil-polluted soil was observed to have an optimal reduction/loss of 97% within the 56th day of the investigation. This study has further revealed that the microbiome of plants pre-exposed to crude oil pollution could serve as a reservoir for mining group of bacterial with broad catabolic potentials for eco-recovery and waste treatment purposes.

Objective: The principle goal of this examination is to screen bacteria having the ability to debase Low density polyethylene. Bacteria were disengaged from different plastic dump locales. Methods: The secludes acquired were screened for their capacity to use polyethylene as sole carbon source in the mineral salt medium (MSM). Among the isolated strains, Pseudomonas putida and Pseudomonas fluorescens could able to utilize Low Density Polyethylene (LDPE) as carbon source. Further to the preliminary screening, gene specific primers for alkane monooxygenase was synthesized and gradient PCR was performed to determine the presence of alkane monooxygenase gene in the bacterial isolates. Results: Among the four bacteria selected, three bacteria such as Pseudomonas putida and Pseudomonas fluorescens could able to express ALKB gene. Hence, those two bacteria may produce the key enzyme alkane monoxygenase, which is a crucial enzyme for the biotransformation of many xenobiotic compounds including LDPE. Conclusion: From this investigation it is inferred that organisms local to soil can possibly degradeplastic with proper method of time. Keywords: Low density polyethylene; Biodegradation; Bacteria; alkane monoxygenase; invitro Biodegradation assay

The repertoire of nucleobase methylation in DNA and RNA, introduced by chemical agents or enzymes, is large. Most methylation can be reversed either directly by restoration of the original nucleobase or indirectly by replacement of the methylated nucleobase with an unmodified nucleobase. In many direct and indirect demethylation reactions, ALKBH (AlkB homolog) and TET (ten eleven translocation) hydroxylases play a role. Here, we suggest a chemical classification of methylation types. We then discuss pathways for removal, emphasizing oxidation reactions. We highlight the recently expanded repertoire of ALKBH- and TET-catalyzed reactions and describe the discovery of a TET-like protein that resembles the hydroxylases but uses an alternative co-factor and catalyzes glyceryl transfer rather than hydroxylation.

The application of restriction analysis of amplification products of the genes rpoC and alkB, encoding the synthesis of the DNA-dependent RNA polymerase β'-subunit and alkane-1-monooxygenase, respectively, for species identification of R. erythropolis and R. qingshengii was studied. Restriction of the rpoC amplicons (1236 bp) by the enzymes AatII and SacI resulted in formation of the fragments differentiating the closely related species R. erythropolis and R. qingshengii from other members of the genus Rhodococcus. At the same time, R. erythropolis and R. qingshengii differed in the number and nucleotide sequences of the genes encoding the synthesis of alkane-1-monooxygenases (alkB genes). Unlike R. erythropolis, which has four alkB copies in the genome, an additional fifth copy of this determinant was revealed in the chromosome of R. qingshengii. Its amplification may be used to identify these microorganisms by the results of polymerase chain reaction. It was also shown that the most pronounced difference between R. erythropolis and R. qingshengii occurred in the alkB3 nucleotide sequences (~95% identity). The presence of the unique site for the MfeI restriction endonuclease in the product of alkB3 amplification in R. qingshengii may be used to determine their taxonomic position. Polymerase chain reaction, which makes it possible to reveal the alkB5 genes, a unique feature of R. qingshengii, and restriction analysis of the rpoC and alkB3 genetic determinants are rapid diagnostic techniques not requiring additional expense for sequence analysis.

AlkB and its human homologue AlkBH2 are Fe(II)- and 2-oxoglutarate (2OG)-dependent oxygenases that repair alkylated DNA bases occurring as a consequence of reactions with mutagenic agents. We used molecular dynamics (MD) and combined quantum mechanics/molecular mechanics (QM/MM) methods to investigate how structural dynamics influences the selectivity and mechanisms of the AlkB- and AlkBH2-catalyzed demethylation of 3-methylcytosine (m3C) in single (ssDNA) and double (dsDNA) stranded DNA. Dynamics studies reveal the importance of the flexibility in both the protein and DNA components in determining the preferences of AlkB for ssDNA and of AlkBH2 for dsDNA. Correlated motions, including of a hydrophobic β-hairpin, are involved in substrate binding in AlkBH2–dsDNA. The calculations reveal that 2OG rearrangement prior to binding of dioxygen to the active site Fe is preferred over a ferryl rearrangement to form a catalytically productive Fe(IV)=O intermediate. Hydrogen atom transfer proceeds via a σ-channel in AlkBH2–dsDNA and AlkB–dsDNA; in AlkB–ssDNA, there is a competition between σ- and π-channels, implying that the nature of the complexed DNA has potential to alter molecular orbital interactions during the substrate oxidation. Our results reveal the importance of the overall protein–DNA complex in determining selectivity and how the nature of the substrate impacts the mechanism., Our study explores the effects of structural variations and the nature of the protein complexed DNA on the dynamics and mechanisms of 2-oxoglutarate- and iron-dependent DNA damage repair enzymes.

Aliphatic synthetic intermediates with high added value are generally produced from alkane sources (e.g., petroleum) by inert carbon-hydrogen (C-H) bond activation using classical chemical methods (i.e. high temperature, rare metals). As an alternative approach for these reactions, alkane monooxygenase from Pseudomonas putida (alkB) is able to catalyze the difficult terminal oxyfunctionalization of alkanes selectively and under mild conditions. Herein, we report an electrosynthetic system using an alkB biocathode which produces alcohols, epoxides, and sulfoxides through bioelectrochemical hydroxylation, epoxidation, sulfoxidation, and demethylation. The capacity of the alkB binding pocket to protect internal functional groups is also demonstrated. By coupling our alkB biocathode with a hydrogenase bioanode and using H2 as a clean fuel source, we have developed and characterized a series of enzymatic fuel cells capable of oxyfunctionalization while simultaneously producing electricity.

N6-methyladenosine (m6A) is the most prevalent internal RNA modification, especially within eukaryotic messenger RNAs (mRNAs). m6A modifications of RNA regulate splicing, translocation, stability, and translation into proteins. m6A modifications are catalyzed by RNA methyltransferases, such as METTL3, METTL14, and WTAP (writers); the modifications are removed by the demethylases fat mass and obesity-associated protein (FTO) and ALKBH5 (ALKB homolog 5) (erasers); and the modifications are recognized by m6A-binding proteins, such as YTHDF domain-containing proteins and IGF2BPs (readers). Abnormal changes in the m6A levels of these genes are closely related to tumor occurrence and development. In this paper, we review the role of m6A in human cancer and summarize its prospective applications in cancer. Keywords: N6-methyladenosine, m6A, RNA methylation, epigenetic, cancer

The establishment, detection, and alteration or elimination of epigenetic DNA modifications are essential to controlling gene expression ranging from bacteria to mammals. The DNA methylations occurring at cytosine and adenine are carried out by SAM-dependent methyltransferases. Successive oxidations of 5-methylcytosine (5mC) by Tet dioxygenases generate 5-hydroxymethyl (5hmC), 5-formyl (5fC), and 5-carboxyl (5caC) derivatives; thus, DNA elements with multiple methylation sites can have a wide range of modification states. In contrast, oxidation of N6-methyladenine by homologs of Escherichia coli AlkB removes the methyl group directly. Both Tet and AlkB enzymes are 2-oxoglutarate- and Fe(II)-dependent dioxygenases. DNA-binding proteins decode the modification status of specific genomic regions. This article centers on two families of sequence-specific transcription factors: bZIP (basic leucine-zipper) proteins, exemplified by the AP-1 and CEBPβ recognition of 5mC; and bHLH (basic helix-loop-helix) proteins, exemplified by MAX and TCF4 recognition of 5caC. We discuss the impact of template strand DNA modification on the activities of DNA and RNA polymerases, and the varied tendencies of modifications to alter base pairing and their interactions with DNA repair enzymes.

Three-methyl cytosine (3meC) are toxic DNA lesions, blocking base pairing. Bacteria and humans express members of the AlkB enzymes family, which directly remove 3meC. However, other organisms, including budding yeast, lack this class of enzymes. It remains an unanswered evolutionary question as to how yeast repairs 3meC, particularly in single-stranded DNA. The yeast Shu complex, a conserved homologous recombination factor, aids in preventing replication-associated mutagenesis from DNA base damaging agents such as methyl methanesulfonate (MMS). We found that MMS-treated Shu complex-deficient cells exhibit a genome-wide increase in A:T and G:C substitutions mutations. The G:C substitutions displayed transcriptional and replicational asymmetries consistent with mutations resulting from 3meC. Ectopic expression of a human AlkB homolog in Shu-deficient yeast rescues MMS-induced growth defects and increased mutagenesis. Thus, our work identifies a novel homologous recombination-based mechanism mediated by the Shu complex for coping with alkylation adducts.

DNA alkylation is used as the key epigenetic mark in eukaryotes, however, most alkylation in DNA can result in deleterious effects. Therefore, this process needs to be tightly regulated. The enzymes of the AlkB and Ten-Eleven Translocation (TET) families are members of the Fe and alpha-ketoglutarate-dependent superfamily of enzymes that are tasked with dealkylating DNA and RNA in cells. Members of these families span all species and are an integral part of transcriptional regulation. While both families catalyze oxidative dealkylation of various bases, each has specific preference for alkylated base type as well as distinct catalytic mechanisms. This perspective aims to provide an overview of computational work carried out to investigate several members of these enzyme families including AlkB, ALKB Homolog 2, ALKB Homolog 3 and Ten-Eleven Translocate 2. Insights into structural details, mutagenesis studies, reaction path analysis, electronic structure features in the active site, and substrate preferences are presented and discussed.

Site-specific DNA methylation plays an important role in epigenetic regulation of gene expression. Chemical methylation of DNA, including the formation of various methylated nitrogenous bases, leads to the formation of genotoxic modifications that impair DNA functions. Despite the fact that different pathways give rise to methyl groups in DNA, the main pathway for their removal is oxidative demethylation, which is catalyzed by nonheme Fe(II)/α-ketoglutarate–dependent DNA dioxygenases. DNA dioxygenases share a common catalytic mechanism of the oxidation of the alkyl groups on nitrogenous bases in nucleic acids. This review presents generalized data on the catalytic mechanism of action of DNA dioxygenases and on the participation of typical representatives of this superfamily, such as prokaryotic enzyme AlkB and eukaryotic enzymes ALKBH1–8 and TET1–3, in both processes of direct repair of alkylated DNA adducts and in the removal of an epigenetic mark (5-methylcytosine).

The epigenetic modifications control the pathogenicity of human pathogenic fungi, which have been poorly studied in Mucorales, causative agents of mucormycosis. This order belongs to a group referred to as early-diverging fungi that are characterized by high levels of N6-methyldeoxy adenine (6mA) in their genome with dense 6mA clusters associated with actively expressed genes. AlkB enzymes can act as demethylases of 6mA in DNA, with the most remarkable eukaryotic examples being mammalian ALKBH1 and Caenorhabditis elegans NMAD-1. The Mucor lusitanicus (formerly M. circinelloides f. lusitanicus) genome contains one gene, dmt1, and two genes, dmt2 and dmt3, encoding proteins similar to C. elegans NMAD-1 and ALKBH1, respectively. The function of these three genes was analyzed by the generation of single and double deletion mutants for each gene. Multiple processes were studied in the mutants, but defects were only found in single and double deletion mutants for dmt1. In contrast to the wild-type strain, dmt1 mutants showed an increase in 6mA levels during the dimorphic transition, suggesting that 6mA is associated with dimorphism in M. lusitanicus. Furthermore, the spores of dmt1 mutants challenged with macrophages underwent a reduction in polar growth, suggesting that 6mA also has a role during the spore–macrophage interaction that could be important in the infection process.

The epigenetic modifications control the pathogenicity of human pathogenic fungi, which have been poorly studied in Mucorales; causative agents of mucormycosis. This order belongs to a group referred to as early-diverging fungi that are characterized by high levels of N6-methyldeoxyadenine (6mA) in their genome with dense 6mA clusters associated with actively expressed genes. AlkB enzymes can act as demethylases of 6mA in DNA, with the most remarkable eukaryotic examples being mammalian ALKBH1 and Caenorhabditis elegans NMAD-1. Mucor lusitanicus (formerly M. circinelloides f. lusitanicus) genome contains one gene, dmt1, and two genes, dmt2 and dmt3, encoding proteins homologs to C. elegans NMAD-1 and ALKBH1, respectively. The function of the three genes was analyzed by the generation of single and double deletion mutants for each gene. Multiple processes were studied in the mutants, but defects were only found in single and double deletion mutants for dmt1. In contrast to the wild-type strain, dmt1 mutants showed an increase of 6mA levels during the dimorphic transition, suggesting that 6mA regulates dimorphism in M. lusitanicus. Furthermore, the spores of dmt1 mutants challenged with macrophages underwent a reduction of polar growth, suggesting that 6mA also has a role during the spore-macrophage interaction that could be important in the infection process.

This study investigated indigenous functional microbial communities associated with the degradation of chloroacetamide herbicides acetochlor (ACE), S-metolachlor (S-MET) and their enantiomers in repeatedly treated soils. The results showed that biodegradation was the main process for the degradation of ACE, S-MET and their enantiomers. Eight dominant bacterial genera associated with the degradation were found: Amycolatopsis, Saccharomonospora, Mycoplasma, Myroides, Mycobacterium, Burkholderia, Afipia, and Kribbella. The S-enantiomers of ACE and S-MET were preferentially degraded, which mainly relied on Amycolatopsis, Saccharomonospora and Kribbella for the ACE S-enantiomer and Amycolatopsis and Saccharomonospora for the S-MET S-enantiomer. Importantly, the relative abundances of Amycolatopsis and Saccharomonospora increased by 146.3%–4467.2% in the S-enantiomer treatments of ACE and S-MET compared with the control, which were significantly higher than that in the corresponding R-enantiomer treatments (25.3%–4168.2%). Both metagenomic and qPCR analyses demonstrated that four genes, ppah, alkb, benA, and p450, were the dominant biodegradation genes (BDGs) potentially involved in the preferential degradation of the S-enantiomers of ACE and S-MET. Furthermore, network analysis suggested that Amycolatopsis, Saccharomonospora, Mycoplasma, Myroides, and Mycobacterium were the potential hosts of these four BDGs. Our findings indicated that Amycolatopsis and Saccharomonospora might play pivotal roles in the preferential degradation of the S-enantiomers of ACE and S-MET.

Digestive system cancers are common cancers with high cancer deaths worldwide. They have become a major threat to public health and economic burden. As one of the most universal RNA modifications in eukaryotes, the N6-methyladenosine (m6A) modification is involved in the occurrence, development, prognosis, and treatment response of various cancers, including digestive system cancers. M6A demethylases shape the m6A landscape dynamically, playing important roles in cancers. In addition, accumulating evidence reveal that many environmental toxicants are the established risk factors for digestive system cancers and associated with m6A modification. In this review, we summarize the multiple functions of M6A demethylases (fat mass and obesity-associated protein (FTO), AlkB homolog 5 (ALKBH5) and AlkB homolog 3 (ALKBH3)) in digestive system cancers, which are aberrantly expressed and affect cancer progression. We also discuss the potential roles of m6A demethylases in the assessment of environmental exposure, the signature for prevention and diagnosis of digestive system cancers.

Class-II fumarases (fumarate hydratase, FH) are dual-targeted enzymes occurring in the mitochondria and cytosol of all eukaryotes. They are essential components in the DNA damage response (DDR) and, more specifically, protect cells from DNA double-strand breaks. Similarly, the gram-positive bacterium Bacillus subtilis class-II fumarase, in addition to its role in the tricarboxylic acid cycle, participates in the DDR. Escherichia coli harbors three fumarase genes: class-I fumA and fumB and class-II fumC Notably, class-I fumarases show no sequence similarity to class-II fumarases and are of different evolutionary origin. Strikingly, here we show that E. coli fumarase functions are distributed between class-I fumarases, which participate in the DDR, and the class-II fumarase, which participates in respiration. In E. coli, we discover that the signaling molecule, alpha-ketoglutarate (α-KG), has a function, complementing DNA damage sensitivity of fum-null mutants. Excitingly, we identify the E. coli α-KG-dependent DNA repair enzyme AlkB as the target of this interplay of metabolite signaling. In addition to α-KG, fumarate (fumaric acid) is shown to affect DNA damage repair on two different levels, first by directly inhibiting the DNA damage repair enzyme AlkB demethylase activity, both in vitro and in vivo (countering α-KG). The second is a more global effect on transcription, because fum-null mutants exhibit a decrease in transcription of key DNA damage repair genes. Together, these results show evolutionary adaptable metabolic signaling of the DDR, in which fumarases and different metabolites are recruited regardless of the evolutionary enzyme class performing the function.

Fe2+/2-ketoglutarate-dependent DNA-dioxygenase AlkB from Escherichia coli is able to restore the native structure of alkylated DNA bases. The enzymatic process utilizes the molecular oxygen, and proceeds through a mechanism of oxidative dealkylation. Here, the kinetics of conformational changes of AlkB and DNA substrates in the course of binding steps were studied. Nickel and cobalt divalent ions were used instead of Fe2+ as metal cofactors in order to inhibit the catalytic activity of AlkB and to study certain stages leading to the formation of a catalytically active enzyme–substrate complex.

AlkB family proteins are enzymes that repair alkylated DNA and RNA by oxidative demethylation. Nine homologs have been identified and characterized in mammals. ALKBH1 is conserved among metazoans including Drosophila. Although the ALKBH1 mouse homolog, Alkbh1 functions in neurogenesis, it currently remains unclear whether ALKBH1 plays a role in neuronal disorders induced by ultraviolet-induced DNA damage. We herein demonstrated that the Drosophila ALKBH1 homolog, AlkB contributed to recovery from neuronal disorders induced by ultraviolet damage. The knockdown of AlkB resulted in not only learning defects but also altered crawling behavior in Drosophila larvae after ultraviolet irradiation. A molecular analysis revealed that AlkB contributed to the repair of ultraviolet-induced DNA damage in the central nervous system of larvae. Therefore, we propose that ALKBH1 plays a role in the repair of ultraviolet-induced DNA damage in central nervous system. Ultraviolet-induced DNA damage is involved in the pathogenesis of xeroderma pigmentosum, and has recently been implicated in Parkinson's disease. The present results will contribute to our understanding of neuronal diseases induced by ultraviolet-induced DNA damage.

Aim of present work was to assess in-planta association potential of isolated endophytic bacterial strain Pseudomonas sp. (J10) (KY608252) with two cultivars of Lolium perenne L. (small & jumbo) and Arabidopsis thaliana L. for total petroleum hydrocarbon (TPH) degradation, alkane monooxygenase (alkb) gene expression and phytotoxicity analysis. A plant-microbe phytoremediation system was established to investigate the bacteria's ability to colonize the plant body and quantification of alkb gene to help withstand TPH stress in soil as well as in hydroponics. A real-time PCR method was developed to analyze bacterial colonization and survival within the plant body. Analysis revealed that J10 efficiently colonized all the tested plant species and expressed alkb gene under hydrocarbon stress ranging between 3.7 × 102–3.9 × 106 in A. thaliana and L. perenne (small), respectively. The colonization was more pronounced in soil as compared to hydroponic system. J10 inoculation reduced phytotoxicity and suggested that inoculation had a positive effect on plant growth under stress conditions as compared to control. L. perenne (small) showed significant TPH removal efficiency (45.6%) followed by L. perenne jumbo (24.5%) and A. thaliana (6.2%). In hydroponics, L. perenne (small) degraded about 28.2% TPH followed by L. perenne (jumbo) as 24.4%. Potential of the indigenously isolated plant endophytes may be exploited further for phytoremediation efficiency and industrial applications.

5-Methylcytosine (5mC) in DNA CpG islands is an important epigenetic biomarker for mammalian gene regulation. It is oxidized to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) by the ten-eleven translocation (TET) family enzymes, which are α-ketoglutarate (α-KG)/Fe(II)-dependent dioxygenases. In this work, we demonstrate that the epigenetic marker 5mC is modified to 5hmC, 5fC, and 5caC in vitro by another class of α-KG/Fe(II)-dependent proteins—the DNA repair enzymes in the AlkB family, which include ALKBH2, ALKBH3 in huamn and AlkB in Escherichia coli. Theoretical calculations indicate that these enzymes may bind 5mC in the syn-conformation, placing the methyl group comparable to 3-methylcytosine, the prototypic substrate of AlkB. This is the first demonstration of the AlkB proteins to oxidize a methyl group attached to carbon, instead of nitrogen, on a DNA base. These observations suggest a broader role in epigenetics for these DNA repair proteins.

The annual production of plastics has doubled over the past 15 years and, consequently, a large amount of plastic has accumulated in the environment generating ecological problems. In this study, a Paenibacillus sp. isolate was obtained from a landfill from Brazil and it presented the alkane hydroxylase gene (alkB). Weight loss of low-density polyethylene (LDPE) was measured and a significant difference in final weight compared to initial weight was assessed. Some chemical characteristics, such as bond scissions and formation of new functional groups [carboxylic acids (3300–2500 cm−1), esters (1210–1163 cm−1), and ethers (1075–1020 cm−1)], were detected by Fourier-transform infrared spectroscopy. Bacterial colonization on the plastic surface and physical changes, as formation of cracks and pits, was visualized by scanning electron microscopy. This isolate was susceptible to all the antimicrobials tested. Therefore, this isolate possesses great potential to degrade polyethylene and become an option for LDPE bioremediation.

With the intensive development of the sheep industry and increasing global temperatures, heat stress in sheep has become an increasingly severe and important issue in recent years. The level of N6-methyladenosine (m6A) RNA methylation changes in response to stress plays important roles in stress responses. However, the role of m6A in the heat stress response of sheep remains unclear. To explore this issue, we measured heat stress protein (HSP) expression, liver function indexes, m6A on RNA, m6A-related enzyme expression, and tissue damage in sheep that had been subjected to heat stress. At the transcriptome level, our results showed significant increases in m6A on RNA and increased mRNA levels of HSPs (HSP70, HSP90, and HSP110) and m6A-related enzymes [METTL3 (methyltransferase-like 3), METTL14 (methyltransferase-like 14), WTAP (wilms tumor 1-associated protein), FTO (fat mass and obesity-associated protein), ALKBH5 (alkB homologue 5), YTHDF1-3 (YTH domain family proteins), and YTHDC1-2 (YTH domain-containing proteins)] following heat stress. At the protein level, the expression of METTL3, YTHDF1-2, and YTHDC2 showed no significant differences following heat stress. However, in contrast to its mRNA level after heat stress, the protein expression of YTHDF3 was reduced, while the expression of HSPs (HSP70, HSP90, and HSP110), METTL14, WTAP, FTO, ALKBH5, YTHDF3, and YTHDC1 increased in line with their measured mRNA levels. Histological experiments revealed that heat stress caused varying degrees of damage to sheep liver tissue. Moreover, immunohistochemical staining indicated that the m6A-related enzymes were expressed in sheep hepatocytes, and differences in expression patterns were observed between the control and heat stress groups. In summary, differences in the level of m6A and the expression of m6A-related enzymes in the liver of sheep were observed after heat stress. This indicates that m6A is involved in the regulation of heat stress in sheep. Our findings provide a new avenue for studying the responses to heat stress in sheep.

Studding the diversity of soil indigenous microorganisms, and monitoring effect of contaminants on microbial population, is very critical for understanding microbial activity during bioremediation and selecting successful remediation strategy. To simulate the natural environment, four microcosms were prepared by artificially contaminating clean soil with defined amounts of petroleum hydrocarbons including alkanes mixture (C13–C20), polyaromatic hydrocarbons (PAHs) mixture (anthracene, phenanthrene, fluoranthene, pyrene and benzo (α) pyrene) and both alkanes and PAHs. Contaminants degradation and heterotrophic bacterial count were measured during a 6-month study. Copy number of alkB and C23DO genes was studied using real-time PCR, and bacterial diversity was monitored by 16S rRNA gene PCR and denaturing gradient gel electrophoresis (DGGE). Results indicated that all types of contaminants (except the five ring benzo (α) pyrene) were totally degraded after 6 months and the increase in hydrocarbon degradation rate coincided with the enhancement of total heterotrophic bacterial count in each microcosm. Real-time PCR results showed a significant increase in the copy number of both alkB and C23DO genes in alkane- and PAHs-contaminated microcosm comparing with the control microcosm, indicating selection for special hydrocarbon degraders in hydrocarbon-amended microcosms. The results of DGGE revealed that the type of contaminant in the same soil has a remarkable influence on soil bacterial community structure. Sequencing of DGGE bands suggested that most of the dominant members of the microbial community of contaminated soil are unculturable bacteria from Proteobacteria and the genus Bacillus.

The combination of phage display technology with high-throughput sequencing enables in-depth analysis of library diversity and selection-driven dynamics. We applied short-read sequencing of the mutagenized region on focused display libraries of two homologous nucleic acid modification eraser proteins-AlkB and FTO-biopanned against methylated DNA. This revealed enriched genotypes with small indels and concomitant doubtful amino acid motifs within the FTO library. Nanopore sequencing of the entire display vector showed additional enrichment of large deletions overlooked by region-specific sequencing, and further impacted the interpretation of the obtained amino acid motifs. We could attribute enrichment of these corrupted clones to amplification bias due to arduous FTO display slowing down host cell growth as well as phage production. This amplification bias appeared to be stronger than affinity-based target selection. Recommendations are provided for proper sequence analysis of phage display data, which can improve motive discovery in libraries of proteins that are difficult to display. ispartof: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES vol:22 issue:11 ispartof: location:Switzerland status: published

AlkB homolog 5 (ALKBH5) has been reported as a key m6A demethylase that is involved in development and diseases; however, the function of ALKBH5 in osteogenesis remains unknown. In this study, we report that ALKBH5 mRNA and protein expression were upregulated during osteoblast differentiation and that ALKBH5 knockdown suppressed osteoblast differentiation, mineralization, and the expression of osteogenic biomarkers. Conversely, ALKBH5 overexpression promoted osteogenesis. Moreover, the expression of wild-type ALKBH5, but not the m6A-modified active site mutant ALKBH5, could rescue ALKBH5 knockdown-induced osteogenesis inhibition. Furthermore, knockdown of ALKBH5 significantly impaired the mRNA stability of the transcription factor Runx2, which plays a key role in osteoblast differentiation. Taken together, our results suggest that ALKBH5 promotes osteogenesis through modulating Runx2 mRNA stability.

usc This study assessed the ability of phosphorus (P) fertilizer to remediate the rhizosphere of three wild plant species (Banksia seminuda, a tree; Chloris truncata, a grass; and Hakea prostrata, a shrub) growing in a soil contaminated with total (aliphatic) petroleum hydrocarbon (TPH). Plant growth, photosynthesis (via chlorophyll fluorescence), soil microbial activity, alkane hydroxylase AlkB (aliphatic hydrocarbon-degrading) gene abundance, and TPH removal were evaluated 120 days after planting. Overall, although TPH served as an additional carbon source for soil microorganisms, the presence of TPH in soil resulted in decreased plant growth and photosynthesis. However, growth, photosynthesis, microbial activities, and AlkB gene abundance were enhanced by the application of P fertilizer, thereby increasing TPH removal rates, although the extent and optimum P dosage varied among the plant species. The highest TPH removal (64.66%) was observed in soil planted with the Poaceae species, C. truncata, and amended with 100 mg P kg−1 soil, while H. prostrata showed higher TPH removal compared to the plant belonging to the same Proteaceae family, B. seminuda. The presence of plants resulted in higher AlkB gene abundance and TPH removal relative to the unplanted control. The removal of TPH was associated directly with AlkB gene abundance (R2 > 0.9, p < 0.001), which was affected by plant identity and P levels. The results indicated that an integrated approach involving wild plant species and optimum P amendment, which was determined through experimentation using different plant species, was an efficient way to remediate soil contaminated with TPH. Refereed/Peer-reviewed

Three-methyl cytosine (3meC) are toxic DNA lesions, blocking base pairing. Bacteria and humans, express members of the AlkB enzymes family, which directly remove 3meC. However, other organisms, including budding yeast, lack this class of enzymes. It remains an unanswered evolutionary question as to how yeast repairs 3meC, particularly in single-stranded DNA. The yeast Shu complex, a conserved homologous recombination factor, aids in preventing replication-associated mutagenesis from DNA base damaging agents such as methyl methanesulfonate (MMS). We found that MMS-treated Shu complex-deficient cells, exhibit a genome-wide increase in A:T and G:C substitutions mutations. The G:C substitutions displayed transcriptional and replicational asymmetries consistent with mutations resulting from 3meC. Ectopic expression of a human AlkB homolog in Shu-deficient yeast rescues MMS-induced growth defects and increased mutagenesis. Finally, the Shu complex exhibits increased affinity for 3meC-containing DNA. Thus, our work identifies a novel mechanism for coping with alkylation adducts.

Polystyrene (PS), a major plastic waste, is difficult to biodegrade due to its unique chemical structure that comprises phenyl moieties attached to long linear alkanes. In this study, we investigated the biodegradation of PS by mesophilic bacterial cultures obtained from various soils in common environments. Two new strains, Pseudomonas lini JNU01 and Acinetobacter johnsonii JNU01, were specifically enriched in non-carbonaceous nutrient medium, with PS as the only source of carbon. Their growth after culturing in basal media increased more than 3-fold in the presence of PS. Fourier transform infrared spectroscopy analysis, used to confirm the formation of hydroxyl groups and potentially additional chemical bond groups, showed an increase in the amount of oxidized PS samples. Moreover, field emission scanning electron microcopy analysis confirmed PS biodegradation by biofilms of the screened microbes. Water contact angle measurement additionally offered insights into the increased hydrophilic characteristics of PS films. Bioinformatics and transcriptional analysis of A. johnsonii JNU01 revealed alkane-1-monooxygenase (AlkB) to be involved in PS biodegradation, which was confirmed by the hydroxylation of PS using recombinant AlkB. These results provide significant insights into the discovery of novel functions of Pseudomonas sp. and Acinetobacter sp., as well as their potential as PS decomposers.

DNA alkylation damage, emanating from the exposure to environmental alkylating agents or produced by certain endogenous metabolic processes, affects cell viability and genomic stability. Fe(II)/2-oxoglutarate-dependent dioxygenase enzymes, such as Escherichia coli AlkB, are involved in protecting DNA from alkylation damage. Inspired by the natural product indenone derivatives reported to inhibit this class of enzymes, and a set of 2-chloro-3-amino indenone derivatives was synthesized and screened for their inhibitory properties against AlkB. The synthesis of 2-chloro-3-amino indenone derivatives was achieved from 2,3-dichloro indenones through addition-elimination method using alkyl/aryl amines under catalyst-free conditions. Using an in vitro reconstituted DNA repair assay, we have identified a 2-chloro-3-amino indenone compound 3o to be an inhibitor of AlkB. We have determined the binding affinity, mode of interaction, and kinetic parameters of inhibition of 3o and tested its ability to sensitize cells to methyl methanesulfonate that mainly produce DNA alkylation damage. This study established the potential of indenone-derived compounds as inhibitors of Fe(II)/2-oxoglutarate-dependent dioxygenase AlkB.

Alkylated DNA lesions, induced by both exogenous chemical agents and endogenous metabolites, represent a major form of DNA damage in cells. The repair of alkylation damage is critical in all cells because such damage is cytotoxic and potentially mutagenic. Alkylation chemotherapy is a major therapeutic modality for many tumors, underscoring the importance of the repair pathways in cancer cells. Several different pathways exist for alkylation repair, including base excision and nucleotide excision repair, direct reversal by methyl-guanine methyltransferase (MGMT), and dealkylation by the AlkB homolog (ALKBH) protein family. However, maintaining a proper balance between these pathways is crucial for the favorable response of an organism to alkylating agents. Here, we summarize the progress in the field of DNA alkylation lesion repair and describe the implications for cancer chemotherapy.

DNA and RNA have various methylation modifications or damage that are directly related to some human diseases and physiological regulation. Most of these methylation modifications are reversible and can be dynamically repaired by RNA or DNA demethylases. Over the past few decades, enzymes from the ALKB and TET families have been shown to have the ability to demethylate nucleic acids, which involves intermediates in the oxidative repair process. These intermediates can be accurately captured by advanced methods such as HPLC, LC-MS, TLC, and crystallization, which can significantly promote our understanding of the dynamic mechanism of demethylation. In this review, we discuss recent research advances in this area and raise open questions and constructive opinions about the capture of nucleic acid demethylation intermediates.

Ozdemir, Guven/0000-0002-7577-4233; SAHAR, Umut/0000-0002-2200-6986; Vural, Caner/0000-0003-1400-6377; DIALLO, Mamadou Malick/0000-0001-8264-2960, WOS: 000497848100001, PubMed: 31752596, Biodegradation is a cost-effective process commonly used to eliminate many xenobiotic hydrocarbons such as diesel oils. However, their hydrophobic character reduces the biodegradation efficiency. in order to overcome this hurdle, kurstakins isolated from Bacillus thuringiensis strain 7SA were used as emulsifying agents. the influence of kurstakin molecules on diesel oil degradation by Acinetobacter haemolyticus strain 2SA was evaluated in the presence and absence of the aforementioned lipopeptide. the degradation rates and gene expressions of alkane hydroxylases were evaluated at days 4, 10, 14 and 21. Results showed that kurstakin molecules increased the hydrophobicity of 2SA. Moreover, diesel oil degradation activities were higher in the presence of kurstakin with 29%, 35%, 29% and 23% improvement at 4th, 10th, 14th and 21st day respectively. Statistical analysis indicated that the difference between the degradation rates in the presence and absence of kurstakin was significant with p = 0.03. the detection of three different hydroxylase genes namely alkB, almA and cyp153 in 2SA genome, might have allowed more efficient degradability of alkanes. According to the real-time PCR results, cyp153 was the most induced gene during diesel oil degradation in the presence and absence of kurstakin. Yet, the three genes demonstrated higher levels of expression in the presence of kurstakin when compared to its absence. This study showed that kurstakins enhance the diesel oil biodegradation rate by increasing the hydrophobicity of 2SA. in addition to their anti-fungal activities, kurstakins can be used as biosurfactant to increase biodegradation of diesel oil.

AlkB is a DNA/RNA repair enzyme that removes base alkylations such as N1-methyladenosine (m1A) or N3-methylcytosine (m3C) from DNA and RNA. The AlkB enzyme has been used as a critical tool to facilitate tRNA sequencing and identification of mRNA modifications. As a tool, AlkB mutants with better reactivity and new functionalities are highly desired; however, previous identification of such AlkB mutants was based on the classical approach of targeted mutagenesis. Here, we introduce a high-throughput screening method to evaluate libraries of AlkB variants for demethylation activity on RNA and DNA substrates. This method is based on a fluorogenic RNA aptamer with an internal modified RNA/DNA residue which can block reverse transcription or introduce mutations leading to loss of fluorescence inherent in the cDNA product. Demethylation by an AlkB variant eliminates the blockage or mutation thereby restores the fluorescence signals. We applied our screening method to sites D135 and R210 in the Escherichia coli AlkB protein and identified a variant with improved activity beyond a previously known hyperactive mutant toward N1-methylguanosine (m1G) in RNA. We also applied our method to O6-methylguanosine (O6mG) modified DNA substrates and identified candidate AlkB variants with demethylating activity. Our study provides a high-throughput screening method for in vitro evolution of any demethylase enzyme.

DNA integrity is challenged by both exogenous and endogenous alkylating agents. DNA repair proteins such as Escherichia coli AlkB family of enzymes can repair 1-methyladenine and 3-methylcytosine adducts by oxidative demethylation. Human AlkB homologue 5 (ALKBH5) is RNA N6-methyladenine demethylase and not known to be involved in DNA repair. Herein we show that ALKBH5 also has weak DNA repair activity and it can demethylate DNA 3-methylcytosine. The mutation of the amino acid residues involved in demethylation also abolishes the DNA repair activity of ALKBH5. Overexpression of ALKBH5 decreases the 3-methylcytosine level in genomic DNA and reduces the cytotoxic effects of the DNA damaging alkylating agent methyl methanesulfonate. Thus, demethylation by ALKBH5 might play a supporting role in maintaining genome integrity.

Iron-dependent dioxygenases of the AlkB protein family found in most organisms throughout the tree of life play a major role in oxidative dealkylation processes. Many of these enzymes have attracted the attention of researchers across different fields and have been subjected to thorough biochemical characterization because of their link to human health and disease. For example, several mammalian AlkB homologues are involved in the direct reversal of alkylation damage in DNA, while others have been shown to play a regulatory role in epigenetic or epitranscriptomic nucleic acid methylation or in post-translational modifications such as acetylation of actin filaments. These studies show that that divergence in amino acid sequence and structure leads to different characteristics and substrate specificities. In this review, we aim to summarize current insights in the structural features involved in the substrate selection of AlkB homologues, with focus on nucleic acid interactions. ispartof: Dna Repair vol:96 ispartof: location:Netherlands status: Published online

The AlkB family of non-heme-Fe(II)/2-oxoglutarate(2OG)-dependent oxygenases are essential regulators of RNA epigenetics by serving as erasers of one-carbon marks on RNA with release of formaldehyde (FA). Two major human AlkB family members, FTO and ALKBH5, both act as oxidative demethylases of N6 methyladenosine (m6A) but furnish different major products, N6 hydroxymethyladenosine (hm6A) and adenosine (A), respectively. Here we identify foundational mechanistic differences between FTO and ALKBH5 that promote these distinct biochemical outcomes. In contrast to FTO, which follows a traditional oxidative N-demethylation pathway to catalyze conversion of m6A to hm6A with subsequent slow release of A and FA, we find that ALKBH5 catalyzes a directm6A-to-A transformation with rapid FA release. We identify a catalytic R130/K132/Y139 triad within ALKBH5 that facilitates release of FA via an unprecedented covalent-based demethylation mechanism with direct detection of a covalent intermediate. Importantly, a K132Q mutant furnishes an ALKBH5 enzyme with an m6A demethylation profile that resembles that of FTO, establishing the importance of this residue in the proposed covalent mechanism. Finally, we show that ALKBH5 is an endogenous source of FA in the cell by activity-based sensing of FA fluxes perturbed via ALKBH5 knockdown. This work provides a fundamental biochemical rationale for non-redundant roles of these RNA demethylases beyond different substrate preferences and cellular localization, where m6A demethylation by ALKBH5 versus FTO results in release of FA, an endogenous one-carbon unit but potential genotoxin, at different rates in living systems.Significance StatementNon-heme iron enzymes FTO and ALKBH5 play central roles in epigenetic RNA regulation by catalyzing the oxidation of N6-methyladenosine (m6A) to produce N6-hydroxymethyladenosine (hm6A) and adenosine (A), respectively. Here, we provide a mechanistic rationale for these distinct biochemical outcomes by identifying that ALKBH5 performs m6A demethylation via an unprecedented covalent-based mechanism with concomitant and rapid release of A and formaldehyde (FA), whereas FTO liberates hm6A to release A and FA over longer timescales. This work reveals foundational biochemical differences between these closely related but non-redundant epigenetic enzymes and identifies ALKBH5 as an endogenous source of rapid formaldehyde generation in cells.

Alkb homolog 7 (ALKBH7) is a mitochondrial α-ketoglutarate dioxygenase required for necrotic cell death in response to DNA alkylating agents, but its physiologic role within tissues remains unclear. Herein, we show that ALKBH7 plays a key role in the regulation of dialdehyde metabolism, which impacts cardiac survival in response to ischemia-reperfusion (IR) injury. Using a multi-omics approach, we do not find evidence that ALKBH7 functions as a prolyl-hydroxylase. However, we do find that mice lacking ALKBH7 exhibit a significant increase in glyoxalase I (GLO-1), a dialdehyde detoxifying enzyme. Consistent with increased dialdehyde production, metabolomics analysis reveals rewiring of metabolic pathways related to the toxic glycolytic by-product methylglyoxal (MGO), as well as accelerated glycolysis and elevated levels of MGO protein adducts, in mice lacking ALKBH7. Consistent with roles for both necrosis and glycative stress in cardiac IR injury, hearts from male but not femaleAlkbh7-/-mice are protected against IR, although somewhat unexpectedly this protection does not appear to involve modulation of the mitochondrial permeability transition pore. Highlighting the importance of MGO metabolism for the observed protection, removal of glucose as a metabolic substrate or pharmacologic inhibition of GLO-1 both abrogate cardioprotection in ALKBH7 deficient mice. Integrating these observations, we propose that ALKBH7 plays a role in the regulation of glyoxal metabolism, and that protection against necrosis and IR injury bought on by ALKBH7 deficiency originates from hormetic signaling in response to elevated MGO stress.

Class-II fumarases (Fumarate Hydratase, FH) are dual targeted enzymes, occurring in the mitochondria and cytosol of all eukaryotes. They are essential components in the DNA damage response (DDR) and more specifically, protecting cells from DNA double strand breaks. Similarly, the Gram-positive Bacterium Bacillus subtilis Class-II fumarase, in addition to its role in the TCA cycle, also participates in the DDR. Escherichia coli, harbors three fumarase genes; Class-I fumA and fumB and Class-II fumC. Notably, Class-I fumarases, show no sequence similarity to Class-II fumarases and are of different evolutionary origin. Strikingly, here we show that E. coli fumarase functions are distributed between Class-I fumarases which participate in the DDR, and the Class-II fumarase which participates in respiration. In E. coli, we discover that the signaling molecule, alpha-ketoglutarate (α-KG), has a novel function, complementing DNA damage sensitivity of fum null mutants. Excitingly, we identify the E. coli α-KG dependent DNA repair enzyme AlkB, as the target of this interplay of metabolite signaling. In addition to α-KG, fumarate (fumaric acid) is shown to affect DNA damage repair on two different levels, first by directly inhibiting the DNA damage repair enzyme AlkB demethylase activity, both in vitro and in vivo (countering α-KG). The second is a more global effect on transcription, as fum null mutants exhibit a decrease in transcription of key DNA damage repair genes. Together these results show evolutionary adaptable metabolic signaling of the DDR, in which fumarases and different metabolites are recruited regardless of the evolutionary enzyme Class preforming the function.Significance StatementClass-II fumarases have been shown to participate in cellular respiration and the DNA damage response. Here we show, for the first time, that in the model prokaryote, Escherichia coli, which harbors both Class-I and Class-II fumarases, it is the Class-I fumarases that participate in DNA damage repair by a mechanism which is different than those described for other fumarases. Strikingly, this mechanism employs a novel signaling molecule, alpha-ketoglutarate (α-KG), and its target is the DNA damage repair enzyme AlkB. In addition, we show that fumarase precursor metabolites, fumarate and succinate, can inhibit the α-KG-dependent DNA damage repair enzyme, AlkB, both in vitro and in vivo. This study provides a new perspective on the function and evolution of metabolic signaling.

Background ALK is the key gene controlling rice gelatinization temperature (GT), which is closely associated with the eating and cooking quality (ECQ) in rice (Oryza sativa L.). To date, at least three ALK alleles are thought to be responsible for the diversity of GT among rice cultivars. The ALKc/SSIIai allele with high activity of the soluble starch synthase IIa (SSIIa) controls high GT, but the accurate functional difference between ALKa and ALKb alleles, both controlling low GT, is not clearly elucidated. Thus, we generated rice near-isogenic lines (NILs) by introducing different ALK alleles into the japonica cultivar Nipponbare (Nip) to clarify the discrepant effects of the two low-GT ALK alleles. Results The results showed that the function of two low-GT alleles (ALKa and ALKb) was different, and a much lower GT was observed in NIL(ALKb) rice grains compared with that of Nip(ALKa). Moreover, the starches of NIL(ALKb) grains had a higher degree of branching, higher setback, consistence and higher cool pasting viscosity than those of Nip(ALKa). The lower expression level of ALKb, compared with ALKa, resulted in depleted intermediate chains and increased short chains of amylopectin, thus affected the thermal and pasting properties of NILs’ grains. Also, the data revealed both low-GT alleles were mainly found in temperate japonica, but more ALKb was found in other subpopulations such as indica as compared to ALKa. Conclusions Overall, all the results suggested that the function between two low-GT alleles was different, and the distribution of ALKb was much wider than that of ALKa among the subpopulations of cultivated rice.

The complete genome sequence of Rhodococcus sp. WAY2 (WAY2) consists of a circular chromosome, three linear replicons and a small circular plasmid. The linear replicons contain typical actinobacterial invertron-type telomeres with the central CGTXCGC motif. Comparative phylogenetic analysis of the 16S rRNA gene along with phylogenomic analysis based on the genome-togenome blast distance phylogeny (GBDP) algorithm and digital DNA–DNA hybridization (dDDH) with other Rhodococcus type strains resulted in a clear differentiation of WAY2, which is likely a new species. The genome of WAY2 contains five distinct clusters of bph, etb and nah genes, putatively involved in the degradation of several aromatic compounds. These clusters are distributed throughout the linear plasmids. The high sequence homology of the ring-hydroxylating subunits of these systems with other known enzymes has allowed us to model the range of aromatic substrates they could degrade. Further functional characterization revealed that WAY2 was able to grow with biphenyl, naphthalene and xylene as sole carbon and energy sources, and could oxidize multiple aromatic compounds, including ethylbenzene, phenanthrene, dibenzofuran and toluene. In addition, WAY2 was able to co-metabolize 23 polychlorinated biphenyl congeners, consistent with the five different ring-hydroxylating systems encoded by its genome. WAY2 could also use n-alkanes of various chain-lengths as a sole carbon source, probably due to the presence of alkB and ladA gene copies, which are only found in its chromosome. These results show that WAY2 has a potential to be used for the biodegradation of multiple organic compounds, This research was funded by GREENER-H2020 (EU), grant number 826312, and MICINN/FEDER EU, grant number RTI2018-0933991- B-I00. D.G.-S. was supported by a MECD FPU fellowship program, grant number FPU14/03965. P.S.-L. was supported by the MICINN FPU fellowship program, grant number FPU18/02169. E.B.-R. was supported by the MECD FPU fellowship program, grant number FPU16/05513. J.S., T.C. and O.U. acknowledge Czech Science Foundation grant number 17–00227S, which enabled PCB co-metabolism experiments to be undertaken

AlkB is a bacterial Fe(II)– and 2-oxoglutarate–dependent dioxygenase that repairs a wide range of alkylated nucleobases in DNA and RNA as part of the adaptive response to exogenous nucleic acid–alkylating agents. Although there has been longstanding interest in the structure and specificity of Escherichia coli AlkB and its homologs, difficulties in assaying their repair activities have limited our understanding of their substrate specificities and kinetic mechanisms. Here, we used quantitative kinetic approaches to determine the transient kinetics of recognition and repair of alkylated DNA by AlkB. These experiments revealed that AlkB is a much faster alkylation repair enzyme than previously reported and that it is significantly faster than DNA repair glycosylases that recognize and excise some of the same base lesions. We observed that whereas 1,N(6)-ethenoadenine can be repaired by AlkB with similar efficiencies in both single- and double-stranded DNA, 1-methyladenine is preferentially repaired in single-stranded DNA. Our results lay the groundwork for future studies of AlkB and its human homologs ALKBH2 and ALKBH3.

DNA base repair mechanisms of alkylated DNA bases is an important reaction in chemical biology and particularly in the human body. It is typically catalyzed by an α-ketoglutarate-dependent nonheme iron dioxygenase named the AlkB repair enzyme. In this work we report a detailed computational study into the structure and reactivity of AlkB repair enzymes with alkylated DNA bases. In particular, we investigate the aliphatic hydroxylation and C[double bond, length as m-dash]C epoxidation mechanisms of alkylated DNA bases by a high-valent iron(iv)-oxo intermediate. Our computational studies use quantum mechanics/molecular mechanics methods on full enzymatic structures as well as cluster models on active site systems. The work shows that the iron(iv)-oxo species is rapidly formed after dioxygen binding to an iron(ii) center and passes a bicyclic ring structure as intermediate. Subsequent cluster models explore the mechanism of substrate hydroxylation and epoxidation of alkylated DNA bases. The work shows low energy barriers for substrate activation and consequently energetically feasible pathways are predicted. Overall, the work shows that a high-valent iron(iv)-oxo species can efficiently dealkylate alkylated DNA bases and return them into their original form.

The family of AlkB homolog (ALKBH) proteins, the homologs of Escherichia coli AlkB 2-oxoglutarate (2OG), and Fe(II)-dependent dioxygenase are involved in a number of important regulatory processes in eukaryotic cells including repair of alkylation lesions in DNA, RNA, and nucleoprotein complexes. There are nine human and thirteen Arabidopsis thaliana ALKBH proteins described, which exhibit diversified functions. Among them, human ALKBH5 and FaT mass and Obesity-associated (FTO) protein and Arabidopsis ALKBH9B and ALKBH10B have been recognized as N6 methyladenine (N6 meA) demethylases, the most abundant posttranscriptional modification in mRNA. The FTO protein is reported to be associated with obesity and type 2 diabetes, and involved in multiple other processes, while ALKBH5 is induced by hypoxia. Arabidopsis ALKBH9B is an N6 meA demethylase influencing plant susceptibility to viral infections via m6 A/A ratio control in viral RNA. ALKBH10B has been discovered to be a functional Arabidopsis homolog of FTO; thus, it is also an RNA N6 meA demethylase involved in plant flowering and several other regulatory processes including control of metabolism. High-throughput mass spectrometry showed multiple sites of human ALKBH phosphorylation. In the case of FTO, the type of modified residue decides about the further processing of the protein. This modification may result in subsequent protein ubiquitination and proteolysis, or in the blocking of these processes. However, the impact of phosphorylation on the other ALKBH function and their downstream pathways remains nearly unexplored in both human and Arabidopsis. Therefore, the investigation of evolutionarily conserved functions of ALKBH proteins and their regulatory impact on important cellular processes is clearly called for.

Repair of alkylation damage in DNA is essential for maintaining genome integrity. Escherichia Coli (E.coli) DNA repair enzyme AlkB removes methyl adducts including 1-methyladenine and 3-methylcytosine present in DNA by oxidative demethylation from single-stranded DNA (ssDNA). E. coli single-stranded DNA binding protein (SSB) selectively binds ssDNA in a sequence-independent manner. We have recently shown that AlkB can repair methyl adduct present in SSB-coated ssDNA. In this study, we aimed to elucidate details of AlkB-mediated DNA repair of SSB-bound DNA substrate. Therefore, we generated a structural model of AlkB-SSB-ssDNA and using Molecular Dynamics simulation analysis we show that flexibility of SSB-bound DNA allows AlkB to bind in multiple ways. Our docking analysis of AlkB-SSB-ssDNA structure revealed that the Cyt109 base is present in the hydrophobic cavity of AlkB active site pocket. The characterization of AlkB-SSB interaction pattern would likely to help in understanding the mode of alkylated DNA adduct recognition by AlkB.

The mammalian AlkB homologue-3 (AlkBH3) is a member of the dioxygenase family of enzymes that in humans is involved in DNA dealkylation repair. Because of its role in promoting tumor cell proliferation and metastasis of cancer, extensive efforts are being directed in developing selective inhibitors for AlkBH3. Here we report synthesis, screening and evaluation of panel of arylated indenone derivatives as new class of inhibitors of AlkBH3 DNA repair activity. An efficient synthesis of 2,3-diaryl indenones from 2,3-dibromo indenones was achieved via Suzuki-Miyaura cross-coupling. Using a robust quantitative assay, we have obtained an AlkBH3 inhibitor that display specific binding and competitive mode of inhibition against DNA substrate. Finally, we established that this compound could prevent the proliferation of lung cancer cell line and enhance sensitivity to DNA damaging alkylating agent.

Intrinsically disordered regions (IDRs) of proteins often regulate function through interactions with folded domains. Escherichia coli single-stranded DNA binding protein SSB binds and stabilizes single-stranded DNA (ssDNA). The N-terminal of SSB contains characteristic OB (oligonucleotide/oligosaccharide-binding) fold which binds ssDNA tightly but non-specifically. SSB also forms complexes with a large number proteins via the C-terminal interaction domain consisting mostly of acidic amino acid residues. The amino acid residues located between the OB-fold and C-terminal acidic domain are known to constitute an IDR and no functional significance has been attributed to this region. Although SSB is known to bind many DNA repair protein, it is not known whether it binds to DNA dealkylation repair protein AlkB. Here, we characterize AlkB SSB interaction and demonstrate that SSB binds to AlkB via the IDR. We have established that AlkB-SSB interaction by in vitro pull-down and yeast two-hybrid analysis. We mapped the site of contact to be the residues 152-169 of SSB. Unlike most of the SSB-binding proteins which utilize C-terminal acidic domain for interaction, IDR of SSB is necessary and sufficient for AlkB interaction.

Objective: To isolate potential hydrocarbon degraders from different habitats within the United Arab Emirates (UAE) contaminated with Hydrocarbon Compounds (HC) and assess their degradation potential by a rapid qualitative method. Methods/Analysis: Eight different oil contaminated samples were collected for isolation of Hydrocarbon Degrading Bacteria (HDB) on the surface of nutrient agar plates where the appearance of bacterial colonies was observed and then phenotypically and biochemically characterized. Isolates were screened for HC degradation by the “whole plate diffusion” method after observation of the bacterial growth around the hole. PCR analysis was carried out to detect the key degrading gene, alkane hydroxylase gene (AlkB) in the positive HDB. Findings: Results indicated the recovery of 19 isolates from different HC contaminated samples with nine isolates namely (2A, 1D, So1, S1A, S3, KF1, SO2, AJ1 and 2B) were identified as positive degraders for one or more of the tested hydrocarbon compounds. The majority of these isolates were able to utilize heptane as a sole carbon source for their survival while the isolates SO2, S1A and S3 were the most potent as indicated by their growth around the agar hole-plate. Degradation of hydrocarbon compounds by indigenous microbial communities to marine environment was shown to be successful with a significant biodegradation role can be played by the recovered isolates. PCR analysis of the positive degraders for the presence of AlkB gene showed two groups with different band size products; group 1 (G1) (~330 bp) and group 2 (G2) (multiple of 330 pb). Novelty/Improvements: This study confirmed the HC degradation after detecting the marker AlkB gene in the positive degraders, and the use of th “Hole plate diffusion method” as a rapid qualitative evaluation method for bacterial HC degradation by bacteria. Keywords: AlkB gene, Degradation, Hydrocarbon, Oil Spills, PCR

Nonheme iron dioxygenases catalyze vital reactions for human health particularly related to aging processes. They are involved in the biosynthesis of amino acids, but also the biodegradation of toxic compounds. Typically they react with substrate through oxygen atom transfer, although often with theassistance of a co-substrate like α-ketoglutarate that is converted to succinate and CO2. Many reaction processes catalyzed by the nonheme iron dioxygenases are stereoselective or regiospecific and hence understanding the mechanism and protein involvement in the selectivity is important for the design of biotechnological applications of these enzymes. In this Account I will review recent work of our group on nonheme iron dioxygenases and include background information on their general structure and catalytic cycle. Examples of stereoselective and regiospecific reaction mechanisms we elucidated are for the AlkB repair enzyme, prolyl-4-hydroxylase and the ergothioneine biosynthesis enzyme. Finally, I cover an example where we bioengineered S-phydroxymandelate synthase into the R-p-hydroxymandelate synthase.

N6-methyladenosine (m6A) is the most abundant and reversible post-transcriptional modification in eukaryotic mRNA and long non-coding RNA (lncRNA). The central role of m6A in various physiological processes has generated considerable biological and pharmacological interest. Alkbh5 (AlkB homologue 5) belongs to the AlkB family and is a non-heme Fe(II)/α-ketoglutarate-dependent dioxygenase that selectively catalyzes the oxidative demethylation of m6A. Herein, we report the backbone 1H, 15N, 13C chemical shift assignment of a fully active, 26 kDa construct of human Alkbh5. Experiments were acquired at 25 °C by heteronuclear multidimensional NMR spectroscopy. Collectively, 92% of all backbone resonances were assigned, with 195 out of a possible 212 residues assigned in the 1H–15N TROSY spectrum. Using the program TALOS+, a secondary structure prediction was generated from the assigned backbone resonance that is consistent with the previously reported X-ray structure of the enzyme. The reported assignment will permit investigations of the protein structural dynamics anticipated to provide crucial insight regarding fundamental aspects in the recognition and enzyme regulation processes.