We studied the effects of some nitrogen-containing, heterocyclic, and cyclic compounds on the rate of oxidative deamination of polyamines and putrescine in tissues with a high proliferation rate. For this purpose, the specific activities of the main enzymes of polyamine oxidative degradation - spermine oxidase (SMO), polyamine oxidase (PAO), and diamine oxidase (DAO) were determined using a cell-free test system from regenerating rat liver. The compounds methyl 2-(5-formylfuran-2-yl)benzoate and 2,7-bis-[2-(diethylamino)ethoxy]-9H-fluoren-9-one (and in the form of dihydrochloride) showed mainly activating effect on oxidative degradation of putrescine, spermidine, and spermine, which indirectly indicates their antiproliferative effect. Nitrogen-free compounds inhibited this process, thus exhibiting potentially carcinogenic properties. Correlations were calculated for activity of DAO, PAO, and SMO with 5 topological indices: Wiener (W), Rouvray (R), Balaban (J) in the Trinaistich modification, detour (Ip), and electropy (Ie). The highest dependence was noted for DAO and the Balaban index (R=-0.55), for PAO and the detour index (R=0.78), and for SMO and the electropy index (R=0.53). The remaining dependencies showed insignificant correlation strength.

During the deamination and amination processes of meso-diaminopimelate dehydrogenase (meso-DAPDH) from Symbiobacterium thermophilum (StDAPDH), residue R71 was observed to display distinct functions. H154 has been proposed as a basic residue that facilitates water molecules to attack the D-chiral carbon of meso-DAP during deamination. Inspired by the phenomenon of R71, the effects of H154 during deamination and amination were investigated in this study with the goal of enhancing the amination activities of StDAPDH. Single site saturation mutagenesis indicated that almost all of the H154 mutants completely lost their deamination activity towards meso-DAP. However, some H154 variants showed enhanced kcat/Km values towards pyruvic acid and other bulky 2-keto acids, such as 2-oxovaleric acid, 4-methyl-2-oxopentanoic acid, 2-ketobutyric acid, and 3-methyl-2-oxobutanoic acid. When combined with the previously reported W121L/H227I mutant, triple mutants with significantly improved kcat/Km values (2.4-, 2.5-, 2.5-, and 4.0-fold) towards these 2-keto acids were obtained. Despite previous attempts, mutations at the H154 site did not yield the desired results. Moreover, this study not only recognizes the distinctive impact of H154 on both the deamination and amination reactions, but also provides guidance for further high-throughput screening in protein engineering and understanding the catalytic mechanism of StDAPDH.

Deamination of bases is a form of DNA damage that occurs spontaneously via the hydrolysis and nitrosation of living cells, generating hypoxanthine from adenine. E. coli endonuclease V (eEndoV) cleaves hypoxanthine-containing double-stranded DNA, whereas human endonuclease V (hEndoV) cleaves hypoxanthine-containing RNA; however, hEndoV in vivo function remains unclear. To date, hEndoV has only been examined using hypoxanthine, because it binds closely to the base located at the cleavage site. Here, we examined whether hEndoV cleaves other lesions (e.g., AP site, 6-methyladenine, xanthine) to reveal its function and whether 2\'-nucleoside modification affects its cleavage activity. We observed that hEndoV is hypoxanthine-specific; its activity was the highest with 2\'-OH modification in ribose. The cleavage activity of hEndoV was compared based on its base sequence. We observed that it has specificity for adenine located on the 3\'-end of hypoxanthine at the cleavage site, both before and after cleavage. These data suggest that hEndoV recognizes and cleaves the inosine generated on the poly A tail to maintain RNA quality. Our results provide mechanistic insight into the role of hEndoV in vivo.

Mutations accumulate in the genome of every cell of the body throughout life, causing cancer and other diseases1,2. Most mutations begin as nucleotide mismatches or damage in one of the two strands of the DNA before becoming double-strand mutations if unrepaired or misrepaired3,4. However, current DNA-sequencing technologies cannot accurately resolve these initial single-strand events. Here we develop a single-molecule, long-read sequencing method (Hairpin Duplex Enhanced Fidelity sequencing (HiDEF-seq)) that achieves single-molecule fidelity for base substitutions when present in either one or both DNA strands. HiDEF-seq also detects cytosine deamination-a common type of DNA damage-with single-molecule fidelity. We profiled 134 samples from diverse tissues, including from individuals with cancer predisposition syndromes, and derive from them single-strand mismatch and damage signatures. We find correspondences between these single-strand signatures and known double-strand mutational signatures, which resolves the identity of the initiating lesions. Tumours deficient in both mismatch repair and replicative polymerase proofreading show distinct single-strand mismatch patterns compared to samples that are deficient in only polymerase proofreading. We also define a single-strand damage signature for APOBEC3A. In the mitochondrial genome, our findings support a mutagenic mechanism occurring primarily during replication. As double-strand DNA mutations are only the end point of the mutation process, our approach to detect the initiating single-strand events at single-molecule resolution will enable studies of how mutations arise in a variety of contexts, especially in cancer and ageing.

Multiomics require concerted recording of independent information, ideally from a single experiment. In this study, we introduce RIMS-seq2, a high-throughput technique to simultaneously sequence genomes and overlay methylation information while requiring only a small modification of the experimental protocol for high-throughput DNA sequencing to include a controlled deamination step. Importantly, the rate of deamination of 5-methylcytosine is negligible and thus does not interfere with standard DNA sequencing and data processing. Thus, RIMS-seq2 libraries from whole- or targeted-genome sequencing show the same germline variation calling accuracy and sensitivity compared with standard DNA-seq. Additionally, regional methylation levels provide an accurate map of the human methylome.

D-amino acid oxidase (DAAO)-catalyzed selective oxidative deamination is a very promising process for synthesizing l-amino acids including l-phosphinothricin (l-PPT, a high-efficiency and broad-spectrum herbicide). However, the wild-type DAAO\'s low activity toward unnatural substrates like d-phosphinothricin (d-PPT) hampers its application. Herein, a DAAO from Caenorhabditis elegans (CeDAAO) was screened and engineered to improve the catalytic potential on d-PPT. First, we designed a novel growth selection system, taking into account the intricate relationship between the growth of Escherichia coli (E. coli) and the catalytic mechanism of DAAO. The developed system was used for high-throughput screening of gene libraries, resulting in the discovery of a variant (M6) with significantly increased catalytic activity against d-PPT. The variant displays different catalytic properties on substrates with varying hydrophobicity and hydrophilicity. Analysis using Alphafold2 modeling and molecular dynamic simulations showed that the reason for the enhanced activity was the substrate-binding pocket with enlarged size and suitable charge distribution. Further QM/MM calculations revealed that the crucial factor for enhancing activity lies in reducing the initial energy barrier of the reductive half reaction. Finally, a comprehensive binding-model index to predict the enhanced activity of DAAO toward d-PPT, and an enzymatic deracemization approach was developed, enabling the efficient synthesis of l-PPT with remarkable efficiency.

Over the past decade, the connection between APOBEC3 cytosine deaminases and cancer mutagenesis has become increasingly apparent. This growing awareness has created a need for biochemical tools that can be used to identify and characterize potential inhibitors of this enzyme family. In response to this challenge, we have developed a Real-time APOBEC3-mediated DNA Deamination assay. This assay offers a single-step set-up and real-time fluorescent read-out, and it is capable of providing insights into enzyme kinetics. The assay also offers a high-sensitivity and easily scalable method for identifying APOBEC3 inhibitors. This assay serves as a crucial addition to the existing APOBEC3 biochemical and cellular toolkit and possesses the versatility to be readily adapted into a high-throughput format for inhibitor discovery.

This review aims to highlight the structures of ADAR proteins that have been crucial in the discernment of their functions and are relevant to future therapeutic development. ADAR proteins can correct or diversify genetic information, underscoring their pivotal contribution to protein diversity and the sophistication of neuronal networks. ADAR proteins have numerous functions in RNA editing independent roles and through the mechanisms of A-I RNA editing that continue to be revealed. Provided is a detailed examination of the ADAR family members-ADAR1, ADAR2, and ADAR3-each characterized by distinct isoforms that offer both structural diversity and functional variability, significantly affecting RNA editing mechanisms and exhibiting tissue-specific regulatory patterns, highlighting their shared features, such as double-stranded RNA binding domains (dsRBD) and a catalytic deaminase domain (CDD). Moreover, it explores ADARs\' extensive roles in immunity, RNA interference, and disease modulation, demonstrating their ambivalent nature in both the advancement and inhibition of diseases. Through this comprehensive analysis, the review seeks to underline the potential of targeting ADAR proteins in therapeutic strategies, urging continued investigation into their biological mechanisms and health implications.

Adenosine-to-inosine (A-to-I) editing and N6-methyladenosine (m6A) modifications are pivotal RNA modifications with widespread functional significance in physiological and pathological processes. Although significant effort has been dedicated to developing methodologies for identifying and quantifying these modifications, traditional approaches have often focused on each modification independently, neglecting the potential co-occurrence of A-to-I editing and m6A modifications at the same adenosine residues. This limitation has constrained our understanding of the intricate regulatory mechanisms governing RNA function and the interplay between different types of RNA modifications. To address this gap, we introduced an innovative technique called deamination-assisted reverse transcription stalling (DARTS), specifically designed for the simultaneous quantification of A-to-I editing and m6A at the same RNA sites. DARTS leverages the selective deamination activity of the engineered TadA-TadA8e protein, which converts adenosine residues to inosine, in combination with the unique property of Bst 2.0 DNA polymerase, which stalls when encountering inosine during reverse transcription. This approach enables the accurate quantification of A-to-I editing, m6A, and unmodified adenosine at identical RNA sites. The DARTS method is remarkable for its ability to directly quantify two distinct types of RNA modifications simultaneously, a capability that has remained largely unexplored in the field of RNA biology. By facilitating a comprehensive analysis of the co-occurrence and interaction between A-to-I editing and m6A modifications, DARTS opens new avenues for exploring the complex regulatory networks modulated by different RNA modifications.

The obligate intracellular bacterium, Chlamydia trachomatis, has evolved to depend on its human host for many metabolites, including most amino acids and three of the four nucleotides. Given this, it is not surprising that depletion of a single amino acid in the host cell growth medium blocks chlamydial replication. Paradoxically, supra-normal levels of some amino acids also block productive replication of Chlamydia. Here, we have determined how elevated serine levels, generated by exogenous supplementation, impede chlamydial inclusion development and reduce the generation of infectious progeny. Our findings reveal that human serine racemase, which is broadly expressed in multiple tissues, potentiates the anti-chlamydial effect of elevated serine concentrations. In addition to reversibly converting l-serine to d-serine, serine racemase also deaminates serine via β-elimination. We have determined that d-serine does not directly impact Chlamydia; rather, ammonia generated by serine deamination limits the productive chlamydial replication. Our findings imply that ammonia produced within host cells can traverse the chlamydial inclusion membrane. Further, this property of serine deaminase can be exploited to sensitize Chlamydia to concentrations of doxycycline that are otherwise not bactericidal. Because exogenously elevated levels of serine can be tolerated over extended periods, the broad expression pattern of serine racemase indicates it to be a host enzyme whose activity can be directed against multiple intracellular bacterial pathogens. From a therapeutic perspective, demonstrating host metabolism can be skewed to generate an anti-bacterial metabolite that synergizes with antibiotics, we believe our results provide a new approach to target intracellular pathogens.