Deamination Activity Research Articles

Abstract 2486: APOBEC3B and APOBEC3C-H have opposite associations with DNA instability and immune cell infiltration in breast cancer tumors

Abstract Introduction: APOBEC3 enzymes are strong mutagenic factors because of their cytidine deamination activity. In particular, the detrimentally of tumoral APOBEC3B (A3B) expression to clinical outcomes has been well established for breast cancer. We have previously demonstrated that high tumoral expression of many other APOBEC3s (A3C-H) confers better T cell anti-cancer immunity and survival benefit in breast cancer, an interesting finding given that these enzymes are DNA mutators. To this end, we hypothesized that each APOBEC3 has different activity on genomic instability and anti-cancer immunity in breast cancer tumors. Methods: Transcriptomic and clinical data for primary tumors were obtained from The Cancer Genome Atlas (TCGA). Patients were divided into thirds by gene expression and highest and lowest expressors were compared. Measurements of genomic or molecular features such as tumor mutation burden, neoantigen load, aneuploidy, homologous recombinant deficiency (HRD), intra-tumor heterogeneity (ITH), B and T cell receptor (BCR and TCR) diversity hypervariable complementarity- determining region 3 (CDR3) which are critical component of B cell immunoglobulin heavy chain were calculated using the datasets from previous publications. Results: As expected, tumor with high expression of A3B, DNA mutagenesis enzyme, were strongly associated with genome instability. Compared to tumors with low A3B expression, those with high expression had significantly greater mutation burden (1.9x for median value) and neoantigen load (1.8x), as well as scores for aneuploidy (1.7x), HRD (2.5x), and ITH (2.5x; all p values < 0.01).In contrast to A3B, we found that A3C, A3D, A3G and A3H are predominantly expressed in immune cells. Tumor with high expression of A3C demonstrated lower tumor mutation burden (0.9x) and aneuploidy (0.7x). Neoantigen load, HRD and ITH were significantly decreased (0.7 - 0.9x, all p< 0.001) in high A3D expressors. Although infiltrating leukocytes and their CD8 T cell subset were both decreased in tumors with high A3B expression, interestingly, they were increased 1.5-1.8-fold in tumors with high A3G or A3H expression. We also found that A3G and A3H expression had positive correlation with not only TCR diversity (2.0x and 2.1x for A3G and A3H respectively) and immune cytolytic activity (6.4x and 6.5x), but also both BCR diversity (2.6x and 2.1x) and number of unique CDR3s (6.4x and 4.8x) with all p < 0.001 significances. Conclusion: Our data suggests that APOBEC3B and APOBEC3C, 3D, 3G and 3H demonstrate opposite phenotype regarding DNA instability and immune cell infiltration in breast cancer. Citation Format: Mariko Asaoka, Santosh K. Patnaik, Takashi Ishikawa, Kazuaki Takabe. APOBEC3B and APOBEC3C-H have opposite associations with DNA instability and immune cell infiltration in breast cancer tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2486.

Biochemical changes and amino acid deamination & decarboxylation activities of spoilage microbiota in chill-stored grass carp (Ctenopharyngodon idella) fillets

This study aimed to reveal amino acid deamination and decarboxylation activities of spoilage microbiota in chill-stored grass carp fillets. Results showed that microbial deamination activities of umami/sweet-taste amino acids were higher than that of bitter-taste amino acids. The total deamination activity of tested amino acids decreased during the late period of storage, which inhibited the increase of ammonia in fish flesh. Microbial decarboxylation activity of ornithine was much higher than lysine and histidine, which was consistent with the rapid increase of putrescine in fish fillets. Meanwhile, putrescine could be produced in large quantities through arginine deiminase pathway of spoilage bacteria. Glucose utilization by spoilage microbiota was active during the late period of storage, which was consistent with the rapid consumption of lactate and total sugar in fish flesh. Overall, results of this study could be beneficial for revealing fish spoilage mechanisms and providing theoretical guidance for developing fish preservation technologies.

Characterization of an NAD(P)+-dependent meso-diaminopimelate dehydrogenase from Thermosyntropha lipolytica

meso-Diaminopimelate dehydrogenase (meso-DAPDH) catalyzes the reversible NADP+-dependent oxidative deamination of meso-2,6-diaminopimelate (meso-DAP) to produce l-2-amino-6-oxopimelate. meso-DAPDH is divided into two major clusters, types I and II, based on substrate specificity and structural characteristic. Here, we describe a novel type II meso-DAPDH from Thermosyntropha lipolytica (TlDAPDH). The gene encoding a putative TlDAPDH was expressed in Escherichia coli cells, and then the enzyme was purified 7.3-fold to homogeneity from the crude cell extract. The molecule of TlDAPDH seemed to form a hexamer, which is the typical structural characteristic of type II meso-DAPDHs. The purified enzyme exhibited oxidative deamination activity toward meso-DAP with both NADP+ and NAD+ as coenzymes. TlDAPDH exhibited reductive amination activity of corresponding 2-oxo acid to produce d-amino acid. In particular, the productivities for d-aspartate and d-glutamate have not been reported in the type II enzymes. The optimum pH and temperature for oxidative deamination of meso-DAP were 10.5 and 55°C, respectively. TlDAPDH retained more than 80% of its activity after incubation for 30 min at temperatures between 50°C and 65°C and in the pH range of 4.5–9.5. Moreover, the coenzyme and substrate recognition mechanisms of TlDAPDH were elucidated based on a multiple sequence alignment and the homology model. The results of these analyses suggested that the molecular mechanisms for coenzyme and substrate recognition of TlDAPDH were similar to those of meso-DAPDH from S. thermophilum, which is the representative type II enzyme. Based on the kinetic characteristics and structural comparison, TlDAPDH was considered to be a novel type II meso-DAPDH.

Structure of a tRNA-specific deaminase with compromised deamination activity.

Nucleotide 34 in tRNA is extensively modified to ensure translational fidelity and efficacy in cells. The deamination of adenosine at this site catalyzed by the enzyme TadA gives rise to inosine (I), which serves as a typical example of the wobble hypothesis due to its diverse basepairing capability. However, recent studies have shown that tRNAArgACG in Mycoplasma capricolum contains unmodified adenosine, in order to decode the CGG codon. The structural basis behind the poorly performing enzyme M. capricolum TadA (McTadA) is largely unclear. Here we present the structures of the WT and a mutant form of McTadA determined at high resolutions. Through structural comparison between McTadA and other active TadA enzymes as well as modeling efforts, we found that McTadA presents multiple structural conflicts with RNA substrates and thus offered support to previous studies from a structural perspective. These clashes would potentially lead to reduced substrate binding affinity of McTadA, consistent with our in vitro deamination activity and binding assays. To rescue the deamination activity of McTadA, we carried out two rounds of protein engineering through structure-guided design. The unsuccessful attempts of the activity restoration could be attributed to the altered dimer interface and stereo hindrance from the non-catalytic subunit of McTadA, which could be the inevitable outcome of the natural evolution. Our study provides structural insight into an alternative decoding and evolutionary strategy by a compromised TadA enzyme at a molecular level.

Adar RNA editing-dependent and -independent effects are required for brain and innate immune functions in Drosophila

ADAR RNA editing enzymes are high-affinity dsRNA-binding proteins that deaminate adenosines to inosines in pre-mRNA hairpins and also exert editing-independent effects. We generated a Drosophila AdarE374A mutant strain encoding a catalytically inactive Adar with CRISPR/Cas9. We demonstrate that Adar adenosine deamination activity is necessary for normal locomotion and prevents age-dependent neurodegeneration. The catalytically inactive protein, when expressed at a higher than physiological level, can rescue neurodegeneration in Adar mutants, suggesting also editing-independent effects. Furthermore, loss of Adar RNA editing activity leads to innate immune induction, indicating that Drosophila Adar, despite being the homolog of mammalian ADAR2, also has functions similar to mammalian ADAR1. The innate immune induction in fly Adar mutants is suppressed by silencing of Dicer-2, which has a RNA helicase domain similar to MDA5 that senses unedited dsRNAs in mammalian Adar1 mutants. Our work demonstrates that the single Adar enzyme in Drosophila unexpectedly has dual functions.

High throughput solid-phase screening of bacteria with cyclic amino alcohol deamination activity for enantioselective synthesis of chiral cyclic β-amino alcohols.

To screening of bacteria with cyclic amino alcohol deamination activity for enantioselective synthesis of chiral cyclic β-amino alcohols. A new strain named Arthrobacter sp. TYUT010-15 with the (R)-selective deamination activity of cyclic β-amino alcohol has been isolated from nature via a high throughput solid-phase screening method. The reaction conditions of TYUT010-15 were optimized. Using the resting cell of TYUT010-15 as the catalyst, kinetic resolution of trans-2-aminocyclopentanol, trans-2-aminocyclohexanol and cis-1-amino-2-indanol was carried out to afford (1S, 2S)-trans-2-aminocyclopentanol, (1S, 2S)-trans-2-aminocyclohexanol and (1R, 2S)-cis-1-amino-2-indanol in > 99% ee and 49.6-50% conversion. Four aromatic β-amino alcohols and two amines were also resolved, (S)-β-amino alcohols and (R)-amines were obtained in > 99% ee. Preparation experiment was conducted with 200mM (23.2g L-1) racemic trans-2-aminocyclohexanol, yielding the desired (1S, 2S)-trans-2-aminocyclohexanol in 40% isolated yield, > 99% ee and 5.8g L-1 d-1 space time yields. This study provides a high throughput solid-phase method for screening of bacteria with cyclic amino alcohol deamination activity and a first example for practical preparation of chiral cyclic β-amino alcohol by Arthrobacter sp. TYUT010-15.

Allosteric Regulation of Glutamate Dehydrogenase Deamination Activity

Glutamate dehydrogenase (GDH) is a key enzyme interlinking carbon and nitrogen metabolism. Recent discoveries of the GDH specific role in breast cancer, hyperinsulinism/hyperammonemia syndrome, and neurodegenerative diseases have reinvigorated interest on GDH regulation, which remains poorly understood despite extensive and long standing studies. Notwithstanding the growing evidence of the complexity of the allosteric network behind GDH regulation, a clear understanding of the molecular dynamics involved in the process is limited to the activator Adenosine diphosphate (ADP) and the inhibitor Guanosine triphosphate (GTP). Presently, the identification of further factors involved in the allosteric network is paramount to deepen our understanding of the complex dynamics that regulate GDH enzymatic activity. Combining structural analyses of cryo-electron microscopy data with molecular dynamic simulations, here we show that the cofactor NADH is a key player in the GDH regulation process. Our analysis indicates that, binding to the regulatory sites in proximity of the antenna region, NADH acts as a positive allosteric modulator by enhancing both the affinity of GTP binding and inhibition of GDH catalytic activity. We further show that the binding of GTP to the NADH-bounded GDH produces a local conformational rearrangement that triggers an anticlockwise rotational motion of interlinked alpha-helices with specific tilted helical extension. This structural transition is a fundamental switch in the GDH enzymatic activity. It introduces a torsional stress, and the associated rotational shift in the Rossmann fold closes the catalytic cleft with consequent inhibition of the deamination process. These results shed new light on GDH regulation and may lay new foundation in the design of allosteric agents.

DHX9 interacts with APOBEC3B and attenuates the anti-HBV effect of APOBEC3B

ABSTRACT Hepatitis B virus (HBV) is a partially double-stranded DNA virus that replicates by reverse transcription. We previously demonstrated that the host restriction factor-APOBEC3B (A3B) inhibited HBV replication which was dependent on its deaminase activity during reverse transcription. However, the host factors involved in the process of regulating the anti-HBV function of A3B are less known. In this research, to obtain a comprehensive understanding of the interaction networks of A3B, we conducted coimmunoprecipitation and mass spectrometry to identify A3B-interacting proteins in the presence of HBV. By this approach, we determined that DExD/H-box helicase 9 (DHX9) suppressed the anti-HBV effect of A3B, and this suppression was dependent on their interaction. Although DHX9 did not affect the deamination activity of A3B in vitro assay or the viral DNA editing of A3B in HepG2-NTCP cells that support HBV infection, it inhibited the binding of A3B with pgRNA. These data suggest that DHX9 can interact with A3B and attenuate the anti-HBV efficacy of A3B. Abbreviations: 3D-PCR: differential DNA denaturation PCR; APOBEC3: apolipoprotein B mRNA-editing catalytic polypeptide 3; cccDNA: covalently closed circular DNA; co-IP: coimmunoprecipitation; DDX: DExD-box RNA helicases; HBc: HBV core protein; HBV: hepatitis B virus; HepAD38: HepG2 cell line stably transfected with HBV DNA; HepG2-NTCP: HepG2 cell line stably transfected with Na+/taurocholate cotransporter polypeptide; Huh7: human hepatoma cell line; pgRNA: pregenomic RNA; PPI: protein–protein interactions; RC DNA: relaxed circular DNA.

Growth parameters of Liberibacter crescens suggest ammonium and phosphate as essential molecules in the Liberibacter-plant host interface

BackgroundLiberibacter crescens is the closest cultured relative of four important uncultured crop pathogens. Candidatus. L. asiaticus, L. americanus, L. africanus cause citrus greening disease, while Ca. L. solanacearum causes potato Zebra chip disease. None of the pathogens grows in axenic culture. L. crescens grows in three media: a BM-7, a serum-free Hi® Grace’s Insect Medium (Hi-GI), and a chemically-defined medium called M15. To date, no optimal growth parameters of the model species L. crescens have been reported. Studying the main growth parameters of L. crescens in axenic culture will give us insights into the lifestyle of the Ca. Liberibacter pathogens.ResultsThe evaluation of the growth parameters—pH, aeration, temperature, and buffering capacity—reflects the optimal living conditions of L. crescens. These variables revealed that L. crescens is an aerobic, neutrophilic bacterium, that grows optimally in broth in a pH range of 5.8 to 6.8, in a fully oxygenated environment (250 rpm), at 28 °C, and with monosodium phosphate (10 mM or 11.69 mM) as the preferred buffer for growth. The increase of pH in the external media likely results from the deamination activity within the cell, with the concomitant over-production of ammonium in the external medium.ConclusionL. crescens and the Ca. Liberibacter pathogens are metabolically similar and grow in similar environments—the phloem and the gut of their insect vectors. The evaluation of the growth parameters of L. crescens reveals the lifestyle of Liberibacter, elucidating ammonium and phosphate as essential molecules for colonization within the hosts. Ammonium is the main driver of pH modulation by active deamination of amino acids in the L. crescens amino acid rich media. In plants, excess ammonium induces ionic imbalances, oxidative stress, and pH disturbances across cell membranes, causing stunted root and shoot growth and chlorosis—the common symptoms of HLB-disease. Phosphate, which is also present in Ca. L. asiaticus hosts, is the preferred buffer for the growth of L. crescens. The interplay between ammonium, sucrose, potassium (K+), phosphate, nitrate (NO3−), light and other photosynthates might lead to develop better strategies for disease management.

In vitro ruminal fermentation characteristics of alfalfa silages in response to different pre-ensiling treatments

The present study investigated the in vitro ruminal fermentation of alfalfa silages (AS) that had been produced using different pre-ensiling treatments, i.e., by changing the wilting intensity and dry matter concentration, and adding sucrose, and therefore differed in silage quality and their N fractions. The data were obtained using an in vitro rumen-simulation technique (Rusitec) system, in which the AS were incubated isonitrogenously in quadruplicate. Samples were taken after 2 days (first time point) and 7 days (second time point) of AS incubation, and a variety of fermentation characteristics as well as the degradability of fiber fractions and organic matter were determined. Sucrose addition substantially raised the propionate concentration during both sampling time points from an average of 17.8 to 29.7 mmol/L (P < 0.001), which might be explained by microbial utilization of residual sugars and lactate from the AS. The extraordinary high concentrations of isovalerate and ammonia-N with all AS point to enhanced deamination activity. At the second time point, the n-butyrate concentration increased during the incubation of high-intensity wilted AS (P = 0.007), which might have been caused by the higher hemicellulose degradability that was also observed for these silages (P = 0.002). However, the organic matter degradability decreased (P = 0.035), indicating a lower degradability of other feed fractions. The gas production (P < 0.001) and degradability of organic matter (P = 0.002) and fiber fractions (P < 0.001) decreased from first to second time point, whereas the concentrations of ammonia-N (P = 0.004), acetate (P < 0.001), and isovalerate (P < 0.001) increased. Thus, it seemed that alterations in the Rusitec system and the microbial community occurred, yet it is unclear why the acetate concentration increased, whereas the fiber degradability decreased. The beneficial effects of combining all three pre-ensiling treatments on silage quality, i.e., higher acidification and increased true protein preservation, were not fully transferred to the in vitro ruminal fermentation system, and comprehensive research on pre-ensiling treatments will pave the way for an optimized ruminal N utilization from AS in the future.

APOBEC3A Loop 1 Is a Determinant for Single-Stranded DNA Binding and Deamination.

The apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3 or A3) family of proteins functions in the innate immune system. The A3 proteins are interferon inducible and hypermutate deoxycytidine to deoxyuridine in foreign single-stranded DNA (ssDNA). However, this deaminase activity cannot discriminate between foreign and host ssDNA at the biochemical level, which presents a significant danger when A3 proteins gain access to the nucleus. Interestingly, this A3 capability can be harnessed when coupled with novel CRISPR-Cas9 proteins to create a targeted base editor. Specifically, A3A has been used in vitro to revert mutations associated with disease states. Recent structural studies have shown the importance of loop regions of A3A and A3G in ssDNA recognition and positioning for deamination. In this work, we further examined loop 1 of A3A to determine how it affects substrate selection, as well as the efficiency of deamination, in the hopes of advancing the potential of A3A in base editing technology. We found that mutating residue H29 enhanced deamination activity without changing substrate specificity. Also interestingly, we found that increasing the length of loop 1 decreases substrate specificity. Overall, these results lead to a better understanding of substrate recognition and deamination by A3A and the A3 family of proteins.

Adding to the RNA editing toolbox

Biotechnology A previously developed RNA editing system called REPAIR can base edit A to I in RNA by fusing the adenine deaminase domain of ADAR2 with catalytically dead CRISPR-Cas13. Using directed evolution, Abudayyeh et al. demonstrated that the ADAR2 deaminase domain can be relaxed to accept other bases. This resulted in cytidine deamination activity, expanding the RNA editing toolbox for C-to-U conversion. This system, referred to as RNA Editing for Specific C-to-U Exchange (RESCUE), can edit on endogenous transcripts and enable modulation of posttranslational protein modification such as phosphorylation. Science , this issue p. [382][1] [1]: /lookup/doi/10.1126/science.aax7063

A cytosine deaminase for programmable single-base RNA editing.

Programmable RNA editing enables reversible recoding of RNA information for research and disease treatment. Previously, we developed a programmable adenosine-to-inosine (A-to-I) RNA editing approach by fusing catalytically inactivate RNA-targeting CRISPR-Cas13 (dCas13) with the adenine deaminase domain of ADAR2. Here, we report a cytidine-to-uridine (C-to-U) RNA editor, referred to as RNA Editing for Specific C-to-U Exchange (RESCUE), by directly evolving ADAR2 into a cytidine deaminase. RESCUE doubles the number of mutations targetable by RNA editing and enables modulation of phosphosignaling-relevant residues. We apply RESCUE to drive β-catenin activation and cellular growth. Furthermore, RESCUE retains A-to-I editing activity, enabling multiplexed C-to-U and A-to-I editing through the use of tailored guide RNAs.

Abstract 3495: The CCT chaperonin is a regulator of mutagenic APOBEC3A activity

Abstract APOBEC3 (A3) enzymes deaminate DNA cytosine bases and are implicated as the cause of a prevalent mutational signature found in cancer genomes. Mutation of the cellular genome is presumed to be an off-target activity of the enzymes. A3 enzymes normally function as part of the innate immune system by deaminating viral genomes to restrict infection. Basal expression of A3 enzymes is low in healthy tissues, but is increased by type I interferon signaling. Aside from interferon induction, the mechanisms regulating expression and activity of A3 enzymes are unknown. Thus the circumstances that allow for off-target activity of A3 on cellular DNA, leading to mutations and genome instability, cannot be predicted. We sought to determine protein interactors of A3 enzymes to identify potential regulators of expression and/or activity. Using a doxycycline-inducible expression system, we introduced A3 genes into isogenic cell lines. Each A3 enzyme was immunoprecipitated (IP) and lysates were analyzed by mass spectrometry (MS). MS analysis of A3A interactors revealed all 8 subunits of the TriC/CCT (TCP-1 ring complex or chaperonin containing TCP1) complex. The CCT complex is a molecular chaperone that assists in folding of many newly-synthesized proteins, preventing aggregation and facilitating protein function. Validation of MS results by A3A IP followed by immunoblotting confirmed interaction with CCT complex subunits. Endogenous A3A levels are increased significantly by interferon in peripheral blood mononuclear cells (PBMCs). We performed IP of CCT in PBMCs followed by immunoblot for A3A, which demonstrated an interaction between endogenous A3A and the CCT complex. To evaluate the impact of the CCT-A3A interaction, we used siRNA to knock down the CCT complex. Although CCT is an essential chaperonin, transient knockdown of individual subunits resulted in minimally decreased cell viability. However, knockdown of CCT subunits combined with induction of A3A expression led to significant decrease in cell proliferation and increase in cell death. To determine the mechanism by which CCT knockdown with A3A expression results in cytotoxicity, we performed an in vitro deamination assay on lysates from cells that were transfected with siRNA targeting CCT subunits and/or treated with doxycycline to induce A3A expression. Quantification of deamination activity demonstrated that CCT knockdown resulted in increased in A3A activity relative to expression levels. Finally, we evaluated cancer genome sequences in TCGA and found an enrichment of the mutational signature attributed to A3 deamination in cancers with deleterious mutations in CCT subunit genes. Together, these data suggest that the CCT complex regulates A3A activity, and disruption of CCT function results in increased A3A mutational activity. Citation Format: Abby M. Green, Ariel S. Dineen, Katarzyna Kulej, Julia H. Szeto, Matthew D. Weitzman. The CCT chaperonin is a regulator of mutagenic APOBEC3A activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3495.

Mechanism for APOBEC3G catalytic exclusion of RNA and non-substrate DNA.

The potent antiretroviral protein APOBEC3G (A3G) specifically targets and deaminates deoxycytidine nucleotides, generating deoxyuridine, in single stranded DNA (ssDNA) intermediates produced during HIV replication. A non-catalytic domain in A3G binds strongly to RNA, an interaction crucial for recruitment of A3G to the virion; yet, A3G displays no deamination activity for cytidines in viral RNA. Here, we report NMR and molecular dynamics (MD) simulation analysis for interactions between A3Gctd and multiple substrate or non-substrate DNA and RNA, in combination with deamination assays. NMR ssDNA-binding experiments revealed that the interaction with residues in helix1 and loop1 (T201-L220) distinguishes the binding mode of substrate ssDNA from non-substrate. Using 2′-deoxy-2′-fluorine substituted cytidines, we show that a 2′-endo sugar conformation of the target deoxycytidine is favored for substrate binding and deamination. Trajectories of the MD simulation indicate that a ribose 2′-hydroxyl group destabilizes the π-π stacking of the target cytosine and H257, resulting in dislocation of the target cytosine base from the catalytic position. Interestingly, APOBEC3A, which can deaminate ribocytidines, retains the ribocytidine in the catalytic position throughout the MD simulation. Our results indicate that A3Gctd catalytic selectivity against RNA is dictated by both the sugar conformation and 2′-hydroxyl group.

RNA Exclusion Mechanism of the Cytidine Deaminase APOBEC3G

APOBEC3G (A3G) is a potent antiretroviral protein that specifically targets and deaminates cytidine nucleotides to uracil in single stranded DNA (ssDNA) intermediates produced during HIV replication. A3G displays high selectivity for specific polynucleotide contexts targeting 5′‐CC sites for deamination. A3G binds strongly to RNA via the N‐terminal non‐catalytic domain, which is crucial for recruitment of A3G to the virion, yet A3G displays no deamination activity on the cytidines in single stranded RNA. Our recent structure of A3G bound to ssDNA shows the preferred substrate conformation but does not reveal the mechanisms of substrate selection and non‐substrate exclusion. Here, we report NMR and molecular dynamics (MD) simulation based analysis of the interactions between the C‐terminal catalytic domain of A3G (A3Gctd) and multiple substrate and non‐substrate ssDNAs, which are coupled with deamination assays. The extent of interaction with residues located in helix1 and loop1 (T201‐L220) distinguishes substrate ssDNAs from non‐substrate ssDNAs, revealing the difference between catalytic and non‐catalytic ssDNA‐binding modes of A3Gctd. Using fluorine substituted ribonucleotides we show that 2′‐endo sugar pucker of the target cytidine is an essential factor for effective deamination. Furthermore, MD simulations show that ribose cytidine does not stay at catalytic position during 100ns of simulation time whereas deoxy‐ribose cytidine does. Trajectories of the MD simulation suggest that 2′‐hydroxyl group of RNA forms a hydrogen bond with His257 that chelates Zn2+, which results in loss of two hydrogen bonds between the target cytidine and A3Gctd, dislocating cytosine base from the catalytic position. This study provides mechanistic models of the catalytic selectivity of A3G against RNA.Support or Funding InformationInstitute for Molecular Virology Training Program (NIH grant T32 AI83196) University of MinnesotaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Alternative conformation induced by substrate binding for Arabidopsis thalianaN6-methyl-AMP deaminase.

Adenosine deaminase is involved in adenosine degradation and salvage pathway, and plays important physiological roles in purine metabolism. Recently, a novel type of adenosine deaminase-like protein has been identified, which displays deamination activity toward N6-methyl-adenosine monophosphate but not adenosine or AMP, and was consequently named N6-methyl-AMP deaminase (MAPDA). The underlying structural basis of MAPDA recognition and catalysis is poorly understood. Here, we present the crystal structures of MAPDA from Arabidopsis thaliana in the free and in the ligand-bound forms. The protein contains a conserved (β/α)8 Tim-barrel domain and a typical zinc-binding site, but it also exhibits idiosyncratic local differences for two flexible helices important for substrate binding. The extensive interactions between the N6-methyl-AMP substrate or the inosine monophosphate product and the enzyme were identified, and subsequently evaluated by the deamination activity assays. Importantly, each structure reported here represents a different stage of the catalytic pathway and their structural differences suggested that the enzyme can exist in two distinct conformational states. The open state switches to the closed one upon the binding of ligands, brought about by the two critical helices. Our structural studies provide the first look of this important metabolic enzyme and shed lights on its catalytic pathway, which holds promise for the structure-based drug design for MAPDA-related diseases.

Cytidine deaminase enables Toll-like receptor 8 activation by cytidine or its analogs.

Toll-like receptor 8 (TLR8), a sensor for pathogen-derived single-stranded RNA (ssRNA), binds to uridine (Uri) and ssRNA to induce defense responses. We here show that cytidine (Cyd) with ssRNA also activated TLR8 in peripheral blood leukocytes (PBLs) and a myeloid cell line U937, but not in an embryonic kidney cell line 293T. Cyd deaminase (CDA), an enzyme highly expressed in leukocytes, deaminates Cyd to Uri. CDA expression enabled TLR8 response to Cyd and ssRNA in 293T cells. CDA deficiency and a CDA inhibitor both reduced TLR8 responses to Cyd and ssRNA in U937. The CDA inhibitor also reduced PBL response to Cyd and ssRNA. A Cyd analogue, azacytidine, is used for the therapy of myelodysplastic syndrome and acute myeloid leukemia. Azacytidine with ssRNA induced tumor necrosis factor-α expression in U937 and PBLs in a manner dependent on CDA and TLR8. These results suggest that CDA enables TLR8 activation by Cyd or its analogues with ssRNA through deaminating activity. Nucleoside metabolism might impact TLR8 responses in a variety of situations such as the treatment with nucleoside analogues.

Structural Analysis of the Active Site and DNA Binding of Human Cytidine Deaminase APOBEC3B.

APOBEC3 (A3) proteins, a family of human cytidine deaminases, protect the host from endogenous retro-elements and exogenous viral infections by introducing hypermutations. However, overexpressed A3s can modify genomic DNA to promote tumorigenesis, especially A3B. Despite their overall similarity, A3 proteins have distinct deamination activity. Recently determined A3 structures have revealed the molecular determinants of nucleotide specificity and DNA binding. However, for A3B, the structural basis for regulation of deamination activity and the role of active site loops in coordinating DNA had remained unknown. Using advanced molecular modeling followed by experimental mutational analysis and dynamics simulations, we investigated the molecular mechanism of DNA binding by A3B-CTD. We modeled fully native A3B-DNA structure, and we identified Arg211 in loop 1 as the gatekeeper coordinating DNA and critical residue for nucleotide specificity. We also identified a unique autoinhibited conformation in A3B-CTD that restricts access and binding of DNA to the active site. Our results reveal the structural basis for DNA binding and relatively lower catalytic activity of A3B and provide opportunities for rational design of specific inhibitors to benefit cancer therapeutics.

Single-Nucleotide Resolution Analysis of 5-Hydroxymethylcytosine in DNA by Enzyme-Mediated Deamination in Combination with Sequencing

The report of the existence of 5-hydroxymethylcytosine (hm5C) in mammalian genomes is a milestone discovery. hm5C is now generally viewed as the sixth base of DNA with important functions on epigenetic regulation. The in-depth investigation of the biological functions of hm5C requires elucidating the distribution patterns of hm5C in genomes, better in single-nucleotide resolution. It was reported that the cytosine deaminases of the APOBEC (apolipoprotein B mRNA-editing catalytic polypeptide-like) family are nucleic acid editing enzymes and can deaminate cytosine (C) to form uracil (U). Particularly, a subfamily of APOBEC (APOBEC3A) can efficiently deaminate both C and 5-methylcytosine (m5C). In the current study, we identified that APOBEC3A protein can effectively deaminate C, m5C, and hm5C but shows no observable deamination activity toward glycosylated hm5C (β-glucosyl-5-hydroxymethyl-2'-deoxycytidine, ghm5C) by using the restriction enzyme-based assay and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis. By virtue of the differential deamination activity of APOBEC3A toward C, m5C, and ghm5C in conjugation with sequencing, we developed the single-nucleotide resolution analysis of hm5C in DNA. In this analytical strategy, the original C and m5C in DNA will be deaminated by APOBEC3A to form U and thymine (T), both of which will read as T during sequencing, while ghm5C is resistant to deamination and will read as C during sequencing. Therefore, the remaining C in the sequence context only could come from original hm5C, which offers the single-nucleotide resolution analysis of hm5C in DNA. This APOBEC3A-mediated deamination sequencing (AMD-seq) is straightforward and involves no bisulfite treatment, which avoids the substantial degradation of DNA. Future application of this strategy can be performed for the reliable mapping of hm5C in genome-wide scale at the single-nucleotide resolution.