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Flavins play a central role in metabolism as molecules that catalyze a wide range of redox reactions in living organisms. Several variations in flavin biosynthesis exist among the domains of life, and their analysis has revealed many new structural and mechanistic insights till date. The cytidine triphosphate (CTP)-dependent riboflavin kinase in archaea is one such example. Unlike most kinases that use adenosine triphosphate, archaeal riboflavin kinases utilize CTP to phosphorylate riboflavin and produce flavin mononucleotide. In this study, we present the characterization of a new mesophilic archaeal CTP-utilizing riboflavin kinase homologue from Methanococcus maripaludis (MmpRibK), which is linked closely in sequence to the previously characterized thermophilic Methanocaldococcus jannaschii homologue. We reconstitute the activity of MmpRibK, determine its kinetic parameters and molecular factors that contribute to its unique properties, and finally establish the residues that improve its thermostability using computation and a series of experiments. Our work advances the molecular understanding of flavin biosynthesis in archaea by the characterization of the first mesophilic CTP-dependent riboflavin kinase. Finally, it validates the role of salt bridges and rigidifying amino acid residues in imparting thermostability to this unique structural fold that characterizes archaeal riboflavin kinase enzymes, with implications in enzyme engineering and biotechnological applications.

Molnupiravir is an orally available antiviral drug candidate currently in phase III trials for the treatment of patients with COVID-19. Molnupiravir increases the frequency of viral RNA mutations and impairs SARS-CoV-2 replication in animal models and in humans. Here, we establish the molecular mechanisms underlying molnupiravir-induced RNA mutagenesis by the viral RNA-dependent RNA polymerase (RdRp). Biochemical assays show that the RdRp uses the active form of molnupiravir, β-d-N4-hydroxycytidine (NHC) triphosphate, as a substrate instead of cytidine triphosphate or uridine triphosphate. When the RdRp uses the resulting RNA as a template, NHC directs incorporation of either G or A, leading to mutated RNA products. Structural analysis of RdRp–RNA complexes that contain mutagenesis products shows that NHC can form stable base pairs with either G or A in the RdRp active center, explaining how the polymerase escapes proofreading and synthesizes mutated RNA. This two-step mutagenesis mechanism probably applies to various viral polymerases and can explain the broad-spectrum antiviral activity of molnupiravir., Quantitative biochemical assays and high-resolution cryo-EM analysis reveal how the COVID-19 antiviral drug candidate molnupiravir causes lethal viral mutagenesis by the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2.

Summary Cereal grain endosperms are an important source of human nutrition. Nuclear division in early endosperm development plays a major role in determining seed size; however, this development is not well understood. We identified the rice mutant endospermless 2 (enl2), which shows defects in the early stages of endosperm development. These phenotypes arise from mutations in OsCTPS1 that encodes a cytidine triphosphate synthase (CTPS). Both wild‐type and mutant endosperms were normal at 8 h after pollination (HAP). In contrast, at 24 HAP, enl2 endosperm had approximately 10–16 clumped nuclei while wild‐type nuclei had increased in number and migrated to the endosperm periphery. Staining of microtubules in endosperm at 24 HAP revealed that wild‐type nuclei were evenly distributed by microtubules while the enl2‐2 nuclei were tightly packed due to their reduction in microtubule association. In addition, OsCTPS1 interacts with tubulins; thus, these observations suggest that OsCTPS1 may be involved in microtubule formation. OsCTPS1 transiently formed macromolecular structures in the endosperm during early developmental stages, further supporting the idea that OsCTPS1 may function as a structural component during endosperm development. Finally, overexpression of OsCTPS1 increased seed weight by promoting endosperm nuclear division, suggesting that this trait could be used to increase grain yield.

DDX3X is a human DEAD-box RNA helicase implicated in many important cellular processes. In addition to the RecA-like catalytic core, DDX3X contains N- and C-terminal domains. The ancillary domains of DEAD-box RNA helicases have been shown to modulate their interactions with RNA and nucleotide substrates. Here, with the goal of understanding the role of N- and C-terminal domains of DDX3X on the DDX3X catalytic activity, we examined the interactions of RNA substrates and nucleotides with a DDX3X construct possessing the entire N-terminal domain and the catalytic core but lacking 80 residues from its C-terminal domain. Next, we compared our results with previously investigated DDX3X constructs. Our data show that the C-terminal truncated DDX3X does not bind to a blunt-ended double-helix RNA. This conclusion agrees with the data obtained on the wild-type LAF-1 protein, the DDX3X ortholog in Caenorhabditis elegans, and disagrees with the data obtained on the minimally active DDX3X construct, which misses 131 residues from its N-terminal domain and 80 residues from its C-terminal domain. The minimally active DDX3X construct was able to bind to the blunt-ended RNA construct. Combined, the previous studies and our results indicate that the N-terminal of DDX3X modulates the choice of DDX3X–RNA substrates. Furthermore, a previous study showed that the wild-type DDX3X construct hydrolyzes all four nucleotides and deoxynucleotides, both in the presence and absence of RNA. The C-terminal truncated DDX3X investigated here hydrolyzes only cytidine triphosphate (CTP) in the absence of RNA and CTP, adenosine triphosphate (ATP), and deoxyribose adenosine triphosphate (dATP) in the presence of RNA. Hence, the C-terminal truncated DDX3X has a more stringent nucleotide specificity than wild-type DDX3X.

Molecular cell 81(19), 3992 - 4007.e10 (2021). doi:10.1016/j.molcel.2021.09.004, ParB-like CTPases mediate the segregation of bacterial chromosomes and low-copy number plasmids. They act as DNA-sliding clamps that are loaded at parS motifs in the centromere of target DNA molecules and spread laterally to form large nucleoprotein complexes serving as docking points for the DNA segregation machinery. Here, we solve crystal structures of ParB in the pre- and post-hydrolysis state and illuminate the catalytic mechanism of nucleotide hydrolysis. Moreover, we identify conformational changes that underlie the CTP- and parS-dependent closure of ParB clamps. The study of CTPase-deficient ParB variants reveals that CTP hydrolysis serves to limit the sliding time of ParB clamps and thus drives the establishment of a well-defined ParB diffusion gradient across the centromere whose dynamics are critical for DNA segregation. These findings clarify the role of the ParB CTPase cycle in partition complex assembly and function and thus advance our understanding of this prototypic CTP-dependent molecular switch., Published by Elsevier, New York, NY

Trypanosomiasis is a major illness affecting camels in tropical and subtropical regions. Comparisons of camel and Trypanosoma evansi genomes can lead to the discovery of new drug targets for treating Trypanosoma infections. The synthesis pathways of cytosine, cytidine, cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP) deoxycytidine, deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP), and deoxycytidine triphosphate (dCTP) were compared in the dromedary camel (Camelus dromedarius) and T. evansi. None of the enzymes involved in cytosine pathway were detected in camels and T. evansi. Notably, cytidine kinase (CK) and 5′-nucleotidase, which interconverts cytidine to CMP, were not detected in T. evansi but were present in camels. UMP/CMP kinase was not predicted in T. evansi. Therefore, the presence of enzymes involved in the CTP synthesis cascade was not predicted in T. evansi. CMP synthesis might also be encoded by other enzymes, e.g., purine nucleotides kinases. Both camel and T. evansi share an efficient enzyme system for converting CDP to CTP. In conclusion, CTP synthase is important for homeostasis of cytosine nucleotides in T. evansi and could be a potential drug target against the parasite. In addition, the inhibition of UMP synthesis might contribute to parasite death as it is a shared source for CTP synthesis.

Noroviruses are the main causative agents of acute viral gastroenteritis worldwide. However, no vaccine or specific antiviral treatment is available, imposing a heavy global health burden. The nucleoside analogue 2’-fluoro-2’-deoxycytidine (2’-FdC) has been reported to have broad antiviral activity. Here, we report that 2’-FdC significantly inhibits murine norovirus replication in macrophages. This effect was partially reversed by exogenous supplementation of cytidine triphosphate. The combination of 2’-FdC with mycophenolic acid, ribavirin or favipiravir (T705) exerts synergistic antiviral effects. These results indicate that 2’-FdC is a potential candidate for antiviral drug development against norovirus infection. Electronic supplementary material The online version of this article (10.1007/s00705-020-04759-4) contains supplementary material, which is available to authorized users.

The interplay between E2 and E3 enzymes regulates the polyubiquitination of substrates in eukaryotes. Among the several RING-domain E3 ligases in humans, many utilize two distinct E2s for polyubiquitination. For example, the cell cycle regulatory E3, human anaphase-promoting complex/cyclosome (APC/C), relies on UBE2C to prime substrates with ubiquitin (Ub) and on UBE2S to extend polyubiquitin chains. However, the potential coordination between these steps in ubiquitin chain formation remains undefined. While numerous studies have unveiled how RING E3s stimulate individual E2s for Ub transfer, here we change perspective to describe a case where the chain-elongating E2 UBE2S feeds back and directly stimulates the E3 APC/C to promote substrate priming and subsequent multiubiquitination by UBE2C. Our work reveals an unexpected model for the mechanisms of RING E3-dependent ubiquitination and for the diverse and complex interrelationship between components of the ubiquitination cascade.

RNA polymerases (RNAPs) transcribe genes through a cycle of recruitment to promoter DNA, initiation, elongation, and termination. After termination, RNAP is thought to initiate the next round of transcription by detaching from DNA and rebinding a new promoter. Here we use single-molecule fluorescence microscopy to observe individual RNAP molecules after transcript release at a terminator. Following termination, RNAP almost always remains bound to DNA and sometimes exhibits one-dimensional sliding over thousands of basepairs. Unexpectedly, the DNA-bound RNAP often restarts transcription, usually in reverse direction, thus producing an antisense transcript. Furthermore, we report evidence of this secondary initiation in live cells, using genome-wide RNA sequencing. These findings reveal an alternative transcription cycle that allows RNAP to reinitiate without dissociating from DNA, which is likely to have important implications for gene regulation., In the canonical bacterial transcription, both nascent transcript and polymerase dissociate from template DNA. By employing multi-color single-molecule fluorescence imaging, here the authors show that RNA polymerases remain bound to DNA after the transcript release.

Gold nanodevices have attracted extensive interest in the detection of specific targets within cells. However, constructing gold sensing devices that can be activated by the simulation of remote applications remains a huge challenge. Here, we report a Au nanoparticle (AuNP)-capped cage fluorescent biosensor based on controlled-release and Exonuclease III (Exo III) assisted cyclic enzymatic amplification that can be activated by adenosine triphosphate (ATP). In the system, AuNPs were used as the building blocks to cap the pores of Au nanocages (AuNCs) loaded with Rhodamine B (RhB) molecules through the hybridization of DNA. The RhB fluorescent molecules were finally released with the help of Exo III in the presence of ATP for detection purposes. Ultimately, the biosensor leads to a wide linear ATP detection range from 1.0 × 10-9 to 1.0 × 10-7 M with a limit of detection (LOD) down to 0.88 nM. In addition, it also has good selectivity for ATP to distinguish between ATP and ATP analogues such as cytidine triphosphate (CTP), guanosine triphosphate (GTP), and uridine triphosphate (UTP). Therefore, as a convenient and sensitive biosensor, it is expected to be widely used in the biomedical field.

Significance An effective immune response depends on the proliferation of T cells, a process that requires the enzyme CTP synthase 1 (CTPS1). Individuals lacking CTPS1 due to a rare genetic disorder exhibit severe immunodeficiencies but lack other major clinical consequences; the requirement for CTP synthase outside of the immune response is met by a second isoform, CTPS2. Inhibiting CTPS1 without affecting CTPS2 is therefore a promising strategy for treating autoimmune disorders and T cell cancers while avoiding off-target effects. We characterize both CTPS1-selective and nonselective inhibitors. Structures of CTPS bound to inhibitors reveal the mechanisms of inhibition and CTPS1 selectivity. Differences in product feedback inhibition between CTPS1 and CTPS2 explain how CTPS1 may sustain enzymatic activity required for T cell proliferation., Cytidine triphosphate synthase 1 (CTPS1) is necessary for an effective immune response, as revealed by severe immunodeficiency in CTPS1-deficient individuals [E. Martin et al.], [Nature] [510], [288–292] ([2014]). CTPS1 expression is up-regulated in activated lymphocytes to expand CTP pools [E. Martin et al.], [Nature] [510], [288–292] ([2014]), satisfying increased demand for nucleic acid and lipid synthesis [L. D. Fairbanks, M. Bofill, K. Ruckemann, H. A. Simmonds], [J. Biol. Chem. ] [270], [29682–29689] ([1995]). Demand for CTP in other tissues is met by the CTPS2 isoform and nucleoside salvage pathways [E. Martin et al.], [Nature] [510], [288–292] ([2014]). Selective inhibition of the proliferative CTPS1 isoform is therefore desirable in the treatment of immune disorders and lymphocyte cancers, but little is known about differences in regulation of the isoforms or mechanisms of known inhibitors. We show that CTP regulates both isoforms by binding in two sites that clash with substrates. CTPS1 is less sensitive to CTP feedback inhibition, consistent with its role in increasing CTP levels in proliferation. We also characterize recently reported small-molecule inhibitors, both CTPS1 selective and nonselective. Cryo-electron microscopy (cryo-EM) structures reveal these inhibitors mimic CTP binding in one inhibitory site, where a single amino acid substitution explains selectivity for CTPS1. The inhibitors bind to CTPS assembled into large-scale filaments, which for CTPS1 normally represents a hyperactive form of the enzyme [E. M. Lynch et al.], [Nat. Struct. Mol. Biol.] [24], [507–514] ([2017]). This highlights the utility of cryo-EM in drug discovery, particularly for cases in which targets form large multimeric assemblies not amenable to structure determination by other techniques. Both inhibitors also inhibit the proliferation of human primary T cells. The mechanisms of selective inhibition of CTPS1 lay the foundation for the design of immunosuppressive therapies.

Proper chromosome segregation is essential in all living organisms. The ParA-ParB-parS system is widely employed for chromosome segregation in bacteria. Previously, we showed that Caulobacter crescentus ParB requires cytidine triphosphate to escape the nucleation site parS and spread by sliding to the neighboring DNA 1. Here, we provide the structural basis for this transition from nucleation to spreading by solving co-crystal structures of a C-terminal domain truncated C. crescentus ParB with parS and with a CTP analog. Nucleating ParB is an open clamp, in which parS is captured at the DNA-binding domain (the DNA-gate). Upon binding CTP, the N-terminal domain (NTD) self-dimerizes to close the NTD-gate of the clamp. The DNA-gate also closes, thus driving parS into a compartment between the DNA-gate and the C-terminal domain. CTP hydrolysis and/or the release of hydrolytic products are likely associated with re-opening of the gates to release DNA and to recycle ParB. Overall, we suggest a CTP-operated gating mechanism that regulates ParB nucleation, spreading, and recycling.

ParABS partition systems, comprising the centromere-like DNA sequence parS, the parS-binding ParB-CTPase, and the nucleoid-binding ParA-ATPase, ensure faithful segregation of bacterial chromosomes and low-copy-number plasmids. F-plasmid partition complexes containing ParBF and parSF move by generating and following a local concentration gradient of nucleoid-bound ParAF. However, the process through which ParBF activates ParAF-ATPase has not been defined. We studied CTP- and parSF-modulated ParAF–ParBF complex assembly, in which DNA-bound ParAF-ATP dimers are activated for ATP hydrolysis by interacting with two ParBF N-terminal domains. CTP or parSF enhances the ATPase rate without significantly accelerating ParAF–ParBF complex assembly. Together, parSF and CTP accelerate ParAF–ParBF assembly without further significant increase in ATPase rate. Magnetic-tweezers experiments showed that CTP promotes multiple ParBF loading onto parSF-containing DNA, generating condensed partition complex-like assemblies. We propose that ParBF in the partition complex adopts a conformation that enhances ParBF–ParBF and ParAF–ParBF interactions promoting efficient partitioning.

5-aza-cytidine (5-aza-C) has been shown to be a potent human immunodeficiency virus type 1 (HIV-1) mutagen that induces G-to-C hypermutagenesis by incorporation of the reduced form (i.e., 5-aza-dC, 5-aza-dCTP). Evidence to date suggests that this lethal mutagenesis is the primary antiretroviral mechanism for 5-aza-C. To investigate the breadth of application of 5-aza-C as an antiretroviral mutagen, we have conducted a comparative, parallel analysis of the antiviral mechanism of 5-aza-C between HIV-1 and gammaretroviruses – i.e., murine leukemia virus (MuLV) and feline leukemia virus (FeLV). Intriguingly, in contrast to the hallmark G-to-C hypermutagenesis observed with HIV-1, MuLV and FeLV did not reveal the presence of a significant increase in mutational burden, particularly that of G-to-C transversion mutations. The effect of 5-aza-dCTP on DNA synthesis revealed that while HIV-1 RT was not inhibited by 5-aza-dCTP even at 100 µM, 5-aza-dCTP was incorporated and significantly inhibited MuLV RT, generating pause sites and reducing the fully extended product. 5-aza-dCTP was found to be incorporated into DNA by MuLV RT or HIV-1 RT, but only acted as a non-obligate chain terminator for MuLV RT. This biochemical data provides an independent line of experimental evidence in support of the conclusion that HIV-1 and MuLV have distinct primary mechanisms of antiretroviral action with 5-aza-C. Taken together, our data provides striking evidence that an antiretroviral mutagen can have strong potency via distinct mechanisms of action among closely related viruses, unlinking antiviral activity from antiviral mechanism of action.

3'-Deoxy-3',4'-didehydro-cytidine triphosphate (ddhCTP) is a novel antiviral molecule produced by the enzyme viperin as part of the innate immune response. ddhCTP has been shown to act as an obligate chain terminator of flavivirus and SARS-CoV-2 RNA-dependent RNA polymerases; however, further biophysical studies have been precluded by limited access to this promising antiviral. Herein, we report a robust and scalable synthesis of ddhCTP as well as the mono- and diphosphates ddhCMP and ddhCDP, respectively. Identification of a 2'-silyl ether protection strategy enabled selective synthesis and facile purification of the 5'-triphosphate, culminating in the preparation of ddhCTP on a gram scale.

Background Effective concentrations of antiretrovirals in the female genital tract (FGT) are critical for suppression of viral shedding or effective preexposure prophylaxis. The disposition of tenofovir diphosphate (TFV-DP) and emtricitabine triphosphate (FTC-TP) in the FGT have been previously described. Despite widespread use, however, lamivudine triphosphate (3TC-TP) exposure in the FGT is unknown. Depot medroxyprogesterone acetate (DMPA) and vaginal dysbiosis have been implicated in increased risk of human immunodeficiency virus (HIV) acquisition, but whether they alter TFV-DP or 3TC-TP exposure, and therefore compromise prevention efficacy, is unknown. Methods Fifty premenopausal women living with HIV in Kampala, Uganda, and receiving daily tenofovir disoproxil fumarate/lamivudine were recruited. Ectocervical biopsies were obtained for quantification of TFV-DP and 3TC-TP using liquid chromatography–mass spectrometry. 16S ribosomal RNA gene sequencing was performed on DNA extracted from vaginal swabs. Wilcoxon rank-sum was used to test for differences between contraceptive groups. Results 3TC-TP concentrations were on average 17-fold greater than TFV-DP concentrations in cervical tissues. TFV-DP concentrations in cervical biopsies were 76% greater in DMPA users compared with women using nonhormonal contraception (n = 23 per group). Abundance of Lactobacillus in vaginal swabs was correlated with 3TC-TP concentrations in cervical tissues. Conclusions We found that TFV-DP concentrations were significantly greater in DMPA users compared with women using nonhormonal contraception, suggesting that prevention efficacy is unlikely to be compromised by DMPA use. Similar to reports of FTC-TP, 3TC-TP exposure was significantly greater than TFV-DP in cervical tissue and was correlated with abundance of Lactobacillus. These data support lamivudine as an option for preexposure prophylaxis. Clinical Trials Registration NCT03377608.

CMP-N-acetylneuraminate synthetase (CMAS) is a key enzyme in the sialic acid incorporation pathway and plays a crucial role in the virulence and survival of several pathogenic bacteria. Here, the structural and functional properties of CMAS from the pathogenic bacterium Vibrio cholerae are reported. Upon CDP binding, a partial domain closure is observed that was previously unreported in homologous structures. Kinetic studies reveal that the enzyme shows substrate promiscuity and can activate both Neu5Ac and Neu5Gc., Several pathogenic bacteria utilize sialic acid, including host-derived N-acetylneuraminic acid (Neu5Ac), in at least two ways: they use it as a nutrient source and as a host-evasion strategy by coating themselves with Neu5Ac. Given the significant role of sialic acid in pathogenesis and host-gut colonization by various pathogenic bacteria, including Neisseria meningitidis, Haemophilus influenzae, Pasteurella multocida and Vibrio cholerae, several enzymes of the sialic acid catabolic, biosynthetic and incorporation pathways are considered to be potential drug targets. In this work, findings on the structural and functional characterization of CMP-N-acetylneuraminate synthetase (CMAS), a key enzyme in the incorporation pathway, from Vibrio cholerae are reported. CMAS catalyzes the synthesis of CMP-sialic acid by utilizing CTP and sialic acid. Crystal structures of the apo and the CDP-bound forms of the enzyme were determined, which allowed the identification of the metal cofactor Mg2+ in the active site interacting with CDP and the invariant Asp215 residue. While open and closed structural forms of the enzyme from eukaryotic and other bacterial species have already been characterized, a partially closed structure of V. cholerae CMAS (VcCMAS) observed upon CDP binding, representing an intermediate state, is reported here. The kinetic data suggest that VcCMAS is capable of activating the two most common sialic acid derivatives, Neu5Ac and Neu5Gc. Amino-acid sequence and structural comparison of the active site of VcCMAS with those of eukaryotic and other bacterial counterparts reveal a diverse hydrophobic pocket that interacts with the C5 substituents of sialic acid. Analyses of the thermodynamic signatures obtained from the binding of the nucleotide (CTP) and the product (CMP-sialic acid) to VcCMAS provide fundamental information on the energetics of the binding process.

The leading cause of bacterial gastroenteritis, Campylobacter jejuni, is a Gram-negative pathogen that contains a unique O-methyl phosphoramidate (MeOPN) on its capsular polysaccharide. Previously, MeOPN has been linked to the evasion of host immune responses and serum resistance. Despite the involvement of MeOPN in pathogenicity, the complete biosynthesis of this modification is unknown; however, the first four enzymatic steps have been elucidated. The second enzyme in this pathway, Cj1416, is a CTP:phosphoglutamine cytididylyltransferase that catalyzes the displacement of pyrophosphate from MgCTP by L-glutamine phosphate to form CDP-L-glutamine. Initially, Cj1416 was predicted to use phosphoramidate to form cytidine diphosphoramidate but no activity was detected with MgATP as a substrate. However, in the presence of MnCTP, Cj1416 can directly catalyze the formation of cytidine diphosphoramidate from phosphoramidate and MnCTP. Here we characterize the manganese-induced promiscuity of Cj1416. In the presence of Mn(2+), Cj1416 catalyzes the formation of 12 different reaction products using L-glutamine phosphate, phosphoramidate, methyl phosphate, methyl phosphonate, phosphate, arsenate, ethanolamine phosphate, glycerol-1-phosphate, glycerol-2-phosphate, serinol phosphate, L-serine phosphate or 3-phospho-D-glycerate as the nucleophile to displace pyrophosphate from CTP.

Flavins play a central role in cellular metabolism as molecules that catalyze a wide range of oxidation-reduction reactions in living organisms. Several interesting variations in flavin biosynthesis exist among the domains of life, and the analysis of enzymes on this pathway have put forth many unique structural and mechanistic insights till date. The CTP-dependent riboflavin kinase in archaea is one such example - unlike most kinase enzymes that use adenosine triphosphate to conduct phosphorylation reactions, riboflavin kinases from archaea utilizes cytidine triphosphate (CTP) to phosphorylate riboflavin to produce flavin mononucleotide (FMN). In this study, we present the characterization of a new mesophilic archaeal riboflavin kinase homolog from Methanococcus maripaludis (MmpRibK), which is linked closely in sequence to the previously characterized thermophilic homolog from Methanocaldococcus jannaschii (MjRibK). We reconstitute the activity of the CTP-dependent MmpRibK, determine its kinetic parameters, and analyse the molecular factors that contribute to the uncommon properties of this class of enzymes. Specifically, we probe the flexibility of MmpRibK and MjRibK under varying temperatures and the role of a metal ion for substrate binding and catalysis using molecular dynamics simulation and a series of experiments. Furthermore, based on the high degree of sequence similarity between the mesophilic MmpRibK and the thermophilic MjRibK, we use comparative analysis and site-directed mutagenesis to establish a set of the residues that are responsible for the thermostability of the enzyme without any loss in activity or substrate specificity. Our work contributes to the molecular understanding of flavin biosynthesis in archaea through the characterization of the first mesophilic CTP-dependent riboflavin kinase. Finally, it validates the role of salt bridges and rigidifying amino acid residues in imparting thermostability to enzymes, with implications in enzyme engineering and biotechnological applications.

Nicotinamide adenine dinucleotide (NAD) and its reduced form are indispensable cofactors in life. Diverse NAD mimics have been developed for applications in chemical and biological sciences. Nicotinamide cytosine dinucleotide (NCD) has emerged as a non-natural cofactor to mediate redox transformations, while cells are fed with chemically synthesized NCD. Here, we create NCD synthetase (NcdS) by reprograming the substrate binding pockets of nicotinic acid mononucleotide (NaMN) adenylyltransferase to favor cytidine triphosphate and nicotinamide mononucleotide over their regular substrates ATP and NaMN, respectively. Overexpression of NcdS alone in the model host Escherichia coli facilitated intracellular production of NCD, and higher NCD levels up to 5.0 mM were achieved upon further pathway regulation. Finally, the non-natural cofactor self-sufficiency was confirmed by mediating an NCD-linked metabolic circuit to convert L-malate into D-lactate. NcdS together with NCD-linked enzymes offer unique tools and opportunities for intriguing studies in chemical biology and synthetic biology., Nicotinamide cytosine dinucleotide is an NAD mimic that can be used to mediate redox reactions. Here the authors design an NCD synthetase for the intracellular production of NCD.

Pyrimidine de novo synthesis is an essential pathway in all organisms. The final and rate-limiting step in the synthesis of the nucleotide cytidine triphosphate (CTP) is catalyzed by CTP synthase (CTPS), and Arabidopsis harbors five isoforms. Single mutant lines defective in each one of the four isoforms do not show apparent phenotypical alterations in comparison to wild-type plants. However, Arabidopsis lines that contain T-DNA insertions in the CTPS2 gene were unable to produce homozygous offspring. Here, we show that CTPS2 exhibits a distinct expression pattern throughout embryo development, and loss-of-function mutants are embryo lethal, as siliques from +/ctps2 plants contained nearly 25% aborted seeds. This phenotype was rescued by complementation with CTPS2 under control of its endogenous promoter. CTPS2::GFP lines revealed expression only in the tip of columella cells in embryo root tips of the heart and later stages. Furthermore, CTPS2 expression in mature roots, most pronounced in the columella cells, shoots, and vasculature tissue of young seedlings, was observed. Filial generations of +/ctps2 plants did not germinate properly, even under external cytidine supply. During embryo development, the CTPS2 expression pattern resembled the established auxin reporter DR5::GFP. Indeed, the cloned promoter region we used in this study possesses a repeat of an auxin response element, and auxin supply increased CTPS2 expression in a cell-type-specific manner. Thus, we conclude that CTPS2 is essential for CTP supply in developing embryos, and loss-of-function mutants in CTPS2 are embryo lethal.

Cytidine triphosphate synthase (CTPS) catalyzes the rate-limiting step of de novo CTP biosynthesis. An intracellular structure of CTPS, the cytoophidium, has been found in many organisms including prokaryotes and eukaryotes. Formation of the cytoophidium has been suggested to regulate the activity and stability of CTPS and may participate in certain physiological events. Herein, we demonstrate that both CTPS1a and CTPS1b in zebrafish are able to form the cytoophidium in cultured cells. A point mutation, H355A, abrogates cytoophidium assembly of zebrafish CTPS1a and CTPS1b. In addition, we show the presence of CTPS cytoophidia in multiple tissues of larval and adult fish under normal conditions, while treatment with a CTPS inhibitor 6-diazo-5-oxo-l-norleucine (DON) can induce more cytoophidia in some tissues. Our findings reveal that forming the CTPS cytoophidium is a natural phenomenon of zebrafish and provide valuable information for future research on the physiological importance of this intracellular structure in vertebrates.

Summary ATP- and GTP-dependent molecular switches are extensively used to control functions of proteins in a wide range of biological processes. However, CTP switches are rarely reported. Here, we report that a nucleoid occlusion protein Noc is a CTPase enzyme whose membrane-binding activity is directly regulated by a CTP switch. In Bacillus subtilis, Noc nucleates on 16 bp NBS sites before associating with neighboring non-specific DNA to form large membrane-associated nucleoprotein complexes to physically occlude assembly of the cell division machinery. By in vitro reconstitution, we show that (1) CTP is required for Noc to form the NBS-dependent nucleoprotein complex, and (2) CTP binding, but not hydrolysis, switches Noc to a membrane-active state. Overall, we suggest that CTP couples membrane-binding activity of Noc to nucleoprotein complex formation to ensure productive recruitment of DNA to the bacterial cell membrane for nucleoid occlusion activity., Graphical abstract, Highlights • CTP is required for Noc to form a higher-order nucleoprotein complex on DNA • CTP binding switches DNA-entrapped Noc to a membrane-active state • CTP hydrolysis likely reverses the association between Noc-DNA and the membrane • The membrane-targeting helix adopts an autoinhibitory conformation in apo-Noc, Jalal et al. report that the nucleoid occlusion protein Noc is a CTPase enzyme whose membrane-binding activity is directly regulated by CTP. CTP binding switches the Noc-DNA complex from a membrane-inactive state to an active state, thus ensuring productive recruitment of DNA to the bacterial cell membrane.

Despite the development of non-radioactive DNA/RNA labelling methods, radiolabelled nucleic acids are commonly used in studies focused on the determination of RNA fate. Nucleic acid fragments with radioactive nucleotide analoguesincorporated into the body or at the 5ʹ or 3ʹ terminus of the molecule can serve as probes in hybridization-based analyses of in vivo degradation and processing of transcripts. Radiolabelled oligoribonucleotides are utilized as substrates in biochemical assays of various RNA metabolic enzymes, such as exo- and endoribonucleases, nucleotidyltransferases or helicases. In some applications, the placement of the label is not a concern, while in other cases it is required that the radioactive mark is located at the 5ʹ- or 3ʹ-end of the molecule. An unsurpassed method for 5ʹ-end RNA labelling employs T4 polynucleotide kinase (PNK) and [γ-32P]ATP. In the case of 3ʹ-end labelling, several different possibilities exist. However, they require the use of costly radionucleotide analogues. Previously, we characterized an untypical nucleotidyltransferase named CutA, which preferentially incorporates cytidines at the 3ʹ-end of RNA substrates. Here, we demonstrate that this unusual feature can be used for the development of a novel, efficient, reproducible and economical method of RNA 3ʹ-end labelling by CutA-mediated cytidine tailing. The labelling efficiency is comparable to that achieved with the most common method applied to date, i.e. [5ʹ-32P]pCp ligation to the RNA 3ʹ-terminus catalysed by T4 RNA ligase I. We show the utility of RNA substrates labelled using our new method in exemplary biochemical assays assessing directionality of two well-known eukaryotic exoribonucleases, namely Dis3 and Xrn1.

The phospholipid biosynthesis of the malaria parasite, Plasmodium falciparum is a key process for its survival and its inhibition is a validated antimalarial therapeutic approach. The second and rate-limiting step of the de novo phosphatidylcholine biosynthesis is catalysed by CTP: phosphocholine cytidylyltransferase (PfCCT), which has a key regulatory function within the pathway. Here, we investigate the functional impact of the key structural differences and their respective role in the structurally unique pseudo-heterodimer PfCCT protein in a heterologous cellular context using the thermosensitive CCT-mutant CHO-MT58 cell line. We found that a Plasmodium-specific lysine-rich insertion within the catalytic domain of PfCCT acts as a nuclear localization signal and its deletion decreases the nuclear propensity of the protein in the model cell line. We further showed that the putative membrane-binding domain also affected the nuclear localization of the protein. Moreover, activation of phosphatidylcholine biosynthesis by phospholipase C treatment induces the partial nuclear-to-cytoplasmic translocation of PfCCT. We additionally investigated the cellular function of several PfCCT truncated constructs in a CHO-MT58 based rescue assay. In absence of the endogenous CCT activity we observed that truncated constructs lacking the lysine-rich insertion, or the membrane-binding domain provided similar cell survival ratio as the full length PfCCT protein.

In the fight against the spread of COVID-19 the emphasis is on vaccination or on reactivating existing drugs used for other purposes. The tight links that necessarily exist between the virus as it multiplies and the metabolism of its host are systematically ignored. Here we show that the metabolism of all cells is coordinated by the availability of a core building block of the cell’s genome, cytidine triphosphate (CTP). This metabolite is also the key to the synthesis of the viral envelope and to the translation of its genome into proteins. This unique role explains why evolution has led to the early emergence in animals of an antiviral immunity enzyme, viperin, that synthesizes a toxic analogue of CTP. The constraints arising from this dependency guide the evolution of the virus. With this in mind, we explored the real-time experiment taking place before our eyes using probabilistic modelling approaches to the molecular evolution of the virus. We have thus followed, almost on a daily basis, the evolution of the composition of the viral genome to link it to the progeny produced over time, particularly in the form of blooms that sparked a firework of viral mutations. Some of those certainly increase the propagation of the virus. This led us to make out the critical role in this evolution of several proteins of the virus, such as its nucleocapsid N, and more generally to begin to understand how the virus ties up the host metabolism to its own benefit. A way for the virus to escape CTP-dependent control in cells would be to infect cells that are not expected to grow, such as neurons. This may account for unexpected body sites of viral development in the present epidemic., Dans la lutte contre la propagation de la COVID-19 l’accent est mis sur la vaccination, d’une part, et sur le redéploiement de traitements utilisés pour d’autres usages, d’autre part. Les liens qui existent nécessairement entre la multiplication du virus et le métabolisme de l’hôte sont systématiquement ignorés. Ici nous montrons que le métabolisme de toutes les cellules est coordonné par l’accessibilité d’un composant central du génome cellulaire, le triphosphate de cytidine (CTP). Ce métabolite est aussi la clé de la synthèse de l’enveloppe virale et de la traduction de son génome en protéines. Ce rôle unique explique pourquoi l’évolution a fait apparaître très tôt chez les animaux une activité enzymatique de l’immunité antivirale, la vipérine, destinée à synthétiser un analogue toxique du CTP. Les contraintes nées de cette dépendance orientent l’évolution du virus. Avec cette servitude à l’esprit, nous avons exploré l’expérience en vraie grandeur qui se déroule sous nos yeux au moyen d’approches de modélisation probabiliste de l’évolution moléculaire du virus. Nous avons ainsi suivi, presque au jour le jour, le devenir de la composition du génome viral pour la relier à la descendance produite au cours du temps, en particulier sous la forme d’efflorescences où apparaît un véritable feu d’artifice de mutations virales. Certaines d’entre elles augmentent certainement la propagation du virus. Cela nous conduit à proposer un rôle important dans cette évolution à certaines protéines du virus, comme celle de la nucléocapside N et plus généralement de commencer à comprendre comment le virus asservit à son bénéfice le métabolisme de l’hôte. L’un des moyens possibles pour le virus d’échapper au contrôle par le CTP serait d’infecter des cellules qui ne se multiplient pas, comme les neurones. Cela pourrait expliquer les sites de développement viral inattendus qu’on observe dans l’épidémie actuelle.

SummaryReactive oxygen species (ROS) produced in chloroplasts cause oxidative damage, but also signal to initiate chloroplast quality control pathways, cell death, and gene expression. The mechanisms behind these signals are largely unknown.The Arabidopsis thaliana plastid ferrochelatase two (fc2) mutant produces the ROS singlet oxygen in chloroplasts that activates such signaling pathways. Here we mapped one fc2 suppressor mutation to CYTIDINE TRIPHOSPHATE SYNTHASE TWO (CTPS2), which encodes one of five enzymes in Arabidopsis necessary for de novo cytoplasmic CTP (and dCTP) synthesis.The ctps2 mutation reduces chloroplast transcripts and DNA content without similarly affecting mitochondria. Chloroplast nucleic acid content and singlet oxygen signaling are restored by exogenous feeding of the dCTP precursor deoxycytidine, suggesting ctps2 blocks signaling by limiting nucleotides for chloroplast genome maintenance.An investigation of CTPS orthologs in Brassicaceae showed CTPS2 is a member of an ancient lineage distinct from CTPS3. Complementation studies confirmed this analysis; CTPS3 was unable to compensate for CTPS2 function in providing nucleotides for chloroplast DNA and signaling.Our studies link cytoplasmic nucleotide metabolism with chloroplast quality control pathways. Such a connection is achieved by a conserved clade of CTPS enzymes that may have evolved specialized functions in providing nucleotides to specific subcellular compartments.

In the context of the COVID-19 pandemic, we describe here the singular metabolic background that constrains enveloped RNA viruses to evolve toward likely attenuation in the long term, possibly after a step of increased pathogenicity. Cytidine triphosphate (CTP) is at the crossroad of the processes allowing SARS-CoV-2 to multiply, because CTP is in demand for four essential metabolic steps. It is a building block of the virus genome, it is required for synthesis of the cytosine-based liponucleotide precursors of the viral envelope, it is a critical building block of the host transfer RNAs synthesis and it is required for synthesis of dolichol-phosphate, a precursor of viral protein glycosylation. The CCA 3′-end of all the transfer RNAs required to translate the RNA genome and further transcripts into the proteins used to build active virus copies is not coded in the human genome. It must be synthesized de novo from CTP and ATP. Furthermore, intermediary metabolism is built on compulsory steps of synthesis and salvage of cytosine-based metabolites via uridine triphosphate that keep limiting CTP availability. As a consequence, accidental replication errors tend to replace cytosine by uracil in the genome, unless recombination events allow the sequence to return to its ancestral sequences. We document some of the consequences of this situation in the function of viral proteins. This unique metabolic setup allowed us to highlight and provide a raison d’être to viperin, an enzyme of innate antiviral immunity, which synthesizes 3ʹ-deoxy-3′,4ʹ-didehydro-CTP as an extremely efficient antiviral nucleotide.

Most cells respond to viral infections by activating innate immune pathways that lead to the induction of antiviral restriction factors. One such factor, viperin, was discovered almost two decades ago based on its induction during viral infection. Since then, viperin has been shown to possess activity against numerous viruses via multiple proposed mechanisms. Most recently, however, viperin was demonstrated to catalyze the conversion of cytidine triphosphate (CTP) to 3′-deoxy-3′,4′-didehydro-CTP (ddhCTP), a previously unknown ribonucleotide. Incorporation of ddhCTP causes premature termination of RNA synthesis by the RNA-dependent RNA polymerase of some viruses. To date, production of ddhCTP by viperin represents the only activity of viperin that links its enzymatic activity directly to an antiviral mechanism in human cells. This review examines the multiple antiviral mechanisms and biological functions attributed to viperin.

In 2010, cytidine 5′-triphosphate synthase (CTPS) was reported to form the filamentous or serpentine structure in Drosophila, which we termed the cytoophidium. In the last decade, CTPS filaments/cytoophidia have been found in bacteria, budding yeast, human cells, mice, fission yeast, plants, and archaea, indicating that this mechanism is highly conserved in evolution. In addition to CTPS, other metabolic enzymes have been identified to have the characteristics of forming cytoophidia or similar advanced structures, demonstrating that this is a basic strategy of cells. Nevertheless, our understanding of the physiological function of the cytoophidium remains incomplete and elusive. Here, we took the larva of Drosophila melanogaster as a model to systematically describe the localization and distribution of cytoophidia in different tissues during larval development. We found that the distribution pattern of CTPS cytoophidia is dynamic and heterogenic in larval tissues. Our study provides a road map for further understanding of the function and regulatory mechanism of cytoophidia.

Stochastic transition of cancer cells between drug-sensitive and drug-tolerant persister phenotypes has been proposed to play a key role in non-genetic resistance to therapy. Yet, we show here that cancer cells actually possess a highly stable inherited chance to persist (CTP) during therapy. This CTP is non-stochastic, determined pre-treatment and has a unimodal distribution ranging from 0 to almost 100%. Notably, CTP is drug specific. We found that differential serine/threonine phosphorylation of the insulin receptor substrate 1 (IRS1) protein determines the CTP of lung and of head and neck cancer cells under epidermal growth factor receptor inhibition, both in vitro and in vivo. Indeed, the first-in-class IRS1 inhibitor NT219 was highly synergistic with anti-epidermal growth factor receptor therapy across multiple in vitro and in vivo models. Elucidation of drug-specific mechanisms that determine the degree and stability of cellular CTP may establish a framework for the elimination of cancer persisters, using new rationally designed drug combinations. Straussman and colleagues undertake clonal analyses and show that drug tolerance to EGFR therapy in lung cancer cell populations is an inherited continuous trait that is determined by IRS1 phosphorylation.

A number of Plasmodium falciparum transferase enzymes are being actively studied as targets for potential antimalarial agents. Farnesytransferases catalyze the attachment of farnesyl hydrocarbon chains to protein substrates. The attached farnesyl chains can then act as hydrophobic anchors to hold the relevant proteins to the surface of cell membranes. Therefore, inhibition of farnesyltransferases should disrupt the function of several parasite membrane-bound proteins. In a similar fashion, inhibitors of P. falciparum N-myristoyltransferase should disrupt the functions of parasite membrane-bound proteins that rely on the myristate moiety as a hydrophobic anchor. Phosphoethanolamine methyltransferase catalyzes the formation of phosphocholine from phosphoethanolamine, and is part of the pathway leading to the synthesis of phosphatidylcholine—an important component of membranes. CTP:phosphocholine cytidyltransferase is also present in the biosynthetic pathway to phosphatidylcholine, and catalyzes the reaction of cytidine triphosphate with phosphocholine to produce CDP-choline. Inhibition of either or both of these enzymes is likely to disrupt the formation of parasite membranes.

Under metabolic stress, cellular components can assemble into distinct membraneless organelles for adaptation. One such example is cytidine 5'-triphosphate synthase (CTPS, for which there are CTPS1 and CTPS2 forms in mammals), which forms filamentous structures under glutamine deprivation. We have previously demonstrated that histidine (His)-mediated methylation regulates the formation of CTPS filaments to suppress enzymatic activity and preserve the CTPS protein under glutamine deprivation, which promotes cancer cell growth after stress alleviation. However, it remains unclear where and how these enigmatic structures are assembled. Using CTPS-APEX2-mediated in vivo proximity labeling, we found that synaptosome-associated protein 29 (SNAP29) regulates the spatiotemporal filament assembly of CTPS along the cytokeratin network in a keratin 8 (KRT8)-dependent manner. Knockdown of SNAP29 interfered with assembly and relaxed the filament-induced suppression of CTPS enzymatic activity. Furthermore, APEX2 proximity labeling of keratin 18 (KRT18) revealed a spatiotemporal association of SNAP29 with cytokeratin in response to stress. Super-resolution imaging suggests that during CTPS filament formation, SNAP29 interacts with CTPS along the cytokeratin network. This study links the cytokeratin network to the regulation of metabolism by compartmentalization of metabolic enzymes during nutrient deprivation.

A simple and fast method of preparation of non-natural nicotinamide adenine dinucleotide (NAD) analog nicotinamide cytosine dinucleotide (NCD) was developed. NCD synthetase (NcdS-1) was developed from nicotinic acid mononucleotide adenylyltransferase (NadD) with higher catalytic efficiency towards nicotinamide mononucleotide (NMN) and cytidine triphosphate (CTP). Reaction composition was optimized with inorganic pyrophosphatase (PPase) from E. coli to achieve near quantitative conversion of substrates. A Scalable purification procedure was determined to achieve a fast 4.19 g NCD preparation of 96 % purity.

Dethiobiotin synthetase fromMycobacterium tuberculosis(MtDTBS) is a promising antituberculosis drug target. Small-molecule inhibitors that targetMtDTBS provide a route towards new therapeutics for the treatment of antibiotic-resistant tuberculosis. Adenosine diphosphate (ADP) is an inhibitor ofMtDTBS; however, structural studies into its mechanism of inhibition have been unsuccessful owing to competitive binding to the enzyme by crystallographic precipitants such as citrate and sulfate. Here, a crystallographic technique termed precipitant–ligand exchange has been developed to exchange protein-bound precipitants with ligands of interest. Proof of concept for the exchange method was demonstrated using cytidine triphosphate (CTP), which adopted the same binding mechanism as that obtained with traditional crystal-soaking techniques. Precipitant–ligand exchange also yielded the previously intractable structure ofMtDTBS in complex with ADP solved to 2.4 Å resolution. This result demonstrates the utility of precipitant–ligand exchange, which may be widely applicable to protein crystallography.

Compartmentation of enzymes via filamentation has arisen as a mechanism for the regulation of metabolism. In 2010, three groups independently reported that CTP synthase (CTPS) can assemble into a filamentous structure termed the cytoophidium. In searching for CTPS-interacting proteins, here we perform a yeast two-hybrid screening of Drosophila proteins and identify a putative CTPS-interacting protein, △1-pyrroline-5-carboxylate synthase (P5CS). Using the Drosophila follicle cell as the in vivo model, we confirm that P5CS forms cytoophidia, which are associated with CTPS cytoophidia. Overexpression of P5CS increases the length of CTPS cytoophidia. Conversely, filamentation of CTPS affects the morphology of P5CS cytoophidia. Finally, in vitro analyses confirm the filament-forming property of P5CS. Our work links CTPS with P5CS, two enzymes involved in the rate-limiting steps in pyrimidine and proline biosynthesis, respectively.

While most of the articles in this issue review the workings of integral membrane enzymes, in this review, we describe the catalytic mechanism of an enzyme that contains a soluble catalytic domain but appears to catalyze its reaction on the membrane surface, anchored and assisted by a separate regulatory amphipathic helical domain and inter-domain linker. Membrane partitioning of CTP: phosphocholine cytidylyltransferase (CCT), a key regulatory enzyme of phosphatidylcholine metabolism, is regulated chiefly by changes in membrane phospholipid composition, and boosts the enzyme's catalytic efficiency >200-fold. Catalytic enhancement by membrane binding involves the displacement of an auto-inhibitory helix from the active site entrance-way and promotion of a new conformational ensemble for the inter-domain, allosteric linker that has an active role in the catalytic cycle. We describe the evidence for close contact between membrane lipid, a compact allosteric linker, and the CCT active site, and discuss potential ways that this interaction enhances catalysis.

In all living organisms, it is essential to transmit genetic information faithfully to the next generation. The SMC-ParAB-parS system is widely employed for chromosome segregation in bacteria. A DNA-binding protein ParB nucleates on parS sites and must associate with neighboring DNA, a process known as spreading, to enable efficient chromosome segregation. Despite its importance, how the initial few ParB molecules nucleating at parS sites recruit hundreds of further ParB to spread is not fully understood. Here, we reconstitute a parS-dependent ParB spreading event using purified proteins from Caulobacter crescentus and show that CTP is required for spreading. We further show that ParB spreading requires a closed DNA substrate, and a DNA-binding transcriptional regulator can act as a roadblock to attenuate spreading unidirectionally in vitro. Our biochemical reconstitutions recapitulate many observed in vivo properties of ParB and opens up avenues to investigate the interactions between ParB-parS with ParA and SMC.

Bacterial lactate racemase is a nickel-dependent enzyme that contains a cofactor, nickel pyridinium-3,5-bisthiocarboxylic acid mononucleotide, hereafter named nickel-pincer nucleotide (NPN). The LarC enzyme from the bacterium Lactobacillus plantarum participates in NPN biosynthesis by inserting nickel ion into pyridinium-3,5-bisthiocarboxylic acid mononucleotide. This reaction, known in organometallic chemistry as a cyclometalation, is characterized by the formation of new metal–carbon and metal–sulfur σ bonds. LarC is therefore the first cyclometallase identified in nature, but the molecular mechanism of LarC-catalyzed cyclometalation is unknown. Here, we show that LarC activity requires Mn(2+)-dependent CTP hydrolysis. The crystal structure of the C-terminal domain of LarC at 1.85 Å resolution revealed a hexameric ferredoxin-like fold and an unprecedented CTP-binding pocket. The loss-of-function of LarC variants with alanine variants of acidic residues leads us to propose a carboxylate-assisted mechanism for nickel insertion. This work also demonstrates the in vitro synthesis and purification of the NPN cofactor, opening new opportunities for the study of this intriguing cofactor and of NPN-utilizing enzymes.

Cytidine triphosphate (CTP) synthase 1 is responsible for the catalytic conversion of uridine triphosphate (UTP) to CTP in lymphocytes. This reaction is important for the biosynthesis of nucleic acids, playing a key role in lymphocyte function and turnover. Lack of this enzyme due to mutation in CTPS gene is associated with impaired capacity of activated T and B-lymphocytes to proliferate in response to antigen-induced activation. Patients with this mutation all have ancestors from the North West of England and present in the first decade of life with severe acute and chronic herpes virus infections and recurrent infections with encapsulated bacteria. There is a high risk of EBV-driven lymphoma. This is a potentially life-threatening primary immune deficiency (PID) and long-term survival has not been reported. We report on 11 patients with CTPS1 mutations who underwent hematopoietic stem cell transplant (HSCT) at three UK centres (London, Manchester and Newcastle). The molecular diagnosis was made pre-HSCT in six patients and retrospectively in five patients who had undergone HSCT for previously undiagnosed life-threatening PID. All patients had the same homozygous NM_001905.3: c.1692-1G>C mutation (rs145092287) mutation and all the parents were heterozygous carriers. This mutation is known to lead to an abnormal transcript lacking exon 18 and complete lack of protein expression. Six patients had an affected 39 sibling. Patient 1 had 2 affected siblings who died of infection and hemophagocytic lymphohistiocytosis (HLH) in early childhood and patient 2 also had a sibling who died of overwhelming VZV and EBV infections before diagnosis. Pre-transplant features are summarized in Table 1. Systemic viral infections were seen in all patients, with acute and chronic EBV infections in nine patients, four suffering from EBV-driven lymphoproliferative disease (LPD) (two CNS and one lung) and one with EBV-driven HLH. Four patients had severe chickenpox. Five patients presented with chronic diarrhea. Four patients with EBV-driven LPD received rituximab and three EBV-specific cytotoxic T-lymphocytes (CTLs) (two pre-transplant and one post-transplant). The age of the patients at transplant ranged from 15 months to 17 years. The source of stem cells was peripheral blood (PB) in seven patients, bone marrow (BM) in three and cord blood (CB) in one. Eight received matched (10/10) donor stem cells, two had 1/10 mismatches and one received a haploidentical 6/10 transplant from his father as there were no suitable matched donor (Table 2). All but one received conditioning with fludarabine in combination with treosulfan (n=3), melphalan (n=4), treosulfan and thiotepa (n=1, the non-reduced intensity conditioning (RIC) protocol) or low dose busulfan (n=2)3 one received treosulfan and cyclophosphamide. All but one patient received serotherapy with alemtuzumab (n=8) or anti T cell globulin (ATG) (n=2). CD34+ cell dose ranged from 0.4 - 16.1 x 106/kg. Neutrophil engraftment (≥0.5 x 109 51 /L) occurred between day 11- 23 post-transplant. Ten had 100% CD3 donor engraftment and one had 76% in last follow up. Eight patients are alive with post-HSCT follow-up of 8 months to 22 years. All surviving patients have discontinued immunosuppression and six are off immunoglobulin replacement therapy. Patient 4 died 76 days post-HSCT following EBV CNS reactivation leading to obstructive hydrocephalus. She was treated with external ventricular drainage and three doses of 2 x 106 55 EBV-CTLs but died of multi-organ failure 4 56 . Patient 7 developed intractable gut inflammation due to a combination of adenovirus and CMV colitis and chronic graft versus host disease (cGvHD) and died at day +290 despite treatment with ganciclovir and cidofovir for the viral infections and corticosteroids, ciclosporin, tacrolimus and infliximab as anti-inflammatory drugs. Patient 8 developed mild acute skin GvHD post-transplant, but later suffered from intractable chronic skin GvHD (but no gut or liver disease) unresponsive to corticosteroids and extracorporeal photophoresis. Twelve months post-transplantation he developed a tremor and altered mental state progressing to coma and died 15 months post-transplant. Brain biopsy and magnetic resonance (MR) scan performed prior to death were indicative of progressive multifocal leukoencephalopathy (PML), although no JC, BK or astrovirus were isolated from cerebrospinal fluid (CSF) or brain tissue. CTPS1 deficiency is a serious life-limiting immunodeficiency with a high risk of death from VZV and EBV in the first decade of life. Seven (41%) of 17 patients worldwide with CTPS1 deficiency have died, three post and four prior to HSCT (one from fulminant VZV and the other three from fulminant EBV LPD). These deaths were prior to clinical availability of rituximab. However, mild clinical phenotype has previously been described. HSCT can be curative but is not without potential risks and complications. Kucuk et al also reported two siblings with this mutation who had successful transplantation. The current report is the largest series of children with CTPS1 deficiency who have undergone HSCT.The overall survival post-HSCT in this series was 72% which is slightly lower than seen after HSCT for other combined PIDs. Pre-transplantation, extra-nodal EBV-driven LPD of the brain and lung as well as fulminant VZV pneumonitis requiring ventilation in intensive care are major risks. The post-transplantation course can be challenging to even the specialist centres because of re-activation of EBV and other herpes viruses, particularly in the brain and gastrointestinal tract, but also because of the risk of unusually severe skin and gut cGvHD, the latter which may be partly fuelled by ongoing underlying low grade chronic viral infection. Both patients that developed fatal GvHD had transplants from 9/10 matched unrelated donors. Viral reactivation can be observed prior to donor T-lymphocyte engraftment post-transplant and perhaps virus-specific CTLs may be helpful in some of these patients. There is no doubt that HSCT can be curative in this condition and we recommend early transplantation before the onset of end-organ damage and for patients without fully matching family or unrelated donors, the consideration of ex-vivo T-cell depletion strategies.

Viral infections continue to represent major challenges to public health, and an enhanced mechanistic understanding of the processes that contribute to viral life cycles is necessary for the development of new therapeutic strategies1. Viperin, a member of the radical S-adenosyl-l-methionine (SAM) superfamily of enzymes, is an interferon-inducible protein implicated in the inhibition of replication of a broad range of RNA and DNA viruses, including dengue virus, West Nile virus, hepatitis C virus, influenza A virus, rabies virus2 and HIV3,4. Viperin has been suggested to elicit these broad antiviral activities through interactions with a large number of functionally unrelated host and viral proteins3,4. Here we demonstrate that viperin catalyses the conversion of cytidine triphosphate (CTP) to 3ʹ-deoxy-3′,4ʹ-didehydro-CTP (ddhCTP), a previously undescribed biologically relevant molecule, via a SAM-dependent radical mechanism. We show that mammalian cells expressing viperin and macrophages stimulated with IFNα produce substantial quantities of ddhCTP. We also establish that ddhCTP acts as a chain terminator for the RNA-dependent RNA polymerases from multiple members of the Flavivirus genus, and show that ddhCTP directly inhibits replication of Zika virus in vivo. These findings suggest a partially unifying mechanism for the broad antiviral effects of viperin that is based on the intrinsic enzymatic properties of the protein and involves the generation of a naturally occurring replication-chain terminator encoded by mammalian genomes. Viperin inhibits the replication of various viruses by catalysing the conversion of CTP to ddhCTP, which is a unique nucleotide that functions as replication-chain terminator of RNA-dependent RNA polymerases.

The metabolic enzyme cytidine triphosphate synthase has recently been found to form micrometer-sized filamentous structures termed cytoophidia, which are evolutionarily conserved across prokaryotes and eukaryotes. The cytoophidium represents a novel type of membraneless organelle and behaves dynamically inside the cell. The question of how cytoophidia transport is mediated, however, remains unanswered. For the first time, we detected in this study the active transport of cytoophidia, taking advantage of the fission yeast Schizosaccharomyces pombe as an excellent model for studying membraneless organelles. We demonstrated that actin filaments, not microtubules, are responsible for this transport. Furthermore, we determined that Myo52, a type of myosin V, is required for the active transport of cytoophidia. These results reveal the major players critical to the dynamics of cytoophidia and extend our understanding of intracellular transport of membraneless organelles.—Li, H., Ye, F., Ren, J.-Y., Wang, P.-Y., Du, L.-L., Liu, J.-L. Active transport of cytoophidia in Schizosaccharomyces pombe.

The cytoophidium, a filamentous structure formed by metabolic enzymes, has emerged as a novel regulatory machinery for certain proteins. The rate-limiting enzymes of de novo CTP and GTP synthesis, cytidine triphosphate synthase (CTPS) and inosine monophosphate dehydrogenase (IMPDH), are the most characterized cytoophidium-forming enzymes in mammalian models. Although the assembly of CTPS cytoophidia has been demonstrated in various organisms including multiple human cancers, a systemic survey for the presence of CTPS cytoophidia in mammalian tissues in normal physiological conditions has not yet been reported. Herein, we examine major organs of adult mouse and observe that CTPS cytoophidia are displayed by a specific thymocyte population ranging between DN3 to early DP stages. Most of these cytoophidium-presenting cells have both CTPS and IMPDH cytoophidia and undergo rapid cell proliferation. In addition, we show that cytoophidium formation is associated with active glycolytic metabolism as the cytoophidium-presenting cells exhibit higher levels of c-Myc, phospho-Akt and PFK. Inhibition of glycolysis with 2DG, however, disrupts most of cytoophidium structures and impairs cell proliferation. Our findings not only indicate that the regulation of CTPS and IMPDH cytoophidia are correlated with the metabolic switch triggered by pre-TCR signaling, but also suggest physiological roles of the cytoophidium in thymocyte development.

Many enveloped viruses bud from cholesterol-rich lipid rafts on the cell membrane. Depleting cellular cholesterol impedes this process and results in viral particles with reduced viability. Viperin (Virus Inhibitory Protein, Endoplasmic Reticulum-associated, Interferon iNducible) is an endoplasmic reticulum membrane-associated enzyme that exerts broad-ranging antiviral effects, including inhibiting the budding of some enveloped viruses. However, the relationship between viperin expression and the retarded budding of virus particles from lipid rafts on the cell membrane is unclear. Here, we investigated the effect of viperin expression on cholesterol biosynthesis using transiently expressed genes in the human cell line human embryonic kidney 293T (HEK293T). We found that viperin expression reduces cholesterol levels by 20% to 30% in these cells. Following this observation, a proteomic screen of the viperin interactome identified several cholesterol biosynthetic enzymes among the top hits, including lanosterol synthase (LS) and squalene monooxygenase (SM), which are enzymes that catalyze key steps in establishing the sterol carbon skeleton. Coimmunoprecipitation experiments confirmed that viperin, LS, and SM form a complex at the endoplasmic reticulum membrane. While coexpression of viperin was found to significantly inhibit the specific activity of LS in HEK293T cell lysates, coexpression of viperin had no effect on the specific activity of SM, although did reduce SM protein levels by approximately 30%. Despite these inhibitory effects, the coexpression of neither LS nor SM was able to reverse the viperin-induced depletion of cellular cholesterol levels, possibly because viperin is highly expressed in transfected HEK293T cells. Our results establish a link between viperin expression and downregulation of cholesterol biosynthesis that helps explain viperin's antiviral effects against enveloped viruses.

The divergence of archaea, bacteria and eukaryotes was a fundamental step in evolution. One marker of this event is a major difference in membrane lipid chemistry between these kingdoms. Whereas the membranes of bacteria and eukaryotes primarily consist of straight fatty acids ester-bonded to glycerol-3-phosphate, archaeal phospholipids consist of isoprenoid chains ether-bonded to glycerol-1-phosphate. Notably, the mechanisms underlying the biosynthesis of these lipids remain elusive. Here, we report the structure of the CDP-archaeol synthase (CarS) of Aeropyrum pernix (ApCarS) in the CTP- and Mg(2+)-bound state at a resolution of 2.4 Å. The enzyme comprises a transmembrane domain with five helices and cytoplasmic loops that together form a large charged cavity providing a binding site for CTP. Identification of the binding location of CTP and Mg(2+) enabled modeling of the specific lipophilic substrate-binding site, which was supported by site-directed mutagenesis, substrate-binding affinity analyses, and enzyme assays. We propose that archaeol binds within two hydrophobic membrane-embedded grooves formed by the flexible transmembrane helix 5 (TM5), together with TM1 and TM4. Collectively, structural comparisons and analyses, combined with functional studies, not only elucidated the mechanism governing the biosynthesis of phospholipids with ether-bonded isoprenoid chains by CTP transferase, but also provided insights into the evolution of this enzyme superfamily from archaea to bacteria and eukaryotes.Cell Research advance online publication 29 September 2017; doi:10.1038/cr.2017.122.

To develop a new one-pot polyphosphate kinase (PPK) system with low cost and high efficiency for ATP regeneration in industrial CTP production. We developed a new one-pot PPK system by applying a three-enzyme cascade (CMK, NDK and PPK) with an in vitro polyP-based ATP regeneration system. The PPK was selected from twenty sources, and was made solvable by fusion expressing with soluble protein and constructing polycistronic plasmids, or co-expressing with molecular chaperones GroES/EL. Activities of other enzymes were optimized by employing fusion expression, tac-pBAD system, Rosetta host and codon optimization. After 24 h, the concentration of CDP and CTP reached 3.8 ± 0.2 and 6.9 ± 0.3 mM l−1 respectively with a yield of approximately 79%. The molar conversion rate of CTP was 51%, and its yield and conversion rate increased 100% from the traditional system. A new one-pot ATP regeneration system applying polyphosphate kinase for CTP production was developed.

Cytidine triphosphate synthase 1 (CTPS1) is an enzyme expressed in activated lymphocytes that catalyzes the conversion of uridine triphosphate (UTP) to cytidine triphosphate (CTP) with ATP-dependent amination, using either L-glutamine or ammonia as the nitrogen source. Since CTP plays an important role in DNA/RNA synthesis, phospholipid synthesis, and protein sialyation, CTPS1-inhibition is expected to control lymphocyte proliferation and size expansion in inflammatory diseases. In contrast, CTPS2, an isozyme of CTPS1 possessing 74% amino acid sequence homology, is expressed in normal lymphocytes. Thus, CTPS1-selective inhibition is important to avoid undesirable side effects. Here, we report the discovery of CTpep-3: Ac-FRLGLLKAFRRLF-OH from random peptide libraries displayed on T7 phage, which exhibited CTPS1-selective binding with a KD value of 210nM in SPR analysis and CTPS1-selective inhibition with an IC50 value of 110nM in the enzyme assay. Furthermore, two fundamentally different approaches, enzyme inhibition assay and HDX-MS, provided the same conclusion that CTpep-3 acts by binding to the amidoligase (ALase) domain on CTPS1. To our knowledge, CTpep-3 is the first CTPS1-selective inhibitor.

Intracellular compartmentation is a key strategy for the functioning of a cell. In 2010, several studies revealed that the metabolic enzyme CTP synthase (CTPS) can form filamentous structures termed cytoophidia in prokaryotic and eukaryotic cells. However, recent structural studies showed that CTPS only forms inactive product-bound filaments in bacteria while forming active substrate-bound filaments in eukaryotic cells. In this study, using negative staining and cryo-electron microscopy, we demonstrate that Drosophila CTPS, whether in substrate-bound or product-bound form, can form filaments. Our results challenge the previous model and indicate that substrate-bound and product-bound filaments can coexist in the same species. We speculate that the ability to switch between active and inactive cytoophidia in the same cells provides an additional layer of metabolic regulation.

Three sets of 7‐deazaadenine and cytosine nucleosides and nucleoside triphosphates bearing either unsubstituted ferrocene, octamethylferrocene and ferrocenecarboxamide linked through an alkyne tether to position 7 or 5, respectively, were designed and synthesized. The modified dNFcXTPs were good substrates for KOD XL DNA polymerase in primer extension and were used for enzymatic synthesis of redox‐labelled DNA probes. Square‐wave voltammetry showed that the octamethylferrocene oxidation potential was shifted to lower values, whilst the ferrocenecarboxamide was shifted to higher potentials, as compared to ferrocene. Tailed PEX products containing different ratios of Fc‐labelled A (dAFc) and FcPa‐labelled C (dCFcPa) were synthesized and hybridized with capture oligonucleotides immobilized on gold electrodes to study the electrochemistry of the redox‐labelled DNA. Clearly distinguishable, fully orthogonal and ratiometric peaks were observed for the dAFc and dCFcPa bases in DNA, demonstrating their potential for use in redox coding of nucleobases and for the direct electrochemical measurement of the relative ratio of nucleobases in an unknown sequence of DNA., Orthogonal redox labelling of nucleobases with substituted ferrocenes with tuned redox potential was developed for redox coding of DNA bases.

SummaryDuring cell division, newly replicated DNA is actively segregated to the daughter cells. In most bacteria, this process involves the DNA-binding protein ParB, which condenses the centromeric regions of sister DNA molecules into kinetochore-like structures that recruit the DNA partition ATPase ParA and the prokaroytic SMC/condensin complex. Here, we report the crystal structure of a ParB-like protein (PadC) that emerges to tightly bind the ribonucleotide CTP. The CTP-binding pocket of PadC is conserved in ParB and composed of signature motifs known to be essential for ParB function. We find that ParB indeed interacts with CTP and requires nucleotide binding for DNA condensation in vivo. We further show that CTP-binding modulates the affinity of ParB for centromeric parS sites, whereas parS recognition stimulates its CTPase activity. ParB proteins thus emerge as a new class of CTP-dependent molecular switches that act in concert with ATPases and GTPases to control fundamental cellular functions.