Chloroflexus aurantiacus acetyl-CoA carboxylase evolves fused biotin carboxylase and biotin carboxyl carrier protein to complete carboxylation activity.

Acetyl-CoA carboxylases (ACCs) convert acetyl-CoA to malonyl-CoA, a key step in fatty acid biosynthesis and autotrophic carbon fixation pathways. Three functionally distinct components, biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT), are either separated or partially fused in different combinations, forming heteromeric ACCs. However, an ACC with fused BC-BCCP and separate CT has not been identified, leaving its catalytic mechanism unclear. Here, we identify two BC isoforms (BC1 and BC2) from Chloroflexus aurantiacus, a filamentous anoxygenic phototroph that employs 3-hydroxypropionate (3-HP) bi-cycle rather than Calvin cycle for autotrophic carbon fixation. We reveal that BC1 possesses fused BC and BCCP domains, where BCCP could be biotinylated by E. coli or C. aurantiacus BirA on Lys553 residue. Crystal structures of BC1 and BC2 at 3.2 Å and 3.0 Å resolutions, respectively, further reveal a tetramer of two BC1-BC homodimers, and a BC2 homodimer, all exhibiting similar BC architectures. The two BC1-BC homodimers are connected by an eight-stranded ß-barrel of the partially resolved BCCP domain. Disruption of ß-barrel results in dissociation of the tetramer into dimers in solution and decreased biotin carboxylase activity. Biotinylation of the BCCP domain further promotes BC1 and CTß-CTα interactions to form an enzymatically active ACC, which converts acetyl-CoA to malonyl-CoA in vitro and produces 3-HP via co-expression with a recombinant malonyl-CoA reductase in E. coli cells. This study revealed a heteromeric ACC that evolves fused BC-BCCP but separate CTα and CTß to complete ACC activity.IMPORTANCEAcetyl-CoA carboxylase (ACC) catalyzes the rate-limiting step in fatty acid biosynthesis and autotrophic carbon fixation pathways across a wide range of organisms, making them attractive targets for drug discovery against various infections and diseases. Although structural studies on homomeric ACCs, which consist of a single protein with three subunits, have revealed the "swing domain model" where the biotin carboxyl carrier protein (BCCP) domain translocates between biotin carboxylase (BC) and carboxyltransferase (CT) active sites to facilitate the reaction, our understanding of the subunit composition and catalytic mechanism in heteromeric ACCs remains limited. Here, we identify a novel ACC from an ancient anoxygenic photosynthetic bacterium Chloroflexus aurantiacus, it evolves fused BC and BCCP domain, but separate CT components to form an enzymatically active ACC, which converts acetyl-CoA to malonyl-CoA in vitro and produces 3-hydroxypropionate (3-HP) via co-expression with recombinant malonyl-CoA reductase in E. coli cells. These findings expand the diversity and molecular evolution of heteromeric ACCs and provide a structural basis for potential applications in 3-HP biosynthesis.

Natural product guvermectin inhibits guanosine 5'-monophosphate synthetase and confers broad-spectrum antibacterial activity.

Bacterial diseases caused substantial yield losses worldwide, with the rise of antibiotic resistance, there is a critical need for alternative antibacterial compounds. Natural products (NPs) from microorganisms have emerged as promising candidates due to their potential as cost-effective and environmentally friendly bactericides. However, the precise mechanisms underlying the antibacterial activity of many NPs, including Guvermectin (GV), remain poorly understood. Here, we sought to explore how GV interacts with Guanosine 5'-monophosphate synthetase (GMPs), an enzyme crucial in bacterial guanine synthesis. We employed a combination of biochemical and genetic approaches, enzyme activity assays, site-directed mutagenesis, bio-layer interferometry, and molecular docking assays to assess GV's antibacterial activity and its mechanism targeting GMPs. The results showed that GV effectively inhibits GMPs, disrupting bacterial guanine synthesis. This was confirmed through drug-resistant assays and direct enzyme inhibition studies. Bio-layer interferometry assays demonstrated specific binding of GV to GMPs, with dependency on Xanthosine 5'-monophosphate. Site-directed mutagenesis identified key residues crucial for the GV-GMP interaction. This study elucidates the antibacterial mechanism of GV, highlighting its potential as a biocontrol agent in agriculture. These findings contribute to the development of novel antibacterial agents and underscore the importance of exploring natural products for agricultural disease management.

AviTrap: A novel solution to achieve complete biotinylation.

Site specific biotinylation of AviTagged recombinant proteins using BirA enzyme is a widely used protein labeling technology. However, due to the incomplete biotinylation reactions and the lack of a purification method specific for the biotinylated proteins, it is challenging to purify the biotinylated sample when mixed with the non-biotinylated byproduct. Here, we have developed a monoclonal antibody that specifically recognizes the non-biotinylated AviTag but not the biotinylated sequence. After a ten-minute incubation with the resin that is conjugated with the antibody, the non-biotinylated AviTagged protein is trapped on the resin while the fully biotinylated material freely passes through. Therefore, our AviTrap (anti-AviTag antibody conjugated resin) provides an efficient solution for enriching biotinylated AviTagged proteins via a simple one-step purification.

Dynamic Cytoophidia during Late-Stage Drosophila Oogenesis.

CTP synthase (CTPS) catalyzes the final step of de novo synthesis of CTP. CTPS was first discovered to form filamentous structures termed cytoophidia in Drosophila ovarian cells. Subsequent studies have shown that cytoophidia are widely present in cells of three life domains. In the Drosophila ovary model, our previous studies mainly focused on the early and middle stages, with less involvement in the later stages. In this work, we focus on the later stages of female germline cells in Drosophila. We use live-cell imaging to capture the continuous dynamics of cytoophidia in Stages 10-12. We notice the heterogeneity of cytoophidia in the two types of germline cells (nurse cells and oocytes), manifested in significant differences in morphology, distribution, and dynamics. Surprisingly, we also find that neighboring nurse cells in the same egg chamber exhibit multiple dynamic patterns of cytoophidia over time. Although the described dynamics may be influenced by the in vitro incubation conditions, our observation provides an initial understanding of the dynamics of cytoophidia during late-stage Drosophila oogenesis.

Identification of G-quadruplex-interacting proteins in living cells using an artificial G4-targeting biotin ligase.

G-quadruplexes (G4s) are noncanonical nucleic acid structures pivotal to cellular processes and disease pathways. Deciphering G4-interacting proteins is imperative for unraveling G4's biological significance. In this study, we developed a G4-targeting biotin ligase named G4PID, meticulously assessing its binding affinity and specificity both in vitro and in vivo. Capitalizing on G4PID, we devised a tailored approach termed G-quadruplex-interacting proteins specific biotin-ligation procedure (PLGPB) to precisely profile G4-interacting proteins. Implementing this innovative strategy in live cells, we unveiled a cohort of 149 potential G4-interacting proteins, which exhibiting multifaceted functionalities. We then substantiate the directly binding affinity of 7 candidate G4-interacting-proteins (SF3B4, FBL, PP1G, BCL7C, NDUV1, ILF3, GAR1) in vitro. Remarkably, we verified that splicing factor 3B subunit 4 (SF3B4) binds preferentially to the G4-rich 3' splice site and the corresponding splicing sites are modulated by the G4 stabilizer PDS, indicating the regulating role of G4s in mRNA splicing procedure. The PLGPB strategy could biotinylate multiple proteins simultaneously, which providing an opportunity to map G4-interacting proteins network in living cells.

Profiling the interactome of oligonucleotide drugs by proximity biotinylation.

Drug-ID is a novel method applying proximity biotinylation to identify drug-protein interactions inside living cells. The covalent conjugation of a drug with a biotin ligase enables targeted biotinylation and identification of the drug-bound proteome. We established Drug-ID for two small-molecule drugs, JQ1 and SAHA, and applied it for RNaseH-recruiting antisense oligonucleotides (ASOs). Drug-ID profiles the drug-protein interactome de novo under native conditions, directly inside living cells and at pharmacologically effective drug concentrations. It requires minimal amounts of cell material and might even become applicable in vivo. We studied the dose-dependent aggregation of ASOs and the effect of different wing chemistries (locked nucleic acid, 2'-methoxyethyl and 2'-Fluoro) and ASO lengths on the interactome. Finally, we demonstrate the detection of stress-induced, intracellular interactome changes (actinomycin D treatment) with an in situ variant of the approach, which uses a recombinant biotin ligase and does not require genetic manipulation of the target cell.

Enhanced theanine production with reduced ATP supply by alginate entrapped Escherichia coli co-expressing γ-glutamylmethylamide synthetase and polyphosphate kinase.

L-theanine is an amino acid with a unique flavor and many therapeutic effects. Its enzymatic synthesis has been actively studied and γ-Glutamylmethylamide synthetase (GMAS) is one of the promising enzymes in the biological synthesis of theanine. However, the theanine biosynthetic pathway with GMAS is highly ATP-dependent and the supply of external ATP was needed to achieve high concentration of theanine production. As a result, this study aimed to investigate polyphosphate kinase 2 (PPK2) as ATP regeneration system with hexametaphosphate. Furthermore, the alginate entrapment method was employed to immobilize whole cells containing both gmas and ppk2 together resulting in enhanced reusability of the theanine production system with reduced supply of ATP. After immobilization, theanine production was increased to 239 mM (41.6 g/L) with a conversion rate of 79.7% using 15 mM ATP and the reusability was enhanced, maintaining a 100% conversion rate up to the fifth cycles and 60% of conversion up to eighth cycles. It could increase long-term storage property for future uses up to 35 days with 75% activity of initial activity. Overall, immobilization of both production and cofactor regeneration system could increase the stability and reusability of theanine production system.

Biotin protein ligase as you like it: Either extraordinarily specific or promiscuous protein biotinylation.

Biotin (vitamin H or B7) is a coenzyme essential for all forms of life. Biotin has biological activity only when covalently attached to a few key metabolic enzyme proteins. Most organisms have only one attachment enzyme, biotin protein ligase (BPL), which attaches biotin to all target proteins. The sequences of these proteins and their substrate proteins are strongly conserved throughout biology. Structures of both the biotin ligase- and biotin-acceptor domains of mammals, plants, several bacterial species, and archaea have been determined. These, together with mutational analyses of ligases and their protein substrates, illustrate the exceptional specificity of this protein modification. For example, the Escherichia coli BPL biotinylates only one of the >4000 cellular proteins. Several bifunctional bacterial biotin ligases transcriptionally regulate biotin synthesis and/or transport in concert with biotinylation. The human BPL has been demonstrated to play an important role in that mutations in the BPL encoding gene cause one form of the disease, biotin-responsive multiple carboxylase deficiency. Promiscuous mutant versions of several BPL enzymes release biotinoyl-AMP, the active intermediate of the ligase reaction, to solvent. The released biotinoyl-AMP acts as a chemical biotinylation reagent that modifies lysine residues of neighboring proteins in vivo. This proximity-dependent biotinylation (called BioID) approach has been heavily utilized in cell biology.

Fortuitous In Vitro Compound Degradation Produces a Tractable Hit against Mycobacterium tuberculosis Dethiobiotin Synthetase: A Cautionary Tale of What Goes In Does Not Always Come Out.

We previously reported potent ligands and inhibitors of Mycobacterium tuberculosis dethiobiotin synthetase (MtDTBS), a promising target for antituberculosis drug development (Schumann et al., ACS Chem Biol. 2021, 16, 2339-2347); here, the unconventional origin of the fragment compound they were derived from is described for the first time. Compound 1 (9b-hydroxy-6b,7,8,9,9a,9b-hexahydrocyclopenta[3,4]cyclobuta[1,2-c]chromen-6(6aH)-one), identified by an in silico fragment screen, was subsequently shown by surface plasmon resonance to have dose-responsive binding (KD = 0.6 mM). Clear electron density was revealed in the DAPA substrate binding pocket when 1 was soaked into MtDTBS crystals, but the density was inconsistent with the structure of 1. Here, we show that the lactone of 1 hydrolyzes to a carboxylic acid (2) under basic conditions, including those of the crystallography soak, with a subsequent ring opening of the component cyclobutane ring forming a cyclopentylacetic acid (3). Crystals soaked directly with authentic 3 produced an electron density that matched that of crystals soaked with presumed 1, confirming the identity of the bound ligand. The synthetic utility of fortuitously formed 3 enabled the subsequent compound development of nanomolar inhibitors. Our findings represent an example of chemical modification within drug discovery assays and demonstrate the value of high-resolution structural data in the fragment hit validation process.

GART Functions as a Novel Methyltransferase in the RUVBL1/ß-Catenin Signaling Pathway to Promote Tumor Stemness in Colorectal Cancer.

Tumor stemness is associated with the recurrence and incurability of colorectal cancer (CRC), which lacks effective therapeutic targets and drugs. Glycinamide ribonucleotide transformylase (GART) fulfills an important role in numerous types of malignancies. The present study aims to identify the underlying mechanism through which GART may promote CRC stemness, as to developing novel therapeutic methods. An elevated level of GART is associated with poor outcomes in CRC patients and promotes the proliferation and migration of CRC cells. CD133+ cells with increased GART expression possess higher tumorigenic and proliferative capabilities both in vitro and in vivo. GART is identified to have a novel methyltransferase function, whose enzymatic activity center is located at the E948 site. GART also enhances the stability of RuvB-like AAA ATPase 1 (RUVBL1) through methylating its K7 site, which consequently aberrantly activates the Wnt/ß-catenin signaling pathway to induce tumor stemness. Pemetrexed (PEM), a compound targeting GART, combined with other chemotherapy drugs greatly suppresses tumor growth both in a PDX model and in CRC patients. The present study demonstrates a novel methyltransferase function of GART and the role of the GART/RUVBL1/ß-catenin signaling axis in promoting CRC stemness. PEM may be a promising therapeutic agent for the treatment of CRC.

Rational Engineering of (S)-Norcoclaurine Synthase for Efficient Benzylisoquinoline Alkaloids Biosynthesis.

(S)-Norcoclaurine is synthesized in vivo through a metabolic pathway that ends with (S)-norcoclaurine synthase (NCS). The former constitutes the scaffold for the biosynthesis of all benzylisoquinoline alkaloids (BIAs), including many drugs such as the opiates morphine and codeine and the semi-synthetic opioids oxycodone, hydrocodone, and hydromorphone. Unfortunately, the only source of complex BIAs is the opium poppy, leaving the drug supply dependent on poppy crops. Therefore, the bioproduction of (S)-norcoclaurine in heterologous hosts, such as bacteria or yeast, is an intense area of research nowadays. The efficiency of (S)-norcoclaurine biosynthesis is strongly dependent on the catalytic efficiency of NCS. Therefore, we identified vital NCS rate-enhancing mutations through the rational transition-state macrodipole stabilization method at the Quantum Mechanics/Molecular Mechanics (QM/MM) level. The results are a step forward for obtaining NCS variants able to biosynthesize (S)-norcoclaurine on a large scale.

Differential roles of CTP synthetases CTPS1 and CTPS2 in cell proliferation.

The CTP nucleotide is a key precursor of nucleic acids metabolism essential for DNA replication. De novo CTP production relies on CTP synthetases 1 and 2 (CTPS1 and CTPS2) that catalyze the conversion of UTP into CTP. CTP synthetase activity is high in proliferating cells including cancer cells; however, the respective roles of CTPS1 and CTPS2 in cell proliferation are not known. By inactivation of CTPS1 and/or CTPS2 and complementation experiments, we showed that both CTPS1 and CTPS2 are differentially required for cell proliferation. CTPS1 was more efficient in promoting proliferation than CTPS2, in association with a higher intrinsic enzymatic activity that was more resistant to inhibition by 3-deaza-uridine, an UTP analog. The contribution of CTPS2 to cell proliferation was modest when CTPS1 was expressed but essential in absence of CTPS1. Public databases analysis of more than 1,000 inactivated cancer cell lines for CTPS1 or CTPS2 confirmed that cell growth is highly dependent of CTPS1 but less or not of CTPS2. Therefore, our results demonstrate that CTPS1 is the main contributor to cell proliferation.

A functional genetic screen for metabolic proteins unveils GART and the de novo purine biosynthetic pathway as novel targets for the treatment of luminal A ERα expressing primary and metastatic invasive ductal carcinoma.

Targeting tumor cell metabolism is a new frontier in cancer management. Thus, metabolic pathway inhibitors could be used as anti-estrogen receptor α (ERα) breast cancer (BC) drugs. Here, the interplay among metabolic enzyme(s), the ERα levels and cell proliferation was studied. siRNA-based screen directed against different metabolic proteins in MCF10a, MCF-7 and MCF-7 cells genetically resistant to endocrine therapy (ET) drugs and metabolomic analyses in numerous BC cell lines unveil that the inhibition of GART, a key enzyme in the purine de novo biosynthetic pathway, induces ERα degradation and prevent BC cell proliferation. We report here that a reduced GART expression correlates with a longer relapse-free-survival (RFS) in women with ERα-positive BCs. ERα-expressing luminal A invasive ductal carcinomas (IDCs) are sensitive to GART inhibition and GART expression is increased in receptor-positive IDCs of high grade and stage and plays a role in the development of ET resistance. Accordingly, GART inhibition reduces ERα stability and cell proliferation in IDC luminal A cells where it deregulates 17ß-estradiol (E2):ERα signaling to cell proliferation. Moreover, the GART inhibitor lometrexol (LMX) and drugs approved for clinical treatment of primary and metastatic BC (4OH-tamoxifen and the CDK4/CDK6 inhibitors) exert synergic antiproliferative effects in BC cells. In conclusion, GART inhibition by LMX or other inhibitors of the de novo purine biosynthetic pathway could be a novel effective strategy for the treatment of primary and metastatic BCs.

A metal-dependent conformational change provides a structural basis for the inhibition of CTP synthase by gemcitabine-5'-triphosphate.

CTP synthases (CTPS) catalyze the de novo production of CTP using UTP, ATP, and l-glutamine with the anticancer drug metabolite gemcitabine-5'-triphosphate (dF-dCTP) being one of its most potent nucleotide inhibitors. To delineate the structural origins of this inhibition, we solved the structures of Escherichia coli CTPS (ecCTPS) in complex with CTP (2.0 Å), 2'-ribo-F-dCTP (2.0 Å), 2'-arabino-F-CTP (2.4 Å), dF-dCTP (2.3 Å), dF-dCTP and ADP (2.1 Å), and dF-dCTP and ATP (2.1 Å). These structures revealed that the increased binding affinities observed for inhibitors bearing the 2'-F-arabino group (dF-dCTP and F-araCTP), relative to CTP and F-dCTP, arise from interactions between the inhibitor's fluorine atom exploiting a conserved hydrophobic pocket formed by F227 and an interdigitating loop from an adjacent subunit (Q114-V115-I116). Intriguingly, crystal structures of ecCTPS•dF-dCTP complexes in the presence of select monovalent and divalent cations demonstrated that the in crystallo tetrameric assembly of wild-type ecCTPS was induced into a conformation similar to inhibitory ecCTPS filaments solely through the binding of Na+ -, Mg2+ -, or Mn2+ •dF-dCTP. However, in the presence of potassium, the dF-dCTP-bound structure is demetalated and in the low-affinity, non-filamentous conformation, like the conformation seen when bound to CTP and the other nucleotide analogues. Additionally, CTP can also induce the filament-competent conformation linked to high-affinity dF-dCTP binding in the presence of high concentrations of Mg2+ . This metal-dependent, compacted CTP pocket conformation therefore furnishes the binding environment responsible for the tight binding of dF-dCTP and provides insights for further inhibitor design.

Venae spermaticae post aures: The early modern angiology-neurology of virility.

The famous discussion of Scythian cross-dressers in Hippocrates' Airs Waters Places (Aer.) 22 puzzled perhaps most medieval and Renaissance medical authorities. The text wrestled with a pre-Hippocratic, encephalocentric theory of spermatogenesis. Modern reception of the convoluted hypothesis put forward here gradually distilled three etiologies of failing virility: impotence, subfertility, and unmanliness. A gradual shift is discernable from increasingly Galenic neuro-andrological theories (sixteenth century) to neuropsychiatric (late-seventeenth through eighteenth century), phrenological and psychopathological (early- and late-nineteenth century), and finally early psycho-endocrinological (early-twentieth century) ideas about masculinity. Aer. 22 was a ubiquitously recurring reference across all of these episodes, indeed well beyond medicine, rendering it a highly sensitive index of change in neurodevelopmental and neuropsychiatric thinking. The pre-Enlightenment, neurology-centric onset of this extended modern history of sexual/gender medicine is briefly discussed, as well as its phrenological afterlife.

STAMP: Spatio-Temporal Association Mapping of Proteins.

We describe a tool, Spatio-Temporal Association Mapping of Proteins (STAMP), for identifying protein interactomes via proximity labeling. For a proof-of-principle study, we use cytidine 5'-triphosphate synthase (CTPS) as an example. CTPS, a metabolic enzyme, forms filamentous structures termed cytoophidia in various tissues. We apply STAMP to a variety of developmental stages and tissues in Drosophila including adult ovaries. Using a cell-specific GAL4 driver, we verify that TurboID can biotinylate the bait protein CTPS, making possible the identification of protein-protein interactions (PPIs) in individual cells. Using the wild-type and mutant CTPS as bait proteins, STAMP results in two distinct sets of proximate proteomes. Our results suggest that STAMP is a feasible tool to catch in vivo PPIs in situ at a defined spatiotemporal resolution.

Cytoophidia safeguard binucleation of Drosophila male accessory gland cells.

Although most cells are mononuclear, the nucleus can exist in the form of binucleate or even multinucleate to respond to different physiological processes. The male accessory gland of Drosophila is the organ that produces semen, and its main cells are binucleate. Here we observe that CTP synthase (CTPS) forms filamentous cytoophidia in binuclear main cells, primarily located at the cell boundary. In CTPSH355A, a point mutation that destroys the formation of cytoophidia, we find that the nucleation mode of the main cells changes, including mononucleates and vertical distribution of binucleates. Although the overexpression of CTPSH355A can restore the level of CTPS protein, it will neither form cytoophidia nor eliminate the abnormal nucleation pattern. Therefore, our data indicate that there is an unexpected functional link between the formation of cytoophidia and the maintenance of binucleation in Drosophila main cells.

Discovery and Optimization of Potent and Orally Available CTP Synthetase Inhibitors for Use in Treatment of Diseases Driven by Aberrant Immune Cell Proliferation.

Herein, we report the discovery of a first-in-class chemotype 2-(alkylsulfonamido)thiazol-4-yl)acetamides that act as pan-selective inhibitors of cytidine 5'-triphosphate synthetase (CTPS1/2), critical enzymes in the de novo pyrimidine synthesis pathway. Weak inhibitors identified from a high-throughput screening of 240K compounds have been optimized to a potent, orally active agent, compound 27, which has shown significant pharmacological responses at 10 mg/kg dose BID in a well-established animal model of inflammation.

Super-Resolution Imaging Reveals Dynamic Reticular Cytoophidia.

CTP synthase (CTPS) can form filamentous structures termed cytoophidia in cells in all three domains of life. In order to study the mesoscale structure of cytoophidia, we perform fluorescence recovery after photobleaching (FRAP) and stimulated emission depletion (STED) microscopy in human cells. By using an EGFP dimeric tag as a tool to explore the physical properties of cytoophidia, we find that cytoophidia are dynamic and reticular. The reticular structure of CTPS cytoophidia may provide space for other components, such as IMPDH. In addition, we observe CTPS granules with tentacles.

Asymmetric inheritance of cytoophidia could contribute to determine cell fate and plasticity: The onset of alternative differentiation patterns in daughter cells may rely on the acquisition of either CTPS or IMPDH cytoophidia: The onset of alternative differentiation patterns in daughter cells may rely on the acquisition of either CTPS or IMPDH cytoophidia.

Two enzymes involved in the synthesis of pyrimidine and purine nucleotides, CTP synthase (CTPS) and IMP dehydrogenase (IMPDH), can assemble into a single or very few large filaments called rods and rings (RR) or cytoophidia. Most recently, asymmetric cytoplasmic distribution of organelles during cell division has been described as a decisive event in hematopoietic stem cell fate. We propose that cytoophidia, which could be considered as membrane-less organelles, may also be distributed asymmetrically during mammalian cell division as previously described for Schizosaccharomyces pombe. Furthermore, because each type of nucleotide intervenes in distinct processes (e.g., membrane synthesis, glycosylation, and G protein-signaling), alterations in the rate of synthesis of specific nucleotide types could influence cell differentiation in multiple ways. Therefore, we hypothesize that whether a daughter cell inherits or not CTPS or IMPDH filaments determines its fate and that this asymmetric inheritance, together with the dynamic nature of these structures enables plasticity in a cell population.