Biosynthetic Enzymes Research Articles

From Starfish to Gibberellins: Biosynthesis and Regulation of Plant Hormones.

I grew up with laboratory glassware and microscopes as treasures from a young age. I was a member of the Chemistry Club in junior high school, and when I visited RIKEN with club members, I wished to become an organic chemist in the future. I received my doctoral degree through the study of the spawning inhibitor of starfish. I became a researcher at RIKEN and identified the chemical structure of a mating pheromone of a yeast. As a plant biochemist, I studied a cell-free system of gibberellins at the University of Göttingen and tried to identify the gibberellin biosynthetic pathways in plants and clone gibberellin biosynthetic enzyme genes to understand the light regulation of plant growth. I also worked on biosynthetic enzymes of abscisic acid, indole acetic acid, and brassinosteroids. I developed a special interest in the oxygenases of plant hormone biosynthesis, cytochrome P450 monooxygenases, 2-oxoglutartae-dependent dioxygenase, molybdenum cofactor-containing oxidase, and flavin-containing monooxygenase.

Mushroom-Mediated Redox Reactions.

The application of biocatalysts in organic synthesis has grown significantly in recent years, and both academia and industry are continuously searching for novel biocatalysts capable of performing challenging chemical reactions. Mushroom is a rich source of ligninolytic and secondary metabolite biosynthetic enzymes, and therefore was consider as promising biocatalysts for organic synthesis. This review focused on the broad utilization potential of the mushroom-based biocatalysts and highlights key advances in mushroom-mediated organic transformations. It mainly includes reduction of ketone and carboxylic acids, hydroxylation of aromatic and aliphatic compounds, epoxidation of olefin, oxidative cleavage of alkene, and other uncommon reactions catalyzed by the whole-cells or purified enzymes of mushroom-origin. Overall, a comprehensive overview of the applications of mushroom as biocatalyst in organic synthesis is provided, which puts this versatile microorganism in the spotlight of further research.

Biosynthetic Incorporation of Non-native Aryl Acid Building Blocks into Peptide Products Using Engineered Adenylation Domains.

Nonribosomal peptides (NRPs), one of the most widespread secondary metabolites in nature, with therapeutically significant activities, are biosynthesized by modular nonribosomal peptide synthetases (NRPSs). Aryl acids contribute to the structural diversity of NRPs as well as nonproteinogenic amino acids and keto acids. We previously confirmed that a single Asn-to-Gly substitution in the 2,3-dihydroxybenzoic acid-activating adenylation (A) domain EntE involved in enterobactin biosynthesis accepts monosubstituted benzoic acid derivatives with nitro, cyano, bromo, and iodo functionalities at the 2 or 3 positions. Here, we showed that the mutant EntE (N235G) accommodates various disubstituted benzoic acid derivatives with halogen, methyl, methoxy, nitro, and cyano functionalities at the 2 and 3 positions and monosubstituted benzoic acid with an alkyne at the 3 position. Structural analysis of the mutant EntE (N235G) with nonhydrolyzable aryl-AMP analogues using 3-chloro-2-methylbenzoic acid and 3-prop-2-ynoxybenzoic acid revealed how bulky 3-chloro-2-methylbenzoic acid and clickable 3-prop-2-ynoxybenzoic acid are recognized by enlarging the substrate-binding pocket of the enzyme. When engineered EntE mutants were coupled with enterobactin and vibriobactin biosynthetic enzymes, 3-hydroxybenzoic acid-, salicylic acid-, and 3-bromo-2-fluorobenzoic acid-containing peptides were produced as early stage intermediates, highlighting the potential of NRP biosynthetic pathway engineering for constructing diverse aryl acid-containing metabolites.

Gibberellin and cytokinin signaling antagonistically control female-germline cell specification in Arabidopsis.

How do growth hormones interact to specify female-germline cell types in flowering plants and control production of the first female-germline cell? Here, we find that gibberellin (GA) biosynthesis and signaling are restricted in ovule primordia, with overexpression of receptors and biosynthetic enzymes resulting in multiple and enlarged megaspore mother cells (MMCs) in Arabidopsis. GA signaling machinery interacts with and promotes the degradation of cytokinin (CK) type-B Arabidopsis response regulators (ARR1/10/12), which also directly interact with DELLA proteins. CK biosynthesis and signaling components are expressed in both MMCs and sporophytic cells, with signaling negatively controlled by GA in ovule primordia, and perturbations leading to the induction of multiple, enlarged MMC-like cells. The vacuolar sorting protein SHRUBBY (SHBY) interacts with GA and CK signaling components to block GA-induced degradation. CK signaling restricts multiple sub-epidermal cells in distal ovule primordia from acquiring MMC identity. By balancing degradation activity, GA and CK signaling antagonistically control female-germline cell specification.

An Ionic Sensor acts in Parallel to dSarm to Promote Neurodegeneration.

How neurons to sense when they are terminally dysfunctional and activate neurodegeneration remains poorly defined. The pro-degenerative NAD + hydrolase dSarm/SARM1 can act as a metabolic sensor by detecting pathological changes in NAD + /NMN and subsequently induce catastrophic axon degeneration. Here we show Drosophila with-no-lysine kinase (dWnk), which can directly sense Cl - , K + and osmotic pressure, is required for neurodegeneration induced by depletion of the NAD + biosynthetic enzyme dNmnat. dWnk functions in parallel to dSarm and acts through the downstream kinase Frayed to promote axon degeneration and neuronal cell death. dWnk and dSarm ultimately converge on the BTB-Back domain molecule Axundead (Axed) to execute neurodegeneration. Our work argues that neurons use direct sensors of both metabolism (dSarm/SARM1) and ionic/osmotic status (dWnk) to evaluate cellular health and, when dysfunctional, promote neurodegeneration though a common axon death signaling molecule, Axundead.

A molecular module improves rice grain quality and yield at high temperature

Abstract Excessive temperatures during grain filling can compromise endosperm starch biosynthesis and decrease grain quality and yield in rice. However, the molecular mechanisms underlying these remain unclear. Here, we show that heat shock protein OsHsp40-1 interacts with and elevates the ATPase activity of OsHsp70-2 in rice. OsHsp40-1 also interacts with the key starch biosynthetic enzymes OsGBSSI and OsPPDKB and thereby enhances their stability and activity, which is essential for maintaining rice quality and grain yield under moderate high-temperature (HT) conditions. Overexpression of OsHsp70-2 and OsHsp40-1 in rice significantly improved grain quality and yield at HT. Furthermore, a haplotype analysis identified favorable alleles of OsHsp70-2 and OsHsp40-1, which could be used for improving thermotolerance in rice. Collectively, our findings reveal a novel mechanism by which OsHsp70-2–OsHsp40-1 module ameliorates the effects of HT on starch biosynthesis, providing a new strategy for genetic improvement of rice quality and yield under HT conditions.

Compensatory mechanisms that maintain androgen production in mice lacking key androgen biosynthetic enzymes

AbstractTestosterone and dihydrotestosterone (DHT) are essential for male development and fertility. In the canonical androgen production pathway, testosterone is produced in the testis by HSD17B3; however, adult male Hsd17b3 knockout (KO) mice continue to produce androgens and are fertile, indicating compensatory mechanisms exist. A second, alternate pathway produces DHT from precursors other than testosterone via 5α‐reductase (SRD5A) activity. We hypothesized that the alternate pathway contributes to androgen bioactivity in Hsd17b3 KO mice. To investigate contributions arising from and interactions between the canonical and alternate pathways, we pharmacologically inhibited SRD5A and ablated Srd5a1 (the predominant SRD5A in the testis) on the background of Hsd17b3 KO mice. Mice with perturbation of either the canonical or both pathways exhibited increased LH, testicular steroidogenic enzyme expression, and normal reproductive tracts and fertility. In the circulation, alternate pathway steroids were increased in the absence of HSD17B3 but were reduced by co‐inhibition of SRD5A1. Mice with perturbations of both pathways produced normal basal levels of intratesticular testosterone, suggesting the action of other unidentified hydroxysteroid dehydrogenase(s). Strikingly, testicular expression of another SRD5A enzyme, Srd5a2, was markedly increased in the absence of Hsd17b3, suggesting a compensatory increase in SRD5A2 to maintain androgen bioactivity during HSD17B3 deficiency. Finally, we observed elevated circulating concentrations of the 11‐keto‐derivative of DHT, suggesting compensatory extra‐gonadal induction of bioactive 11‐keto androgen production. Taken together, we conclude that, in the absence of the canonical pathway of androgen production, multiple intra‐ and extra‐gonadal mechanisms cooperate to maintain testosterone and DHT production, supporting male development and fertility.

Phenylpropanoid pathway mediated the defense response of ‘Korla’ fragrant pear against Alternaria alternata infection

Alternaria alternata has been found to be the dominating pathogenic fungus of harvested ‘Korla’ fragrant pear, and the resulting blackhead disease is a significant factor affecting the storage quality of pears. The present study explored the specific mechanisms by which the phenylpropanoid pathway mediates the defense response to A. alternata infection in pear fruit. In the A. alternata-inoculated group, the fruit exhibited increased activity and gene expression levels of key enzymes (PAL, C4H, and 4CL) as well as higher content of phenolic acids (trans-cinnamic acid, ferulic acid, caffeic acid, p-coumaric acid, and sinapic acid) and total phenol in the general phenylpropanoid pathway. In the mid-to-late storage period, the activity and gene expression levels of key enzymes in the lignin biosynthetic pathway (CCR and CAD) were suppressed, and the content of lignin monomers (sinapyl alcohol, coniferyl alcohol, and p-coumaryl alcohol) and lignin was reduced. Notably, the activity and gene expression levels of flavonoid biosynthetic pathway-related enzymes (CHS and CHI) as well as the content of various flavonoids (naringenin, apigenin, rutin, quercetin, and epicatechin) and total flavonoids continuously increased in response to A. alternata infection in the early to middle stages of storage but declined in the late storage period. In summary, the initial infection of A. alternata infection activated the stress response of pear fruit, particularly the phenylpropanoid–flavonoid branch pathway, to enhance the fruit’s defense against pathogens, but with the prolongation of the infestation time, the fruit could not continuously resist the invasion of pathogens, ultimately leading to the outbreak of disease. The present findings furnish a theoretical foundation further elucidating the interaction between ‘Korla’ fragrant pear and A. alternata and for developing an effective strategy to control the blackhead disease.

Exploring the Effect of Resveratrol, Tyrosol, and Their Derivatives on Platelet-Activating Factor Biosynthesis in U937 Cells.

Platelet-activating factor (PAF) is a potent lipid mediator, involved in thrombosis, inflammation, and atherosclerosis. The protective effect of wine and olive oil against atherosclerotic diseases is largely attributed to their phenolic compounds and mostly to resveratrol and tyrosol. Both compounds have been reported to inhibit PAF biosynthesis in interleukin-1β (IL-1β)-stimulated monocytes and also to attenuate PAF biosynthesis in cell lysates. The aim of this study was to investigate the effects of resveratrol, tyrosol, and their derivatives on unstimulated U937 cells and to explore the intracellular messaging pathways that participate in the activation of PAF biosynthesis in the same cell line. Tyrosol and its derivatives did not exert any substantial effect on PAF biosynthesis. Resveratrol (50 and 100 μM), as well as its methoxy derivative (5-20 μM), caused a reduction in the PAF biosynthetic enzymes' activity by 20-43% after 24 h of incubation. On the other hand, lower resveratrol concentration (10 μM) and higher concentration of the methoxy derivative (50 μM) increased the Ca2+-dependent lyso-PAF acetyltransferase (LysoPAF-ATC) activity by 28-45% after half-hour incubation via p38 mitogen-activated protein kinase (p38-MAPK) action. IL-1β activated PAF biosynthetic pathways via different signaling pathways, with phospholipase C-β (PLC-β) being a key enzyme.

Targeting Candida albicans O-acetyl-L-homoserine sulfhydrylase (Met15p) in antifungal treatment

Fungal infections are a serious threat to public health as they are becoming increasingly frequent. A major problem stems also from a rising fungal resistance to currently available antifungal therapies, therefore novel molecular targets are highly desirable. Exploration of enzymes participating in the biosynthesis pathways of essential amino acids such as L-methionine (L-Met) may provide new insights into pharmaceutical development. The MET15 gene from Candida albicans, encoding O-acetyl-L-homoserine sulfhydrylase (Met15p), an enzyme catalyzing the second step in that pathway, was cloned and expressed in two versions: as N and C-terminal oligo-His-tagged fusion proteins. The recombinant enzymes revealed appropriate activity, and catalyzed conversion of O-acetyl-L-homoserine and a sulfide ion to produce L-homocysteine. A new RP-HPLC-DAD method, using the enzymatic reaction product pre-column derivatization with 5,5’-dithio-bis-(2-nitrobenzoic acid) was developed and used by us to determine Met15p activity. Newly synthesized compounds as well as two commercially available exhibited a Met15p inhibitory effect which was related to antifungal activity. Fungal cells’ sensitivity to inhibitors depending on the presence or absence of L-Met in the medium clearly indicated Met15p targeting. Moreover, the synergistic effect of the first methionine biosynthetic enzyme affecting inhibitor and Met15p inhibitors indicate that methionine biosynthesis pathway enzymes are promising molecular targets.

Proteomic characterization of Mycobacterium tuberculosis subjected to carbon starvation.

Mycobacterium tuberculosis ( Mtb ) is the causative agent of tuberculosis (TB), the leading cause of infectious-disease related deaths worldwide. TB infections present as a spectrum from active to latent disease. In the human host, Mtb faces hostile environments, such as nutrient deprivation, hypoxia, and low pH. Under these conditions, Mtb can enter a dormant, but viable, state characterized by a lack of cell replication and increased resistance to antibiotics. These dormant Mtb pose a major challenge to curing infections and eradicating TB globally. In the current study, we subjected Mtb to carbon starvation (CS), a culture condition that induces growth stasis and mimics nutrient-starved conditions associated with dormancy in vivo . We provide a detailed analysis of the proteome in CS compared to replicating samples. We observed extensive proteomic reprogramming, with 36% of identified proteins significantly altered in CS. Many enzymes involved in oxidative phosphorylation and lipid metabolism were retained or upregulated in CS. The cell wall biosynthetic machinery was present in CS, although numerous changes in the abundance of peptidoglycan, arabinogalactan, and mycolic acid biosynthetic enzymes likely result in pronounced remodeling of the cell wall. Many clinically approved anti-TB drugs target cell wall biosynthesis, and we found that these enzymes were largely retained in CS. Lastly, we compared our results to those of other dormancy models and propose that CS produces a physiologically-distinct state of stasis compared to hypoxia in Mtb .

CNSC-68. SEX DIFFERENCES IN GLYCINE BIOSYNTHESIS DRIVE RESPONSES TO COMBINED NUTRIENT DEPRIVATION AND PHARMACOLOGICAL INHIBITION IN PEDIATRIC HGG

Abstract In children and adults, high-grade gliomas (HGGs) are the most common and deadly primary brain malignancies. Generally, male HGGs occur at higher incidence and demonstrate more rapid proliferation, resulting in shorter overall male survival. Obscurity of molecular mechanisms driving this phenomenon prevents implementation of clinically effective treatments. Recently, a positive correlation between HGG cellular proliferation and metabolic reprogramming toward glycine biosynthesis was identified. By analyzing pediatric and adult HGG patient data, we discovered significantly worse outcomes unique to pediatric males bearing upregulation of serine and glycine biosynthesis transcripts. Previously, we have identified differential flux of glucose into serine and glycine biosynthesis drives sex differences in lung cancer. Therefore, we sought to confirm and target this paradigm in our NF1-/- DNp53 model of HGG. By performing metabolic tracing with [13C]-labeled glucose in male and female cells, we identified higher enrichment of [13C]-labeled serine and glycine in male cells relative to female cells. Through Western blotting, we identify male-specific upregulation of serine and glycine biosynthetic pathway enzymes in transformed cells, supporting our labelling results. Next, we sought to determine if inhibition of de-novo serine and glycine biosynthesis was similarly sex-biased. Utilizing serine-and-glycine-deprived media conditions, we identified a male-biased decrease in proliferation following pharmacological inhibition of de-novo serine and glycine biosynthesis. More precisely, in the absence of exogenous glycine, we discovered that inhibition of de-novo synthesis of glycine from serine drove male-specific decreases in proliferation. Additionally, we identified that transformed female cell robustness against inhibition of glycine biosynthesis is driven by the uptake of exogenous nucleotide precursors. These data confirm that sex differences in de-novo glycine biosynthesis drive sex differences in HGG cellular proliferation. As well, our work suggests that dietary restriction of serine and glycine, in-combination with pharmacological inhibition of de-novo glycine biosynthesis, represents a novel treatment paradigm.

TMET-32. THE ROLE OF HOXC10 IN DRIVING GLIOBLASTOMA AGGRESSIVENESS

Abstract BACKGROUND Glioblastoma (GBM) is an aggressive malignancy, with a tumor doubling time of just 49.6 days. Despite maximal safe surgical resection, radiation, and temozolomide, median survival is only 14.6 months. Homeobox gene, homeobox C10 (HOXC10), a transcription factor of the HOX10 family, is pivotal in neuronal development, upregulated in several cancers, including glioma, and correlates with poor survival. METHODS RNA sequencing analysis was done on our mouse glioma syngeneic cell lines. HOXC10 expression analysis was done on human and mouse GBM cell lines and tissues by immunoblotting and immunohistochemistry, respectively. HOXC10 and p16-/-silencing were done using 4 unique 29mer shRNA constructs in the GFP vector, respectively. Gene Ontology predicted HOXC10 functions. HOXC10 and pyrroline-5-carboxylic acid reductase 1 (PYCR 1), a proline biosynthetic enzyme co-expression was studied using immunofluorescence analysis. RESULTS HOXC10 is highly expressed in human and mouse GBM tissues. HOXC10 was upregulated only in the high-grade/aggressive cell line [EGFRvIII; p16-/- & GFAP Cre] and demonstrated increased expression with p16 deletion in the mouse glioma cell line. Compellingly, TCGA analysis of GBM correlated the negatively prognostic p16 homozygous deletion (HD) with HOXC10 upregulation. Gene Ontology supports the role of HOXC10 for proline biosynthesis and metabolism. Immunofluorescence on mouse GBM sections revealed co-expression of HOXC10 and PYCR1. HOXC10 silencing significantly decreased GBM cell line proliferation, induced apoptosis, and decreased PYCR1 expression in vitro. CONCLUSIONS HOXC10 expression is increased in GBM and with p16 HD. HOXC10 silencing significantly decreased GBM cell line proliferation and decreased PYCR1 expression. HOXC10-mediated proline biosynthesis may present a novel target to improve GBM survival.

Hydroxy-wybutosine tRNA modifications as indicators of disease progression and therapeutic targets in leukaemia.

Therapeutic approaches for acute myeloid leukaemia (AML) and myelodysplastic syndromes (MDS) differ due to distinct diagnostic criteria and treatment strengths. However, reliable biomarkers to differentiate AML from MDS are needed. This study investigated transfer RNA (tRNA) modifications, particularly hydroxy-wybutosine (OHyW), in the transition from MDS to AML. We found a significant decrease in OHyW and its biosynthetic enzyme leucine carboxyl methyltransferase 2 (LCMT2, alias symbol is TYW4) levels in AML compared to MDS. Mass spectrometric analysis revealed distinct tRNA modification patterns, with AML showing decreased OHyW and increased precursor levels, indicating a disrupted biosynthetic pathway. Lower LCMT2 expression correlated with reduced drug sensitivity and limited differentiation potential in AML cell lines. The results highlight the pivotal role of tRNA modifications in the progression from MDS to AML and suggest that targeting LCMT2 may enhance therapeutic outcomes in AML. By understanding these molecular mechanisms, we can develop new diagnostic markers and therapeutic strategies, potentially transforming the clinical management of AML and improving patient outcomes.

Continuous evolution of user-defined genes at 1 million times the genomic mutation rate.

When nature evolves a gene over eons at scale, it produces a diversity of homologous sequences with patterns of conservation and change that contain rich structural, functional, and historical information about the gene. However, natural gene diversity accumulates slowly and likely excludes large regions of functional sequence space, limiting the information that is encoded and extractable. We introduce upgraded orthogonal DNA replication (OrthoRep) systems that radically accelerate the evolution of chosen genes under selection in yeast. When applied to a maladapted biosynthetic enzyme, we obtained collections of extensively diverged sequences with patterns that revealed structural and environmental constraints shaping the enzyme's activity. Our upgraded OrthoRep systems should support the discovery of factors influencing gene evolution, uncover previously unknown regions of fitness landscapes, and find broad applications in biomolecular engineering.

Yeast Synthesis and Herbicidal Activity Evaluation of Aspterric Acid.

Aspterric acid (AA) is a novel natural product herbicide that targets dihydroxyacid dehydratase in plants. In this study, we introduced two distinct AA biosynthesis-related gene clusters into Saccharomyces cerevisiae and screened the core biosynthetic enzyme, sesquiterpene cyclase, from various fungi. The combination of sesquiterpene cyclase from Aspergillus taichungensis IBT 19404 and two cytochrome P450s from Penicillium brasilianum resulted in the optimal AA synthesis efficiency in yeast, with the highest titer of 33.21 mg/L achieved by optimizing fermentation conditions in shake flasks. Moreover, the herbicidal effects of AA on weed germination and growth were evaluated. Notably, AA strongly inhibited the germination of Amaranthus tricolor, Portulaca oleracea, Bidens pilosa, Lolium perenne, and Leptochloa chinensis. Furthermore, AA could also inhibit the shoot and root growth of weeds with a superior inhibitory effect on roots relative to shoots. Our work not only provides a sustainable method for the biosynthesis of AA in yeast but also paves the way for the application of AA as a preemergence herbicide.

Genome-wide characterization of melatonin biosynthetic pathway genes in carnation (Dianthus caryophyllus L.) and their expression analysis in response to exogenous melatonin

Carnation (Dianthus caryophyllus L.) is a prominent floricultural crop valued for its diverse colors, offering significant economic and ornamental value globally. However, the global demand for its flowers makes flower yield an important attribute that relies on the quantity of lateral branches in the crop. Melatonin as a multi-regulatory phytohormone play vital functions in governing plant growth and development. It is synthesized from tryptophan via four key enzymes. Tryptophan decarboxylase (TDC), Tryptamine 5-hydroxylase (T5H), Serotonin N-acetyltransferase (SNAT), and N-Acetylserotonin O-methyltransferase (ASMT). Although the significance of melatonin is recognized, its impact on the growth and development of carnation remains understudied. In the current study, we investigated the effect of exogenous melatonin at different concentrations, on growth pattern of carnation, followed by genome-wide characterization, in-silico analysis and expression profiling of melatonin biosynthetic pathway genes. Results showed increased branching and reduced height with increased melatonin concentrations up to a point. In-silico analysis identified ten genes in the melatonin biosynthetic pathway, including two TDC, two T5H, one SNAT, and five ASMT members. Domain analysis confirmed the presence of characteristic domains such as pyridoxal-dependent decarboxylase, cytochrome P450, Acetyltransferase_1, and O-methyltransferase. Physiochemical properties, gene structure, conserved motifs, promoter regions, gene ontology, synteny, and evolutionary relationships through phylogeny were also analysed. Sub-cellular localization predictions showed distribution of melatonin biosynthetic enzymes across various cellular compartments. Expression analysis of these genes under different exogenous melatonin concentrations (100, 200, 300, 400, 500, and 1000 µM) revealed significant upregulation at 100 µM and 500 µM, while no change was observed at 1000 µM. These findings suggest that optimal exogenous melatonin concentrations enhance the expression of biosynthetic pathway genes ultimately led to increased branching in carnation due to increased endogenous melatonin levels. This study establishes a basis for future functional characterization of melatonin biosynthetic pathway genes to elucidate their roles in carnation growth and development.

Targeting NAD+ Metabolism Vulnerability in FH-Deficient Hereditary Leiomyomatosis and Renal Cell Carcinoma with the novel NAMPT Inhibitor OT-82.

Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) is an inherited cancer syndrome caused by germline pathogenic variants in the fumarate hydratase (FH) gene. Affected individuals are at risk for developing cutaneous and uterine leiomyomas and aggressive FH-deficient renal cell carcinoma (RCC) with a papillary histology. Due to a disrupted TCA cycle, FH-deficient kidney cancers rely on aerobic glycolysis for energy production, potentially creating compensatory metabolic vulnerabilities. This study conducted a high-throughput drug screen in HLRCC cell lines, which identified a critical dependency on nicotinamide adenine dinucleotide (NAD), a redox cofactor produced by the biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT). Human HLRCC tumors and HLRCC-derived cell lines exhibited elevated NAMPT expression compared to controls. FH-deficient HLRCC cells, but not FH-restored HLRCC or normal kidney cells, were sensitive to NAMPT inhibition. HLRCC cell line viability was significantly decreased in both 2D and 3D in vitro cultures in response to the clinically relevant NAMPT inhibitor OT-82. NAMPT inhibition in vitro significantly decreased the total amount of NAD+, NADH, NADP, NADPH, and PAR levels and the effects of NAMPT inhibition could be rescued by the downstream NAD precursor nicotinamide mononucleotide, confirming the on-target activity of OT-82. Moreover, NAMPT inhibition by OT-82 in two HLRCC xenograft models resulted in severely reduced tumor growth. OT-82 treatment of HLRCC xenograft tumors in vivo inhibited glycolytic flux as demonstrated by reduced lactate/pyruvate ratio in hyperpolarized 13C-pyruvate magnetic resonance spectroscopic imaging experiments. Overall, our data define NAMPT inhibition as a potential therapeutic approach for FH-deficient HLRCC-associated renal cell carcinoma.

An ABA biosynthesis enzyme gene OsNCED4 regulates NaCl and cold stress tolerance in rice

Rice (Oryza sativa L.) is susceptible to various abiotic stresses, such as salt, cold, and drought. Therefore, there is an urgent need to explore the relevant genes that enhance tolerance to these stresses. In this study, we identified a gene, OsNCED4 (9-cis-epoxycarotenoid dioxygenase 4), which regulates tolerance to multiple abiotic stresses. OsNCED4 encodes a chloroplast-localized abscisic acid (ABA) biosynthetic enzyme. The expression of OsNCED4 gene was significantly induced by 150 mM NaCl and cold stress. Disruption of OsNCED4 by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9-mediated mutagenesis resulted in significant sensitivity to NaCl and cold stress. The salt and cold sensitivity of osnced4 mutant was due to the reduction of ABA content and the excessive accumulation of reactive oxygen species (ROS) under stress. Moreover, OsNCED4 also regulates drought stress tolerance of rice seedlings. Taken together, these results indicate that OsNCED4 is a new regulator for multiple abiotic stress tolerance in rice, and provided a potential target gene for enhancing multiple stress tolerance in the future.

Combatting melioidosis with chemical synthetic lethality

Burkholderia thailandensis has emerged as a nonpathogenic surrogate for Burkholderia pseudomallei, the causative agent of melioidosis, and an important Gram-negative model bacterium for studying the biosynthesis and regulation of secondary metabolism. We recently reported that subinhibitory concentrations of trimethoprim induce vast changes in both the primary and secondary metabolome of B. thailandensis. In the current work, we show that the folate biosynthetic enzyme FolE2 is permissive under standard growth conditions but essential for B. thailandensis in the presence of subinhibitory doses of trimethoprim. Reasoning that FolE2 may serve as an attractive drug target, we screened for and identified ten inhibitors, including dehydrocostus lactone (DHL), parthenolide, and β-lapachone, all of which are innocuous individually but form a chemical-synthetic lethal combination with subinhibitory doses of trimethoprim. We show that DHL is a mechanism-based inhibitor of FolE2 and capture the structure of the covalently inhibited enzyme using X-ray crystallography. In vitro, the combination of subinhibitory trimethoprim and DHL is more potent than Bactrim, the current standard of care against melioidosis. Moreover, unlike Bactrim, this combination does not affect the growth of most commensal and beneficial gut bacteria tested, thereby providing a degree of specificity against B. pseudomallei. Our work provides a path for identifying antimicrobial drug targets and for utilizing binary combinations of molecules that form a toxic cocktail based on metabolic idiosyncrasies of specific pathogens.