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Interventions that combat obesity and its associated metabolic perturbations may decrease incidence and improve outcomes of endometrial cancer (EC). Potential options for weight loss include pharmacotherapeutic interventions such as tirzepatide, a dual-acting glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP) receptor agonist. Given this, we explored the anti-obesity and anti-tumorigenic effects of tirzepatide in our pre-clinical mouse model of endometrioid EC. Starting at 4 weeks of age, Lkb1 fl/fl p53 fl/fl mice were fed a low-fat diet vs a high-fat diet to generate a lean or obese phenotype. Nine weeks after induction of EC, obese and lean mice were randomized to receive tirzepatide for 4 weeks. Body and tumor weights, tumor transcriptomic and metabolomic profiles, and serum metabolic markers and chemokines were assessed. Both obese and lean mice began to lose body weight after 2 weeks of tirzepatide treatment, ultimately achieving a significant weight loss of 20.1 % in obese mice and 16.8 % in lean mice. Tirzepatide improved obesity-induced serum adiponectin, leptin, GIP, and C-reactive protein levels. Furthermore, tirzepatide relative to vehicle, effectively reduced tumor growth in obese and lean mice, inhibited the ErbB signaling and glycolysis/gluconeogenesis in tumors of obese mice, and increased O-linked glycosylation biosynthesis and phospholipase D signaling in tumors of lean mice. Tirzepatide decreased both mouse weight and tumor growth via effects on metabolic and immune pathways in the EC tumors that differed between obese and lean mice. This novel weight loss treatment deserves further evaluation as an innovative strategy in the management of EC. • Tirzepatide inhibited body weight and tumor growth in obese and lean LKB1 fl/fl p53 fl/fl mice. • Tirzepatide reversed the metabolic disturbances associated with obesity such as glycolysis and fatty acid metabolism. • Tirzepatide improved serum metabolic hormones and inflammatory markers in obese LKB1 fl/fl p53 fl/fl mice. • The antitumor activity of tirzepatide may rely on distinct mechanisms controlling tumor metabolism in obese and lean mice. [ABSTRACT FROM AUTHOR]

Heme is an essential cofactor for multiple cellular processes in most organisms. In developing erythroid cells, the demand for heme synthesis is high, but is significantly lower in non-erythroid cells. While the biosynthesis of heme in metazoans is well understood, the tissue-specific regulation of the pathway is less explored. To better understand this, we analyzed the mitochondrial heme metabolon in erythroid and non-erythroid cell lines from the perspective of ferrochelatase (FECH), the terminal enzyme in the heme biosynthetic pathway. Affinity purification of FLAG-tagged-FECH, together with mass spectrometric analysis, was carried out to identify putative protein partners in human and murine cell lines. Proteins involved in the heme biosynthetic process and mitochondrial organization were identified as the core components of the FECH interactome. Interestingly, in non-erythroid cell lines, the FECH interactome is highly enriched with proteins associated with the tricarboxylic acid (TCA) cycle. Overall, our study shows that the mitochondrial heme metabolon in erythroid and non-erythroid cells has similarities and differences, and suggests new roles for the mitochondrial heme metabolon and heme in regulating metabolic flux and key cellular processes.

SUMMARY: Flavan‐3‐ols are prominent phenolic compounds found abundantly in the young leaves of tea plants. The enzymes involved in flavan‐3‐ol biosynthesis in tea plants have been extensively investigated. However, the localization and associations of these numerous functional enzymes within cells have been largely neglected. In this study, we aimed to investigate the synthesis of flavan‐3‐ols in tea plants, particularly focusing on epigallocatechin gallate. Our analysis involving the DESI‐MSI method to reveal a distinct distribution pattern of B‐ring trihydroxylated flavonoids, primarily concentrated in the outer layer of buds. Subcellular localization showed that CsC4H, CsF3′H, and CsF3′5′H localizes endoplasmic reticulum. Protein–protein interaction studies demonstrated direct associations between CsC4H, CsF3′H, and cytoplasmic enzymes (CHS, CHI, F3H, DFR, FLS, and ANR), highlighting their interactions within the biosynthetic pathway. Notably, CsF3′5′H, the enzyme for B‐ring trihydroxylation, did not directly interact with other enzymes. We identified cytochrome b5 isoform C serving as an essential redox partner, ensuring the proper functioning of CsF3′5′H. Our findings suggest the existence of distinct modules governing the synthesis of different B‐ring hydroxylation compounds. This study provides valuable insights into the mechanisms underlying flavonoid diversity and efficient synthesis and enhances our understanding of the substantial accumulation of B‐ring trihydroxylated flavan‐3‐ols in tea plants. Significance Statement: Our work reveals the mechanism of flavonoid diversity and efficient synthesis in tea plants from the perspective of metabolon. Different metabolons modules provide a new perspective on the scientific problem of why tea plants accumulate high levels of EGCG. The discovery of obligate electron shuttle proteins provides new directions for improving metabolic engineering. [ABSTRACT FROM AUTHOR]

Chalcone synthase (CHS) and chalcone isomerase (CHI) catalyze the first two committed steps of the flavonoid pathway that plays a pivotal role in the growth and reproduction of land plants, including UV protection, pigmentation, symbiotic nitrogen fixation, and pathogen resistance. Based on the obtained X-ray crystal structures of CHS, CHI, and chalcone isomerase-like protein (CHIL) from the same monocotyledon, Panicum virgatum, along with the results of the steady-state kinetics, spectroscopic/thermodynamic analyses, intermolecular interactions, and their effect on each catalytic step are proposed. In addition, PvCHI's unique activity for both naringenin chalcone and isoliquiritigenin was analyzed, and the observed hierarchical activity for those type-I and -II substrates was explained with the intrinsic characteristics of the enzyme and two substrates. The structure of PvCHS complexed with naringenin supports uncompetitive inhibition. PvCHS displays intrinsic catalytic promiscuity, evident from the formation of p-coumaroyltriacetic acid lactone (CTAL) in addition to naringenin chalcone. In the presence of PvCHIL, conversion of p-coumaroyl-CoA to naringenin through PvCHS and PvCHI displayed ~400-fold increased Vmax with reduced formation of CTAL by 70%. Supporting this model, molecular docking, ITC (Isothermal Titration Calorimetry), and FRET (Fluorescence Resonance Energy Transfer) indicated that both PvCHI and PvCHIL interact with PvCHS in a non-competitive manner, indicating the plausible allosteric effect of naringenin on CHS. Significantly, the presence of naringenin increased the affinity between PvCHS and PvCHIL, whereas naringenin chalcone decreased the affinity, indicating a plausible feedback mechanism to minimize spontaneous incorrect stereoisomers. These are the first findings from a three-body system from the same species, indicating the importance of the macromolecular assembly of CHS-CHI-CHIL in determining the amount and type of flavonoids produced in plant cells. [ABSTRACT FROM AUTHOR]

Soybean (Glycine max) produces a class of phenylalanine (Phe) derived specialized metabolites, isoflavonoids. Isoflavonoids are unique to legumes and are involved in defense responses in planta, and they are also necessary for nodule formation with nitrogen-fixing bacteria. Since Phe is a precursor of isoflavonoids, it stands to reason that the synthesis of Phe is coordinated with isoflavonoid production. Two putative AROGENATE DEHYDRATASE (ADT) isoforms were previously co-purified with the soybean isoflavonoid metabolon anchor ISOFLAVONE SYNTHASE2 (GmIFS2), however the GmADT family had not been characterized. Here, we present the identification of the nine member GmADT family. We determined that the GmADTs share sequences required for enzymatic activity and allosteric regulation with other characterized plant ADTs. Furthermore, the GmADTs are differentially expressed, and multiple members have dual substrate specificity, also acting as PREPHENATE DEHYDRATASES. All GmADT isoforms were detected in the stromules of chloroplasts, and they all interact with GmIFS2 in the cytosol. In addition, GmADT12A interacts with multiple other isoflavonoid metabolon members. These data substantiate the involvement of GmADT isoforms in the isoflavonoid metabolon. [ABSTRACT FROM AUTHOR]

Isotope dilution ultrahigh‐performance liquid chromatography coupled to tandem mass spectrometry (UHPLC–MS/MS) is commonly used for trace analysis of polyfluoroalkyl and perfluoroalkyl substances (PFAS) in difficult matrices. Commercial nontargeted analysis of major PFAS where relative concentrations are obtained cost effectively is rapidly emerging and is claimed to provide comparable results to that of absolute quantification using matrix matched calibration and isotope dilution UHPLC–MS/MS. However, this remains to be demonstrated on a large scale. We aimed to assess the performance of a targeted absolute quantification isotope dilution LC–MS/MS assay versus a commercial nontargeted relative quantification assay for detection of three major PFAS in human blood. We evaluated a population‐based cohort of 503 individuals. Correlations were assessed using Spearman's rank correlation coefficients (rho). Precision and bias were assessed using Bland–Altman plots. For perfluorooctane sulfonic acid, the median concentrations were 5.10 ng/mL (interquartile range [IQR] 3.50–7.24 ng/mL), the two assays correlated with rho 0.83. For perfluorooctanoic acid, the median concentrations were 2.14 ng/mL (IQR 1.60–3.0 ng/mL), the two assays correlated with rho 0.92. For perfluorohexanesulfonate, the median concentrations were 5.5 ng/mL (IQR 2.50–11.61 ng/mL), the two assays correlated with rho 0.96. The Bland–Altman statistical test showed agreement of the mean difference for the majority of samples (97–98%) between the two assays. Absolute plasma concentrations of PFAS obtained using matrix matched calibration and isotope dilution UHPLC–MS/MS show agreement with relative plasma concentrations from a nontargeted commercial platform by Metabolon. We observed striking consistency between the two assays when examining the associations of the three PFAS with cholesterol, offering additional confidence in the validity of utilizing the nontargeted approach for correlations with various health phenotypes. [ABSTRACT FROM AUTHOR]

Soybean (Glycine max) produces a class of phenylalanine (Phe) derived specialized metabolites, isoflavonoids. Isoflavonoids are unique to legumes and are involved in defense responses in planta, and they are also necessary for nodule formation with nitrogen-fixing bacteria. Since Phe is a precursor of isoflavonoids, it stands to reason that the synthesis of Phe is coordinated with isoflavonoid production. Two putative AROGENATE DEHYDRATASE (ADT) isoforms were previously co-purified with the soybean isoflavonoid metabolon anchor ISOFLAVONE SYNTHASE2 (GmIFS2), however the GmADT family had not been characterized. Here, we present the identification of the nine member GmADT family. We determined that the GmADTs share sequences required for enzymatic activity and allosteric regulation with other characterized plant ADTs. Furthermore, the GmADTs are differentially expressed, and multiple members have dual substrate specificity, also acting as PREPHENATE DEHYDRATASES. All GmADT isoforms were detected in the stromules of chloroplasts, and they all interact with GmIFS2 in the cytosol. In addition, GmADT12A interacts with multiple other isoflavonoid metabolon members. These data substantiate the involvement of GmADT isoforms in the isoflavonoid metabolon.

Summary: Plant lipids have essential biological roles in plant development and stress responses through their functions in cell membrane formation, energy storage and signalling. Vegetable oil, which is composed mainly of the storage lipid triacylglycerol, also has important applications in food, biofuel and oleochemical industries. Lipid biosynthesis occurs in multiple subcellular compartments and involves the coordinated action of various pathways. Although biochemical and molecular biology research over the last few decades has identified many proteins associated with lipid metabolism, our current understanding of the dynamic protein interactomes involved in lipid biosynthesis, modification and channelling is limited. This review examines advances in the identification and characterization of protein interactomes involved in plant lipid biosynthesis, with a focus on protein complexes consisting of different subunits for sequential reactions such as those in fatty acid biosynthesis and modification, as well as transient or dynamic interactomes formed from enzymes in cooperative pathways such as assemblies of membrane‐bound enzymes for triacylglycerol biosynthesis. We also showcase a selection of representative protein interactome structures predicted using AlphaFold2, and discuss current and prospective strategies involving the use of interactome knowledge in plant lipid biotechnology. Finally, unresolved questions in this research area and possible approaches to address them are also discussed. [ABSTRACT FROM AUTHOR]

SUMMARY: Ammonium in the soil is converted into nitrate by nitrifying bacteria or archaea. While nitrate is readily available for plants, it is prone to leaching and contributes to eutrophication. In addition, when the soil conditions become anaerobic, nitrate can be reduced to nitrous oxide, a powerful greenhouse gas. Therefore, slowing nitrification in agricultural soil offers some benefits by reducing nitrogen loss and decreasing water and air pollution. Since nitrogen is a limiting nutrient for most ecological niches, many plants have evolved specialized compounds that reduce nitrification. One such compound, sorgoleone, which is secreted from the root hair of sorghum, has been relatively well studied due to its allelopathic function, with most enzymes involved in its biosynthesis elucidated. However, the secretion mechanisms remain unknown. Previous studies reported numerous lipidic vesicles in the sorghum root hair and speculated that they are involved in sorgoleone storage or secretion, but their roles remain unclear. Also, the subcellular organelles that are involved in sorgoleone synthesis have not been identified. In the present study, we found that the expression of sorgoleone biosynthesis enzymes is induced in a specific root zone, indicating that the secretion is developmentally regulated. The accumulation of internal vesicles preceded the peak of sorgoleone biosynthesis and secretion, indicating that the vesicles play a role in precursor storage rather than secretion. Moreover, our data suggest that enzymes that catalyze the first three steps, SbDES2, SbDES3, and SbARS1, interact with each other to form a multi‐enzyme complex on the endoplasmic reticulum surface. Significance Statement: This work clarifies the subcellular location and the timing of the biosynthesis of sorgoleone, a phytochemical that functions as a nitrification inhibitor as well as an allelopathic chemical. The results suggest that sorgoleone synthesis happens within a multi‐enzyme complex located in the endoplasmic reticulum at a specific developmental stage. [ABSTRACT FROM AUTHOR]

SUMMARY: Metabolons are temporary structural‐functional complexes of sequential enzymes of a metabolic pathway that are distinct from stable multi‐enzyme complexes. Here we provide a brief history of the study of enzyme–enzyme assemblies with a particular focus on those that mediate substrate channeling in plants. Large numbers of protein complexes have been proposed for both primary and secondary metabolic pathways in plants. However, to date only four substrate channels have been demonstrated. We provide an overview of current knowledge concerning these four metabolons and explain the methodologies that are currently being applied to unravel their functions. Although the assembly of metabolons has been documented to arise through diverse mechanisms, the physical interaction within the characterized plant metabolons all appear to be driven by interaction with structural elements of the cell. We therefore pose the question as to what methodologies could be brought to bear to enhance our knowledge of plant metabolons that assemble via different mechanisms? In addressing this question, we review recent findings in non‐plant systems concerning liquid droplet phase separation and enzyme chemotaxis and propose strategies via which such metabolons could be identified in plants. We additionally discuss the possibilities that could be opened up by novel approaches based on: (i) subcellular‐level mass spectral imaging, (ii) proteomics, and (iii) emergent methods in structural and computational biology. Significance Statement: Metabolite channeling represents an important method by which metabolic flux can be regulated at branchpoints. However, despite considerable interest only four metabolite channels have been categorically demonstrated to date. Here we review the evidence for their operation and detail contemporary and emerging technologies that will aid in their detection and characterization. [ABSTRACT FROM AUTHOR]

Operons represent one of the leading strategies of gene organization in prokaryotes, having a crucial influence on the regulation of gene expression and on bacterial chromosome organization. However, there is no consensus yet on why, how, and when operons are formed and conserved, and many different theories have been proposed. Histidine biosynthesis is a highly studied metabolic pathway, and many of the models suggested to explain operons origin and evolution can be applied to the histidine pathway, making this route an attractive model for the study of operon evolution. Indeed, the organization of his genes in operons can be due to a progressive clustering of biosynthetic genes during evolution, coupled with a horizontal transfer of these gene clusters. The necessity of physical interactions among the His enzymes could also have had a role in favoring gene closeness, of particular importance in extreme environmental conditions. In addition, the presence in this pathway of paralogous genes, heterodimeric enzymes and complex regulatory networks also support other operon evolution hypotheses. It is possible that histidine biosynthesis, and in general all bacterial operons, may result from a mixture of several models, being shaped by different forces and mechanisms during evolution. [ABSTRACT FROM AUTHOR]

The sporopollenin polymer is a major component of the pollen exine. Fatty acid derivatives synthesized in the tapetum are among the precursors of sporopollenin. Progress has been made to understand sporopollenin metabolism in rice; however, the underlying molecular mechanisms remain elusive. We found that OsTKPR2 and OsTKPR1 share a similar expression pattern, and their coding proteins have a similar subcellular localization and enzyme activities towards reduced tetraketide α-pyrone and hydroxylated tetraketide α-pyrone. Unexpectedly, OsTKPR1 pro : OsTKPR2-eGFP could not rescue the phenotype of ostkpr1-4. Three independent ostkpr2 mutant lines generated by CRISPR/Cas9 displayed reduced male fertility to various extents which were correlated with the severity of gene disruptions. Notably, the anther cuticle, Ubisch bodies, and pollen development were affected in the ostkpr2-1 mutant, where a thinner pollen exine was noticed. OsTKPR1 and OsTKPR2 were integrated into a metabolon including OsACOS12 and OsPKS2, which resulted in a significant increased enzymatic efficiency when both OsTKPR1 and OsTKPR2 were present, indicating the mutual dependence of OsTKPR2 and OsTKPR1 for their full biochemical activities. Thus, our results demonstrated that OsTKPR2 is required for anther and pollen development where an OsTKPR2-containing metabolon is functional during rice sporopollenin synthesis. Furthermore, the cooperation and possible functional divergence between OsTKPR2 and OsTKPR1 is also discussed. [ABSTRACT FROM AUTHOR]

Heme is an essential cofactor for multiple cellular processes in most organisms. In developing erythroid cells, the demand for heme synthesis is high, but is significantly lower in non-erythroid cells. While the biosynthesis of heme in metazoans is well understood, the tissue-specific regulation of the pathway is less explored. To better understand this, we analyzed the mitochondrial heme metabolon in erythroid and non-erythroid cell lines from the perspective of ferrochelatase (FECH), the terminal enzyme in the heme biosynthetic pathway. Affinity purification of FLAG-tagged-FECH, together with mass spectrometric analysis, was carried out to identify putative protein partners in human and murine cell lines. Proteins involved in the heme biosynthetic process and mitochondrial organization were identified as the core components of the FECH interactome. Interestingly, in non-erythroid cell lines, the FECH interactome is highly enriched with proteins associated with the tricarboxylic acid (TCA) cycle. Overall, our study shows that the mitochondrial heme metabolon in erythroid and non-erythroid cells has similarities and differences, and suggests new roles for the mitochondrial heme metabolon and heme in regulating metabolic flux and key cellular processes. [ABSTRACT FROM AUTHOR]

White Kwao Krua (Pueraria candollei var. mirifica), a Thai medicinal plant, is a rich source of phytoestrogens, especially isoflavonoids and chromenes. These phytoestrogens are well known; however, their biosynthetic genes remain largely uncharacterized. Cytochrome P450 (P450) is a large protein family that plays a crucial role in the biosynthesis of various compounds in plants, including phytoestrogens. Thus, we focused on P450s involved in the isoflavone hydroxylation that potentially participates in the biosynthesis of miroestrol. Three candidate P450s were isolated from the transcriptome libraries by considering the phylogenetic and expression data of each tissue of P. mirifica. The candidate P450s were functionally characterized both in vitro and in planta. Accordingly, the yeast microsome harboring PmCYP81E63 regiospecifically exhibited either 2′ or 3′ daidzein hydroxylation and genistein hydroxylation. Based on in silico calculation, PmCYP81E63 had higher binding energy with daidzein than with genistein, which supported the in vitro result of the isoflavone specificity. To confirm in planta function, the candidate P450s were then transiently co-expressed with isoflavone-related genes in Nicotiana benthamiana. Despite no daidzein in the infiltrated N. benthamiana leaves, genistein and hydroxygenistein biosynthesis were detectable by liquid Chromatography with tandem mass spectrometry (LC-MS/MS). Additionally, we demonstrated that PmCYP81E63 interacted with several enzymes related to isoflavone biosynthesis using bimolecular fluorescence complementation studies and a yeast two-hybrid analysis, suggesting a scheme of metabolon formation in the pathway. Our findings provide compelling evidence regarding the involvement of PmCYP81E63 in the early step of the proposed miroestrol biosynthesis in P. mirifica. [ABSTRACT FROM AUTHOR]

Glycolysis is a highly conserved metabolic pathway responsible for the anaerobic production of adenosine triphosphate (ATP) from the breakdown of glucose molecules. While serving as a primary metabolic pathway in prokaryotes, glycolysis is also utilized by respiring eukaryotic cells, providing pyruvate to fuel oxidative metabolism. Furthermore, glycolysis is the primary source of ATP production in multiple cellular states (e.g., hypoxia) and is particularly important in maintaining bioenergetic homeostasis in the most abundant cell type in the human body, the erythrocyte. Beyond its role in ATP production, glycolysis also functions as a signaling hub, producing several metabolic intermediates which serve roles in both signaling and metabolic processes. These signals emanating from the glycolytic pathway can profoundly impact cell function, phenotype, and fate and have previously been overlooked. In this review, we will discuss the role of the glycolytic pathway as a source of signaling molecules in eukaryotic cells, emphasizing the newfound recognition of glycolysis' multifaceted nature and its importance in maintaining cellular homeostasis, beyond its traditional role in ATP synthesis., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

This review discusses the intriguing yet controversial concept of metabolons, focusing on the malate dehydrogenase-citrate synthase (MDH-CISY) metabolon as a model. Metabolons are multienzyme complexes composed of enzymes that catalyze sequential reactions in metabolic pathways. Metabolons have been proposed to enhance metabolic pathway efficiency by facilitating substrate channeling. However, there is skepticism about the presence of metabolons and their functionality in physiological conditions in vivo. We address the skepticism by reviewing compelling evidence supporting the existence of the MDH-CISY metabolon and highlighting its potential functions in cellular metabolism. The electrostatic interaction between MDH and CISY and the intermediate oxaloacetate, channeled within the metabolon, has been demonstrated using various experimental techniques, including protein-protein interaction assays, isotope dilution studies, and enzyme coupling assays. Regardless of the wealth of in vitro evidence, further validation is required to elucidate the functionality of MDH-CISY metabolons in living systems using advanced structural and spatial analysis techniques., (© 2024 The Author(s).)

Metabolism within an organism is regulated by various processes, including post-translational modifications (PTMs). These types of chemical modifications alter the molecular, biochemical, and cellular properties of proteins and allow the organism to respond quickly to different environments, energy states, and stresses. Malate dehydrogenase (MDH) is a metabolic enzyme that is conserved in all domains of life and is extensively modified post-translationally. Due to the central role of MDH, its modification can alter metabolic flux, including the Krebs cycle, glycolysis, and lipid and amino acid metabolism. Despite the importance of both MDH and its extensively post-translationally modified landscape, comprehensive characterization of MDH PTMs, and their effects on MDH structure, function, and metabolic flux remains underexplored. Here, we review three types of MDH PTMs - acetylation, ADP-ribosylation, and methylation - and explore what is known in the literature and how these PTMs potentially affect the 3D structure, enzymatic activity, and interactome of MDH. Finally, we briefly discuss the potential involvement of PTMs in the dynamics of metabolons that include MDH., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Metabolite levels measured in the human population are endophenotypes for biological processes. We combined sequencing data for 3,924 (whole-exome sequencing, WES, discovery) and 2,805 (whole-genome sequencing, WGS, replication) donors from a prospective cohort of blood donors in England. We used multiple approaches to select and aggregate rare genetic variants (minor allele frequency [MAF] < 0.1%) in protein-coding regions and tested their associations with 995 metabolites measured in plasma by using ultra-high-performance liquid chromatography–tandem mass spectrometry. We identified 40 novel associations implicating rare coding variants (27 genes and 38 metabolites), of which 28 (15 genes and 28 metabolites) were replicated. We developed algorithms to prioritize putative driver variants at each locus and used mediation and Mendelian randomization analyses to test directionality at associations of metabolite and protein levels at the ACY1 locus. Overall, 66% of reported associations implicate gene targets of approved drugs or bioactive drug-like compounds, contributing to drug targets' validating efforts. [Display omitted] [ABSTRACT FROM AUTHOR]

Over the past fifteen years, we have unveiled a new mechanism by which cells achieve greater efficiency in de novo purine biosynthesis. This mechanism relies on the compartmentalization of de novo purine biosynthetic enzymes into a dynamic complex called the purinosome. In this review, we highlight our current understanding of the purinosome with emphasis on its biophysical properties and function and on the cellular mechanisms that regulate its assembly. We propose a model for functional purinosomes in which they consist of at least ten enzymes that localize near mitochondria and carry out de novo purine biosynthesis by metabolic channeling. We conclude by discussing challenges and opportunities associated with studying the purinosome and analogous metabolons. [ABSTRACT FROM AUTHOR]

Simple Summary: Emerging evidence shows that the organization of the cells into membraneless sub-compartments confers unique properties to cancer cells. The biochemical and biophysical mechanisms underpinning the formation of these compartments, also called biological condensates, enlighten how some mutations and gene expression changes contribute to cancer formation and progression. The cancer state is thought to be maintained by genetic and epigenetic changes that drive a cancer-promoting gene expression program. However, recent results show that cellular states can be also stably maintained by the reorganization of cell structure leading to the formation of biological condensates via the process of liquid–liquid phase separation. Here, we review the data showing cancer-specific biological condensates initiated by mutant oncoproteins, RNA-binding proteins, or lincRNAs that regulate oncogenic gene expression programs and cancer metabolism. Effective anticancer drugs may specifically partition into oncogenic biological condensates (OBC). [ABSTRACT FROM AUTHOR]

Porphyrin and iron are ubiquitous and essential for sustaining life in virtually all living organisms. Unlike iron, which exists in many forms, porphyrin macrocycles are mostly functional as metal complexes. The iron-containing porphyrin, heme, serves as a prosthetic group in a wide array of metabolic pathways; including respiratory cytochromes, hemoglobin, cytochrome P450s, catalases, and other hemoproteins. Despite playing crucial roles in many biological processes, heme, iron, and porphyrin intermediates are potentially cytotoxic. Thus, the intersection of porphyrin and iron metabolism at heme synthesis, and intracellular trafficking of heme and its porphyrin precursors are tightly regulated processes. In this review, we discuss recent advances in understanding the physiological dynamics of eukaryotic ferrochelatase, a mitochondrially localized metalloenzyme. Ferrochelatase catalyzes the terminal step of heme biosynthesis, the insertion of ferrous iron into protoporphyrin IX to produce heme. In most eukaryotes, except plants, ferrochelatase is localized to the mitochondrial matrix, where substrates are delivered and heme is synthesized for trafficking to multiple cellular locales. Herein, we delve into the structural and functional features of ferrochelatase, as well as its metabolic regulation in the mitochondria. We discuss the regulation of ferrochelatase via post-translational modifications, transportation of substrates and product across the mitochondrial membrane, protein-protein interactions, inhibition by small-molecule inhibitors, and ferrochelatase in protozoal parasites. Overall, this review presents insight on mitochondrial heme homeostasis from the perspective of ferrochelatase.

In vivo, as an advanced catalytic strategy, transient non-covalently bound multi-enzyme complexes can be formed to facilitate the relay of substrates, i. e. substrate channeling, between sequential enzymatic reactions and to enhance the throughput of multi-step enzymatic pathways. The human thymidylate synthase and dihydrofolate reductase catalyze two consecutive reactions in the folate metabolism pathway, and experiments have shown that they are very likely to bind in the same multi-enzyme complex in vivo. While reports on the protozoa thymidylate synthase-dihydrofolate reductase bifunctional enzyme give substantial evidences of substrate channeling along a surface "electrostatic highway," attention has not been paid to whether the human thymidylate synthase and dihydrofolate reductase, if they are in contact with each other in the multi-enzyme complex, are capable of substrate channeling employing surface electrostatics. This work utilizes protein-protein docking, electrostatics calculations, and Brownian dynamics to explore the existence and mechanism of the substrate channeling between the human thymidylate synthase and dihydrofolate reductase. The results show that the bound human thymidylate synthase and dihydrofolate reductase are capable of substrate channeling and the formation of the surface "electrostatic highway." The substrate channeling efficiency between the two can be reasonably high and comparable to that of the protozoa.

Flavones predominantly accumulate as O - and C -glycosides in kumquat plants. Two catalytic mechanisms of flavone synthase II (FNSII) support the biosynthesis of glycosyl flavones, one involving flavanone 2-hydroxylase (which generates 2-hydroxyflavanones for C -glycosylation) and another involving the direct catalysis of flavanones to flavones for O -glycosylation. However, FNSII has not yet been characterized in kumquats. In this study, we identified two kumquat FNSII genes (FcFNSII-1 and FcFNSII-2), based on transcriptome and bioinformatics analysis. Data from in vivo and in vitro assays showed that FcFNSII-2 directly synthesized apigenin and acacetin from naringenin and isosakuranetin, respectively, whereas FcFNSII-1 showed no detectable catalytic activities with flavanones. In agreement, transient overexpression of FcFNSII-2 in kumquat peels significantly enhanced the transcription of structural genes of the flavonoid-biosynthesis pathway and the accumulation of several O -glycosyl flavones. Moreover, studying the subcellular localizations of FcFNSII-1 and FcFNSII-2 demonstrated that N-terminal membrane-spanning domains were necessary to ensure endoplasmic reticulum localization and anchoring. Protein–protein interaction analyses, using the split-ubiquitin yeast two-hybrid system and bimolecular fluorescence-complementation assays, revealed that FcFNSII-2 interacted with chalcone synthase 1, chalcone synthase 2, and chalcone isomerase-like proteins. The results provide strong evidence that FcFNSII-2 serves as a nucleation site for an O -glycosyl flavone metabolon that channels flavanones for O -glycosyl flavone biosynthesis in kumquat fruits. They have implications for guiding genetic engineering efforts aimed at enhancing the composition of bioactive flavonoids in kumquat fruits. [ABSTRACT FROM AUTHOR]

Metabolons represent the structural organization of proteins for metabolic or regulatory pathways. Here, the interaction of fumarase FumB, aspartase AspA, and L-tartrate dehydratase TtdAB with the C4-dicarboxylate (C4-DC) transporters DcuA, DcuB, DcuC, and the L-tartrate transporter TtdT of Escherichia coli was tested by a bacterial two-hybrid (BACTH) assay in situ , or by co-chromatography using mSPINE (membrane Streptavidin protein interaction experiment). From the general C4-DC transporters, DcuB interacted with FumB and AspA, DcuA with AspA, whereas DcuC interacted with neither FumB nor AspA. Moreover, TtdT did not interact with TtdAB. The fumB-dcuB , the dcuA-aspA , and the ttdAB-ttdT genes encoding the respective proteins colocalize on the genome and each pair of genes forms cotranscripts, whereas the dcuC gene lies alone. The data suggest the formation of DcuB/FumB and DcuB/AspA metabolons for the uptake of L-malate, or L-aspartate, and their conversion to fumarate for fumarate respiration and excretion of the product succinate. The DcuA/AspA metabolon catalyzes uptake and conversion of L-aspartate to fumarate coupled to succinate excretion. The DcuA/AspA metabolon provides ammonia at the same time for nitrogen assimilation (ammonia shuttle). On the other hand, TtdT and TtdAB are not organized in a metabolon. Reasons for the formation (DcuA/AspA, DcuB/FumB, and DcuB/AspA) or nonformation (DcuC, TtdT, and TtdAB) of metabolons are discussed based on their metabolic roles. [ABSTRACT FROM AUTHOR]

Flavones predominantly accumulate as O- and C-glycosides in kumquat plants. Two catalytic mechanisms of flavone synthase II (FNSII) support the biosynthesis of glycosyl flavones, one involving flavanone 2-hydroxylase (which generates 2-hydroxyflavanones for C-glycosylation) and another involving the direct catalysis of flavanones to flavones for O-glycosylation. However, FNSII has not yet been characterized in kumquats. In this study, we identified two kumquat FNSII genes (FcFNSII-1 and FcFNSII-2), based on transcriptome and bioinformatics analysis. Data from in vivo and in vitro assays showed that FcFNSII-2 directly synthesized apigenin and acacetin from naringenin and isosakuranetin, respectively, whereas FcFNSII-1 showed no detectable catalytic activities with flavanones. In agreement, transient overexpression of FcFNSII-2 in kumquat peels significantly enhanced the transcription of structural genes of the flavonoid-biosynthesis pathway and the accumulation of several O-glycosyl flavones. Moreover, studying the subcellular localizations of FcFNSII-1 and FcFNSII-2 demonstrated that N-terminal membrane-spanning domains were necessary to ensure endoplasmic reticulum localization and anchoring. Protein–protein interaction analyses, using the split-ubiquitin yeast two-hybrid system and bimolecular fluorescence-complementation assays, revealed that FcFNSII-2 interacted with chalcone synthase 1, chalcone synthase 2, and chalcone isomerase-like proteins. The results provide strong evidence that FcFNSII-2 serves as a nucleation site for an O-glycosyl flavone metabolon that channels flavanones for O-glycosyl flavone biosynthesis in kumquat fruits. They have implications for guiding genetic engineering efforts aimed at enhancing the composition of bioactive flavonoids in kumquat fruits.

Heme is an essential cofactor for multiple cellular processes in most organisms. In developing erythroid cells, the demand for heme synthesis is high, but is significantly lower in non-erythroid cells. While the biosynthesis of heme in metazoans is well understood, the tissue-specific regulation of the pathway is less explored. To better understand this, we analyzed the mitochondrial heme metabolon in erythroid and non-erythroid cell lines from the perspective of ferrochelatase (FECH), the terminal enzyme in the heme biosynthetic pathway. Affinity purification of FLAG-tagged-FECH, together with mass spectrometric analysis, was carried out to identify putative protein partners in human and murine cell lines. Proteins involved in the heme biosynthetic process and mitochondrial organization were identified as the core components of the FECH interactome. Interestingly, in non-erythroid cell lines, the FECH interactome is highly enriched with proteins associated with the tricarboxylic acid (TCA) cycle. Overall, our study shows that the mitochondrial heme metabolon in erythroid and non-erythroid cells has similarities and differences, and suggests new roles for the mitochondrial heme metabolon and heme in regulating metabolic flux and key cellular processes.

Significance: Alongside well-characterized permanent multimeric enzymes and multienzyme complexes, relatively unstable transient enzyme–enzyme assemblies, including metabolons, provide an important mechanism for the regulation of energy and redox metabolism. Critical Issues: Despite the fact that enzyme–enzyme assemblies have been proposed for many decades and experimentally analyzed for at least 40 years, there are very few pathways for which unequivocal evidence for the presence of metabolite channeling, the most frequently evoked reason for their formation, has been provided. Further, in contrast to the stronger, permanent interactions for which a deep understanding of the subunit interface exists, the mechanism(s) underlying transient enzyme–enzyme interactions remain poorly studied. Recent Advances: The widespread adoption of proteomic and cell biological approaches to characterize protein–protein interaction is defining an ever-increasing number of enzyme–enzyme assemblies as well as enzyme–protein interactions that likely identify factors which stabilize such complexes. Moreover, the use of microfluidic technologies provided compelling support of a role for substrate-specific chemotaxis in complex assemblies. Future Directions: Embracing current and developing technologies should render the delineation of metabolons from other enzyme–enzyme complexes more facile. In parallel, attempts to confirm that the findings reported in microfluidic systems are, indeed, representative of the cellular situation will be critical to understanding the physiological circumstances requiring and evoking dynamic changes in the levels of the various transient enzyme–enzyme assemblies of the cell. Antioxid. Redox Signal. 35, 788–807. [ABSTRACT FROM AUTHOR]

With the elucidation of myriad anabolic and catabolic enzyme-catalyzed cellular pathways crisscrossing each other, an obvious question arose: how could these networks operate with maximal catalytic efficiency and minimal interference? A logical answer was the postulate of metabolic channeling, which in its simplest embodiment assumes that the product generated by one enzyme passes directly to a second without diffusion into the surrounding medium. This tight coupling of activities might increase a pathway's metabolic flux and/or serve to sequester unstable/toxic/reactive intermediates as well as prevent their access to other networks. Here, we present evidence for this concept, commencing with enzymes that feature a physical molecular tunnel, to multi-enzyme complexes that retain pathway substrates through electrostatics or enclosures, and finally to metabolons that feature collections of enzymes assembled into clusters with variable stoichiometric composition. Lastly, we discuss the advantages of reversibly assembled metabolons in the context of the purinosome, the purine biosynthesis metabolon. The channeling of metabolic intermediates can increase pathway fluxes and protect metabolites. Mechanisms of channeling include direct physical tunnels, electrostatic trapping and enclosures, and membrane-less metabolic compartments or "metabolons." We highlight examples of the above classes, finally focusing on the purinosome to discuss the advantages of dynamically assembled metabolons. [ABSTRACT FROM AUTHOR]

Quinolinic acid (QA) is a key intermediate of nicotinic acid (Niacin) which is an essential human nutrient and widely used in food and pharmaceutical industries. In this study, a quinolinic acid producer was constructed by employing comprehensive engineering strategies. Firstly, the quinolinic acid production was improved by deactivation of NadC (to block the consumption pathway), NadR (to eliminate the repression of L-aspartate oxidase and quinolinate synthase), and PtsG (to slow the glucose utilization rate and achieve a more balanced metabolism, and also to increase the availability of the precursor phosphoenolpyruvate). Further modifications to enhance quinolinic acid production were investigated by increasing the oxaloacetate pool through overproduction of phosphoenolpyruvate carboxylase and deactivation of acetate-producing pathway enzymes. Moreover, quinolinic acid production was accelerated by assembling NadB and NadA as an enzyme complex with the help of peptide-peptide interaction peptides RIAD and RIDD, which resulted in up to 3.7 g/L quinolinic acid being produced from 40 g/L glucose in shake-flask cultures. A quinolinic acid producer was constructed in this study, and these results lay a foundation for further engineering of microbial cell factories to efficiently produce quinolinic acid and subsequently convert this product to nicotinic acid for industrial applications. The authors engineered E. coli MG1655 to produce quinolinic acid, a key precursor of nicotinic acid, by employing comprehensive engineering strategies, including 1) enabling quinolinate production by deactivation of nadC (blocking consumption), nadR (eliminating transcriptional repression), and ptsG (increasing PEP availability for PEPC); 2) improving quinolinate production by overexpression of gene encoding PEPC (raising the OAA pool for aspartate synthesis), and deactivation of by-product acetate producing pathways; 3) accelerating quinolinate production by assembling NadA and NadB as an enzyme complex at a ratio of 2:1 with the help of peptide-peptide interaction peptides RIDD and RIAD. Up to 3.7 g/L quinolinate was produced from 40 g/L glucose in shake-flask cultures. [Display omitted] • Quinolinate producer was constructed by employing comprehensive engineering strategies. • Quinolinate production was improved by assembling NadA and NadB as an enzyme complex at a ratio of 2:1. • Up to 3.7 g/L quinolinate was produced in E. coli strain FZ763 with NadA-RIDD and NadB-RIAD. [ABSTRACT FROM AUTHOR]

Mitochondria are essential organelles because of their function in energy conservation. Here, we show an involvement of mitochondria in phytochrome‐dependent light sensing in fungi. Phytochrome photoreceptors are found in plants, bacteria, and fungi and contain a linear, heme‐derived tetrapyrrole as chromophore. Linearization of heme requires heme oxygenases (HOs) which reside inside chloroplasts in planta. Despite the poor degree of conservation of HOs, we identified two candidates in the fungus Alternaria alternata. Deletion of either one phenocopied phytochrome deletion. The two enzymes had a cooperative effect and physically interacted with phytochrome, suggesting metabolon formation. The metabolon was attached to the surface of mitochondria with a C‐terminal anchor (CTA) sequence in HoxA. The CTA was necessary and sufficient for mitochondrial targeting. The affinity of phytochrome apoprotein to HoxA was 57,000‐fold higher than the affinity of the holoprotein, suggesting a "kiss‐and‐go" mechanism for chromophore loading and a function of mitochondria as assembly platforms for functional phytochrome. Hence, two alternative approaches for chromophore biosynthesis and insertion into phytochrome evolved in plants and fungi. SYNOPSIS: Heme oxygenase activity converts heme into a linear tetrapyrrole for insertion into the light‐sensory phytochrome apoprotein. Here, identification of two fungal heme oxygenases reveals unprecedented mitochondrial involvement in chromophore production. The fungus Alternaria alternata contains two heme oxygenases, HoxA and HoxB.HoxA and HoxB localize to the outer mitochondrial membrane.Both heme oxygenases interact with phytochrome apoprotein and release functional phytochrome after chromophore loading. [ABSTRACT FROM AUTHOR]

The analysis of cryo-electron tomography images of human and rat mitochondria revealed that the mitochondrial matrix is at least as crowded as the cytosol. To mitigate the crowding effects, metabolite transport in the mitochondria primarily occurs through the intermembrane space, which is significantly less crowded. The scientific literature largely ignores how enzyme systems and metabolite transport are organized in the crowded environment of the mitochondrial matrix. Under crowded conditions, multivalent interactions carried out by disordered protein regions (IDRs), may become extremely important. We analyzed the human mitochondrial proteome to determine the presence and physiological significance of IDRs. Despite mitochondrial proteins being generally more ordered than cytosolic or overall proteome proteins, disordered regions plays a significant role in certain mitochondrial compartments and processes. Even in highly ordered enzyme systems, there are proteins with long IDRs. Some IDRs act as binding elements between highly ordered subunits, while the roles of others are not yet established. Mitochondrial systems, like their bacterial ancestors, rely less on IDRs and more on RNA for LLPS compartmentalization. More evolutionarily advanced subsystems that enable mitochondria-cell interactions contain more IDRs. The study highlights the crucial and often overlooked role played by IDRs and non-coding RNAs in mitochondrial organization. [ABSTRACT FROM AUTHOR]

Adaptation to a hypertonic marine environment is one of the major topics in animal physiology research. Marine teleosts lose water osmotically from the gills and compensate for this loss by drinking surrounding seawater and absorbing water from the intestine. This situation is in contrast to that in mammals, which experience a net osmotic loss of water after drinking seawater. Water absorption in fishes is made possible by (1) removal of monovalent ions (desalinization) by the esophagus, (2) removal of divalent ions as carbonate (Mg/CaCO3) precipitates promoted by HCO3− secretion, and (3) facilitation of NaCl and water absorption from diluted seawater by the intestine using a suite of unique transporters. As a result, 70–85% of ingested seawater is absorbed during its passage through the digestive tract. Thus, the digestive tract is an essential organ for marine teleost survival in the hypertonic seawater environment. The eel is a species that has been frequently used for osmoregulation research in laboratories worldwide. The eel possesses many advantages as an experimental animal for osmoregulation studies, one of which is its outstanding euryhalinity, which enables researchers to examine changes in the structure and function of the digestive tract after direct transfer from freshwater to seawater. In recent years, the molecular mechanisms of ion and water transport across epithelial cells (the transcellular route) and through tight junctions (the paracellular route) have been elucidated for the esophagus and intestine. Thanks to the rapid progress in analytical methods for genome databases on teleosts, including the eel, the molecular identities of transporters, channels, pumps and junctional proteins have been clarified at the isoform level. As 10 y have passed since the previous reviews on this subject, it seems relevant and timely to summarize recent progress in research on the molecular mechanisms of water and ion transport in the digestive tract in eels and to compare the mechanisms with those of other teleosts and mammals from comparative and evolutionary viewpoints. We also propose future directions for this research field to achieve integrative understanding of the role of the digestive tract in adaptation to seawater with regard to pathways/mechanisms including the paracellular route, divalent ion absorption, metabolon formation and cellular trafficking of transporters. Notably, some of these have already attracted practical attention in laboratories. [ABSTRACT FROM AUTHOR]

Metabolons are transient multi-protein complexes of sequential enzymes that mediate substrate channeling. They differ from multi-enzyme complexes in that they are dynamic, rather than permanent, and as such have considerably lower dissociation constants. Despite the fact that a huge number of metabolons have been suggested to exist in plants, most of these claims are erroneous as only a handful of these have been proven to channel metabolites. We believe that physical protein–protein interactions between consecutive enzymes of a pathway should rather be called enzyme–enzyme assemblies. In this review, we describe how metabolons are generally assembled by transient interactions and held together by both structural elements and non-covalent interactions. Experimental evidence for their existence comes from protein–protein interaction studies, which indicate that the enzymes physically interact, and direct substrate channeling measurements, which indicate that they functionally interact. Unfortunately, advances in cell biology and proteomics have far outstripped those in classical enzymology and flux measurements, rendering most reports reliant purely on interactome studies. Recent developments in co-fractionation mass spectrometry will likely further exacerbate this bias. Given this, only dynamic enzyme–enzyme assemblies in which both physical and functional interactions have been demonstrated should be termed metabolons. We discuss the level of evidence for the manifold plant pathways that have been postulated to contain metabolons and then list examples in both primary and secondary metabolism for which strong evidence has been provided to support these claims. In doing so, we pay particular attention to experimental and mathematical approaches to study metabolons as well as complexities that arise in attempting to follow them. Finally, we discuss perspectives for improving our understanding of these fascinating but enigmatic interactions.

Isoflavonoid is one of the groups of flavonoids that play pivotal roles in the survival of land plants. Chalcone synthase (CHS), the first enzyme of the isoflavonoid biosynthetic pathway, catalyzes the formation of a common isoflavonoid precursor. We have previously reported that an isozyme of soybean CHS (termed GmCHS1) is a key component of the isoflavonoid metabolon, a protein complex to enhance efficiency of isoflavonoid production. Here, we determined the crystal structure of GmCHS1 as a first step of understanding the metabolon structure, as well as to better understand the catalytic mechanism of GmCHS1. [ABSTRACT FROM AUTHOR]

Flavan-3-ols are prominent phenolic compounds found abundantly in the young leaves of tea plants. The enzymes involved in flavan-3-ol biosynthesis in tea plants have been extensively investigated. However, the localization and associations of these numerous functional enzymes within cells have been largely neglected. In this study, we aimed to investigate the synthesis of flavan-3-ols in tea plants, particularly focusing on epigallocatechin gallate. Our analysis involving the DESI-MSI method to reveal a distinct distribution pattern of B-ring trihydroxylated flavonoids, primarily concentrated in the outer layer of buds. Subcellular localization showed that CsC4H, CsF3'H, and CsF3'5'H localizes endoplasmic reticulum. Protein-protein interaction studies demonstrated direct associations between CsC4H, CsF3'H, and cytoplasmic enzymes (CHS, CHI, F3H, DFR, FLS, and ANR), highlighting their interactions within the biosynthetic pathway. Notably, CsF3'5'H, the enzyme for B-ring trihydroxylation, did not directly interact with other enzymes. We identified cytochrome b 5 isoform C serving as an essential redox partner, ensuring the proper functioning of CsF3'5'H. Our findings suggest the existence of distinct modules governing the synthesis of different B-ring hydroxylation compounds. This study provides valuable insights into the mechanisms underlying flavonoid diversity and efficient synthesis and enhances our understanding of the substantial accumulation of B-ring trihydroxylated flavan-3-ols in tea plants., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Influencing the production of natural products in planta by metabolic engineering approaches has become an increasingly popular means to address a variety of important agricultural, medicinal, and nutraceutical issues. There have been many successes in this area, but also many surprising and unanticipated results. As an understanding of the complexity of metabolism has emerged, it has become increasingly apparent that successfully designing synthetic approaches to influence metabolite production requires knowledge beyond that of just the individual enzymatic steps involved and the order in which they are arranged into a biosynthetic pathway. Although much remains to be learned about the structures of metabolic organization and their impact on flux in living systems, taking the information that is available into account is one step towards helping to overcome the difficulties in generating sufficient yields of current natural products of interest and to accelerate the production of target compounds in the future. In this review, we reflect on current knowledge relating to the use of metabolic engineering of plant natural product pathways to influence the production of compounds of interest and consider the impact that higher levels of organization, such as metabolon formation, may have on these systems.

Despite advances in targeted therapeutics and understanding in molecular mechanisms, metastasis remains a substantial obstacle for cancer treatment. Acquired genetic mutations and transcriptional changes can promote the spread of primary tumor cells to distant tissues. Additionally, recent studies have uncovered that metabolic reprogramming of cancer cells is tightly associated with cancer metastasis. However, whether intracellular metabolism is spatially and temporally regulated for cancer cell migration and invasion is understudied. In this review, we highlight the emergence of a concept, termed “membraneless metabolic compartmentalization,” as one of the critical mechanisms that determines the metastatic capacity of cancer cells. In particular, we focus on the compartmentalization of purine nucleotide metabolism (e.g., ATP and GTP) at the leading edge of migrating cancer cells through the uniquely phase-separated microdomains where dynamic exchange of nucleotide metabolic enzymes takes place. We will discuss how future insights may usher in a novel class of therapeutics specifically targeting the metabolic compartmentalization that drives tumor metastasis.

Key points: According to the HCO3− metabolon hypothesis, direct association of cytosolic carbonic anhydrases (CAs) with the electrogenic Na/HCO3 cotransporter NBCe1‐A speeds transport by regenerating/consuming HCO3−. The present work addresses published discrepancies as to whether cytosolic CAs stimulate NBCe1‐A, heterologously expressed in Xenopus oocytes.We confirm the essential elements of the previous experimental observations, taken as support for the HCO3− metabolon hypothesis.However, using our own experimental protocols or those of others, we find that NBCe1‐A function is unaffected by cytosolic CAs.Previous conclusions that cytosolic CAs do stimulate NBCe1‐A can be explained by an unanticipated stimulatory effect of the CAs on an endogenous Na‐H exchanger.Theoretical analyses show that, although CAs could stimulate non‐HCO3− transporters (e.g. Na‐H exchangers) by accelerating CO2/HCO3−‐mediated buffering of acid‐base equivalents, they could not appreciably affect transport rates of NBCe1 or other transporters carrying HCO3−, CO3=, or NaCO3− ion pairs. The HCO3− metabolon hypothesis predicts that cytosolic carbonic anhydrase (CA) binds to NBCe1‐A, promotes HCO3− replenishment/consumption, and enhances transport. Using a short step‐duration current‐voltage (I–V) protocol with Xenopus oocytes expressing eGFP‐tagged NBCe1‐A, our group reported that neither injecting human CA II (hCA II) nor fusing hCA II to the NBCe1‐A carboxy terminus affects background‐subtracted NBCe1 slope conductance (GNBC), which is a direct measure of NBCe1‐A activity. Others – using bovine CA (bCA), untagged NBCe1‐A, and protocols keeping holding potential (Vh) far from NBCe1‐A's reversal potential (Erev) for prolonged periods – found that bCA increases total membrane current (ΔIm), which apparently supports the metabolon hypothesis. We systematically investigated differences in the two protocols. In oocytes expressing untagged NBCe1‐A, injected with bCA and clamped to −40 mV, CO2/HCO3− exposures markedly decrease Erev, producing large transient outward currents persisting for >10 min and rapid increases in [Na+]i. Although the CA inhibitor ethoxzolamide (EZA) reduces both ΔIm and d[Na+]i/dt, it does not reduce GNBC. In oocytes not expressing NBCe1‐A, CO2/HCO3− triggers rapid increases in [Na+]i that both hCA II and bCA enhance in concentration‐dependent manners. These d[Na+]i/dt increases are inhibited by EZA and blocked by EIPA, a Na‐H exchanger (NHE) inhibitor. In oocytes expressing untagged NBCe1‐A and injected with bCA, EIPA abolishes the EZA‐dependent decreases in ΔIm and d[Na+]i/dt. Thus, CAs/EZA produce their ΔIm and d[Na+]i/dt effects not through NBCe1‐A, but endogenous NHEs. Theoretical considerations argue against a CA stimulation of HCO3− transport, supporting the conclusion that an NBCe1‐A–HCO3− metabolon does not exist in oocytes. Key points: According to the HCO3− metabolon hypothesis, direct association of cytosolic carbonic anhydrases (CAs) with the electrogenic Na/HCO3 cotransporter NBCe1‐A speeds transport by regenerating/consuming HCO3−. The present work addresses published discrepancies as to whether cytosolic CAs stimulate NBCe1‐A, heterologously expressed in Xenopus oocytes.We confirm the essential elements of the previous experimental observations, taken as support for the HCO3− metabolon hypothesis.However, using our own experimental protocols or those of others, we find that NBCe1‐A function is unaffected by cytosolic CAs.Previous conclusions that cytosolic CAs do stimulate NBCe1‐A can be explained by an unanticipated stimulatory effect of the CAs on an endogenous Na‐H exchanger.Theoretical analyses show that, although CAs could stimulate non‐HCO3− transporters (e.g. Na‐H exchangers) by accelerating CO2/HCO3−‐mediated buffering of acid‐base equivalents, they could not appreciably affect transport rates of NBCe1 or other transporters carrying HCO3−, CO3=, or NaCO3− ion pairs. [ABSTRACT FROM AUTHOR]

Despite advances in targeted therapeutics and understanding in molecular mechanisms, metastasis remains a substantial obstacle for cancer treatment. Acquired genetic mutations and transcriptional changes can promote the spread of primary tumor cells to distant tissues. Additionally, recent studies have uncovered that metabolic reprogramming of cancer cells is tightly associated with cancer metastasis. However, whether intracellular metabolism is spatially and temporally regulated for cancer cell migration and invasion is understudied. In this review, we highlight the emergence of a concept, termed "membraneless metabolic compartmentalization, " as one of the critical mechanisms that determines the metastatic capacity of cancer cells. In particular, we focus on the compartmentalization of purine nucleotide metabolism (e.g., ATP and GTP) at the leading edge of migrating cancer cells through the uniquely phase-separated microdomains where dynamic exchange of nucleotide metabolic enzymes takes place. We will discuss how future insights may usher in a novel class of therapeutics specifically targeting the metabolic compartmentalization that drives tumor metastasis. [ABSTRACT FROM AUTHOR]

Protein scaffolding is a natural phenomenon whereby proteins colocalize into macromolecular complexes via specific protein–protein interactions. In the case of metabolic enzymes, protein scaffolding drives metabolic flux through specific pathways by colocalizing enzyme active sites. Synthetic protein scaffolding is increasingly used as a mechanism to improve product specificity and yields in metabolic engineering projects. To date, synthetic scaffolding has focused primarily on soluble enzyme systems, but many metabolic pathways for high-value secondary metabolites depend on membrane-bound enzymes. The compositional diversity of biological membranes and general challenges associated with modifying membrane proteins complicate scaffolding with membrane-requiring enzymes. Several recent studies have introduced new approaches to protein scaffolding at membrane surfaces, with notable success in improving product yields from specific metabolic pathways. Protein scaffolding techniques are increasingly being used to create synthetic protein complexes bound to biological membranes. Scaffolding tags can be used to recruit soluble enzymes or complexes to specific membrane domains. Synthetic transport metabolons, where enzymes are scaffolded to transmembrane transporters, are a powerful means to control metabolite fate upstream of central carbon metabolism. New membrane-integral scaffolding tools facilitate the design of synthetic complexes of membrane-requiring enzymes. [ABSTRACT FROM AUTHOR]

For millennia, humans have used plants for food, raw materials, and medicines, but only within the past two centuries have we begun to connect particular plant metabolites with specific properties and utilities. Since the utility of classical molecular genetics beyond model species is limited, the vast specialized metabolic systems present in the Earth's flora remain largely unstudied. With an explosion in genomics resources and a rapidly expanding toolbox over the past decade, exploration of plant specialized metabolism in nonmodel species is becoming more feasible than ever before. We review the state-of-the-art tools that have enabled this rapid progress. We present recent examples of de novo biosynthetic pathway discovery that employ various innovative approaches. We also draw attention to the higher-order organization of plant specialized metabolism at subcellular, cellular, tissue, interorgan, and interspecies levels, which will have important implications for the future design of comprehensive metabolic engineering strategies. [ABSTRACT FROM AUTHOR]

Summary: Sesame (Sesamum indicum) seeds contain a large number of lignans, phenylpropanoid‐related plant specialized metabolites. (+)‐Sesamin and (+)‐sesamolin are major hydrophobic lignans, whereas (+)‐sesaminol primarily accumulates as a water‐soluble sesaminol triglucoside (STG) with a sugar chain branched via β1→2 and β1→6‐O‐glucosidic linkages [i.e. (+)‐sesaminol 2‐O‐β‐d‐glucosyl‐(1→2)‐O‐β‐d‐glucoside‐(1→6)‐O‐β‐d‐glucoside]. We previously reported that the 2‐O‐glucosylation of (+)‐sesaminol aglycon and β1→6‐O‐glucosylation of (+)‐sesaminol 2‐O‐β‐d‐glucoside (SMG) are mediated by UDP‐sugar‐dependent glucosyltransferases (UGT), UGT71A9 and UGT94D1, respectively. Here we identified a distinct UGT, UGT94AG1, that specifically catalyzes the β1→2‐O‐glucosylation of SMG and (+)‐sesaminol 2‐O‐β‐d‐glucosyl‐(1→6)‐O‐β‐d‐glucoside [termed SDG(β1→6)]. UGT94AG1 was phylogenetically related to glycoside‐specific glycosyltransferases (GGTs) and co‐ordinately expressed with UGT71A9 and UGT94D1 in the seeds. The role of UGT94AG1 in STG biosynthesis was further confirmed by identification of a STG‐deficient sesame mutant that predominantly accumulates SDG(β1→6) due to a destructive insertion in the coding sequence of UGT94AG1. We also identified UGT94AA2 as an alternative UGT potentially involved in sugar–sugar β1→6‐O‐glucosylation, in addition to UGT94D1, during STG biosynthesis. Yeast two‐hybrid assays showed that UGT71A9, UGT94AG1, and UGT94AA2 were found to interact with a membrane‐associated P450 enzyme, CYP81Q1 (piperitol/sesamin synthase), suggesting that these UGTs are components of a membrane‐bound metabolon for STG biosynthesis. A comparison of kinetic parameters of these UGTs further suggested that the main β‐O‐glucosylation sequence of STG biosynthesis is β1→2‐O‐glucosylation of SMG by UGT94AG1 followed by UGT94AA2‐mediated β1→6‐O‐glucosylation. These findings together establish the complete biosynthetic pathway of STG and shed light on the evolvability of regio‐selectivity of sequential glucosylations catalyzed by GGTs. Significance Statement: Sesaminol triglucoside (STG) of sesame seeds has unique bioactivities with potential health benefits, yet the STG biosynthesis pathway remains to be fully established. This study has identified the missing enzyme in STG biosynthesis that catalyzes β1→2‐O‐glucosylation, a regioselective sequential glycosylation of sesame lignan biosynthesis. [ABSTRACT FROM AUTHOR]

Abstract The arrangement of proteins into complexes is a key organizational principle for many cellular functions. Although the topology of many complexes has been systematically analyzed in isolation, their molecular sociology in situ remains elusive. Here, we show that crude cellular extracts of a eukaryotic thermophile, Chaetomium thermophilum, retain basic principles of cellular organization. Using a structural proteomics approach, we simultaneously characterized the abundance, interactions, and structure of a third of the C. thermophilum proteome within these extracts. We identified 27 distinct protein communities that include 108 interconnected complexes, which dynamically associate with each other and functionally benefit from being in close proximity in the cell. Furthermore, we investigated the structure of fatty acid synthase within these extracts by cryoEM and this revealed multiple, flexible states of the enzyme in adaptation to its association with other complexes, thus exemplifying the need for in situ studies. As the components of the captured protein communities are known—at both the protein and complex levels—this study constitutes another step forward toward a molecular understanding of subcellular organization.

Flavonoids are a class of plant specialized metabolites with more than 6,900 known structures and play important roles in plant survival and reproduction. These metabolites are derived from p-coumaroyl-CoA via the sequential actions of a variety of flavonoid enzymes, which have been proposed to form weakly bound, ordered protein complexes termed flavonoid metabolons. This review discusses the impacts of the formation of flavonoid metabolons on the chemodiversity of flavonoids. Specific protein-protein interactions in the metabolons of Arabidopsis thaliana and other plant species have been studied for two decades. In many cases, metabolons are associated with the ER membrane, with ER-bound cytochromes P450 hypothesized to serve as nuclei for metabolon formation. Indeed, cytochromes P450 have been found to be components of flavonoid metabolons in rice, snapdragon, torenia, and soybean. Recent studies illustrate the importance of specific interactions for the efficient production and temporal/spatial distribution of flavonoids. For example, in diverse plant species, catalytically inactive type-IV chalcone isomerase-like protein serves as an enhancer of flavonoid production via its involvement in flavonoid metabolons. In soybean roots, a specific isozyme of chalcone reductase (CHR) interacts with 2-hydroxyisoflavanone synthase, to which chalcone synthase (CHS) can also bind, providing a mechanism to prevent the loss of the unstable CHR substrate during its transfer from CHS to CHR. Thus, diversification in chemical structures and temporal/spatial distribution patterns of flavonoids in plants is likely to be mediated by the formation of specific flavonoid metabolons via specific protein-protein interactions.

Paracoccidioides is a dimorphic fungus, the causative agent of paracoccidioidomycosis. The disease is endemic within Latin America and prevalent in Brazil. The treatment is based on azoles, sulfonamides and amphotericin B. The seeking for new treatment approaches is a real necessity for neglected infections. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an essential glycolytic enzyme, well known for its multitude of functions within cells, therefore categorized as a moonlight protein. To our knowledge, this is the first approach performed on the Paracoccidioides genus regarding the description of PPIs having GAPDH as a target. Here, we show an overview of experimental GAPDH interactome in different phases of Paracoccidioides lutzii and an in silico analysis of 18 proteins partners. GAPDH interacted with 207 proteins in P. lutzii. Several proteins bound to GAPDH in mycelium, transition and yeast phases are common to important pathways such as glycolysis and TCA. We performed a co-immunoprecipitation assay to validate the complex formed by GAPDH with triose phosphate isomerase, enolase, isocitrate lyase and 2-methylcitrate synthase. We found GAPDH participating in complexes with proteins of specific pathways, indicating the existence of a glycolytic and a TCA metabolon in P. lutzii. GAPDH interacted with several proteins that undergoes regulation by nitrosylation. In addition, we modeled the GAPDH 3-D structure, performed molecular dynamics and molecular docking in order to identify the interacting interface between GAPDH and the interacting proteins. Despite the large number of interacting proteins, GAPDH has only four main regions of contact with interacting proteins, reflecting its ancestrality and conservation over evolution.