An enzyme that selectively S-nitrosylates proteins to regulate insulin signaling.

Acyl-coenzyme A (acyl-CoA) species are cofactors for numerous enzymes that acylate thousands of proteins. Here, we describe an enzyme that uses S-nitroso-CoA (SNO-CoA) as its cofactor to S-nitrosylate multiple proteins (SNO-CoA-assisted nitrosylase, SCAN). Separate domains in SCAN mediate SNO-CoA and substrate binding, allowing SCAN to selectively catalyze SNO transfer from SNO-CoA to SCAN to multiple protein targets, including the insulin receptor (INSR) and insulin receptor substrate 1 (IRS1). Insulin-stimulated S-nitrosylation of INSR/IRS1 by SCAN reduces insulin signaling physiologically, whereas increased SCAN activity in obesity causes INSR/IRS1 hypernitrosylation and insulin resistance. SCAN-deficient mice are thus protected from diabetes. In human skeletal muscle and adipose tissue, SCAN expression increases with body mass index and correlates with INSR S-nitrosylation. S-nitrosylation by SCAN/SNO-CoA thus defines a new enzyme class, a unique mode of receptor tyrosine kinase regulation, and a revised paradigm for NO function in physiology and disease.

Strain-level fitness in the gut microbiome is an emergent property of glycans and a single metabolite.

The human gut microbiota resides within a diverse chemical environment challenging our ability to understand the forces shaping this ecosystem. Here, we reveal that fitness of the Bacteroidales, the dominant order of bacteria in the human gut, is an emergent property of glycans and one specific metabolite, butyrate. Distinct sugars serve as strain-variable fitness switches activating context-dependent inhibitory functions of butyrate. Differential fitness effects of butyrate within the Bacteroides are mediated by species-level variation in Acyl-CoA thioesterase activity and nucleotide polymorphisms regulating an Acyl-CoA transferase. Using in vivo multi-omic profiles, we demonstrate Bacteroides fitness in the human gut is associated together, but not independently, with Acyl-CoA transferase expression and butyrate. Our data reveal that each strain of the Bacteroides exists within a unique fitness landscape based on the interaction of chemical components unpredictable by the effect of each part alone mediated by flexibility in the core genome.

Quantitative subcellular acyl-CoA analysis reveals distinct nuclear metabolism and isoleucine-dependent histone propionylation.

Quantitative subcellular metabolomic measurements can explain the roles of metabolites in cellular processes but are subject to multiple confounding factors. We developed stable isotope labeling of essential nutrients in cell culture-subcellular fractionation (SILEC-SF), which uses isotope-labeled internal standard controls that are present throughout fractionation and processing to quantify acyl-coenzyme A (acyl-CoA) thioesters in subcellular compartments by liquid chromatography-mass spectrometry. We tested SILEC-SF in a range of sample types and examined the compartmentalized responses to oxygen tension, cellular differentiation, and nutrient availability. Application of SILEC-SF to the challenging analysis of the nuclear compartment revealed a nuclear acyl-CoA profile distinct from that of the cytosol, with notable nuclear enrichment of propionyl-CoA. Using isotope tracing, we identified the branched chain amino acid isoleucine as a major metabolic source of nuclear propionyl-CoA and histone propionylation, thus revealing a new mechanism of crosstalk between metabolism and the epigenome.

Cytoplasmic SIRT6-mediated ACSL5 deacetylation impedes nonalcoholic fatty liver disease by facilitating hepatic fatty acid oxidation.

Nonalcoholic fatty liver disease (NAFLD) is characterized by excessive hepatic lipid accumulation, which can progress to nonalcoholic steatohepatitis (NASH). Histone deacetylase Sirtuin 6 (SIRT6) regulates NAFLD by regulating metabolism-related gene expression, but an extrachromosomal role for SIRT6 in NAFLD development remains elusive. We investigated whether SIRT6 functions on NAFLD in the cytoplasm. We found that SIRT6 binds saturated fatty acids, especially palmitic acid. This binding leads to its nuclear export, where it deacetylates long-chain acyl-CoA synthase 5 (ACSL5), thereby facilitating fatty acid oxidation. High-fat diet-induced NAFLD is suppressed by ACSL5 hepatic overexpression but is exacerbated by its depletion. As confirmation, overexpression of a deacetylated ACSL5 mimic attenuated NAFLD in Sirt6 liver-specific knockout mice. Moreover, NASH-hepatic tissues from both patients and diet-fed mice exhibited significantly reduced cytoplasmic SIRT6 levels and increased ACSL5 acetylation. The SIRT6/ACSL5 signaling pathway has a critical role in NAFLD progression and might constitute an avenue for therapeutic intervention.

Metabolic regulation of gene expression through histone acylations.

Eight types of short-chain Lys acylations have recently been identified on histones: propionylation, butyrylation, 2-hydroxyisobutyrylation, succinylation, malonylation, glutarylation, crotonylation and ß-hydroxybutyrylation. Emerging evidence suggests that these histone modifications affect gene expression and are structurally and functionally different from the widely studied histone Lys acetylation. In this Review, we discuss the regulation of non-acetyl histone acylation by enzymatic and metabolic mechanisms, the acylation 'reader' proteins that mediate the effects of different acylations and their physiological functions, which include signal-dependent gene activation, spermatogenesis, tissue injury and metabolic stress. We propose a model to explain our present understanding of how differential histone acylation is regulated by the metabolism of the different acyl-CoA forms, which in turn modulates the regulation of gene expression.

SUCLG1 restricts POLRMT succinylation to enhance mitochondrial biogenesis and leukemia progression.

Mitochondria are cellular powerhouses that generate energy through the electron transport chain (ETC). The mitochondrial genome (mtDNA) encodes essential ETC proteins in a compartmentalized manner, however, the mechanism underlying metabolic regulation of mtDNA function remains unknown. Here, we report that expression of tricarboxylic acid cycle enzyme succinate-CoA ligase SUCLG1 strongly correlates with ETC genes across various TCGA cancer transcriptomes. Mechanistically, SUCLG1 restricts succinyl-CoA levels to suppress the succinylation of mitochondrial RNA polymerase (POLRMT). Lysine 622 succinylation disrupts the interaction of POLRMT with mtDNA and mitochondrial transcription factors. SUCLG1-mediated POLRMT hyposuccinylation maintains mtDNA transcription, mitochondrial biogenesis, and leukemia cell proliferation. Specifically, leukemia-promoting FMS-like tyrosine kinase 3 (FLT3) mutations modulate nuclear transcription and upregulate SUCLG1 expression to reduce succinyl-CoA and POLRMT succinylation, resulting in enhanced mitobiogenesis. In line, genetic depletion of POLRMT or SUCLG1 significantly delays disease progression in mouse and humanized leukemia models. Importantly, succinyl-CoA level and POLRMT succinylation are downregulated in FLT3-mutated clinical leukemia samples, linking enhanced mitobiogenesis to cancer progression. Together, SUCLG1 connects succinyl-CoA with POLRMT succinylation to modulate mitochondrial function and cancer development.

Recognition of Histone Crotonylation by Taf14 Links Metabolic State to Gene Expression.

Metabolic signaling to chromatin often underlies how adaptive transcriptional responses are controlled. While intermediary metabolites serve as co-factors for histone-modifying enzymes during metabolic flux, how these modifications contribute to transcriptional responses is poorly understood. Here, we utilize the highly synchronized yeast metabolic cycle (YMC) and find that fatty acid ß-oxidation genes are periodically expressed coincident with the ß-oxidation byproduct histone crotonylation. Specifically, we found that H3K9 crotonylation peaks when H3K9 acetylation declines and energy resources become limited. During this metabolic state, pro-growth gene expression is dampened; however, mutation of the Taf14 YEATS domain, a H3K9 crotonylation reader, results in de-repression of these genes. Conversely, exogenous addition of crotonic acid results in increased histone crotonylation, constitutive repression of pro-growth genes, and disrupted YMC oscillations. Together, our findings expose an unexpected link between metabolic flux and transcription and demonstrate that histone crotonylation and Taf14 participate in the repression of energy-demanding gene expression.

Does chromatin function as a metabolite reservoir?

Alternative histone acylations integrate gene expression with cellular metabolic states. Recent measurements of cellular acyl-coenzyme A (acyl-CoA) pools highlight the potential that histone post-translational modifications (PTMs) contribute directly to the regulation of metabolite pools. A metabolite-centric view throws new light onto roles and evolution of histone PTMs.

Structure and catalytic mechanism of a human triacylglycerol-synthesis enzyme.

Triacylglycerols store metabolic energy in organisms and have industrial uses as foods and fuels. Excessive accumulation of triacylglycerols in humans causes obesity and is associated with metabolic diseases1. Triacylglycerol synthesis is catalysed by acyl-CoA diacylglycerol acyltransferase (DGAT) enzymes2-4, the structures and catalytic mechanisms of which remain unknown. Here we determined the structure of dimeric human DGAT1, a member of the membrane-bound O-acyltransferase (MBOAT) family, by cryo-electron microscopy at approximately 3.0 Å resolution. DGAT1 forms a homodimer through N-terminal segments and a hydrophobic interface, with putative active sites within the membrane region. A structure obtained with oleoyl-CoA substrate resolved at approximately 3.2 Å shows that the CoA moiety binds DGAT1 on the cytosolic side and the acyl group lies deep within a hydrophobic channel, positioning the acyl-CoA thioester bond near an invariant catalytic histidine residue. The reaction centre is located inside a large cavity, which opens laterally to the membrane bilayer, providing lipid access to the active site. A lipid-like density-possibly representing an acyl-acceptor molecule-is located within the reaction centre, orthogonal to acyl-CoA. Insights provided by the DGAT1 structures, together with mutagenesis and functional studies, provide the basis for a model of the catalysis of triacylglycerol synthesis by DGAT.

Structure and mechanism of human diacylglycerol O-acyltransferase 1.

Diacylglycerol O-acyltransferase 1 (DGAT1) synthesizes triacylglycerides and is required for dietary fat absorption and fat storage in humans1. DGAT1 belongs to the membrane-bound O-acyltransferase (MBOAT) superfamily, members of which are found in all kingdoms of life and are involved in the acylation of lipids and proteins2,3. How human DGAT1 and other mammalian members of the MBOAT family recognize their substrates and catalyse their reactions is unknown. The absence of three-dimensional structures also hampers rational targeting of DGAT1 for therapeutic purposes. Here we present the cryo-electron microscopy structure of human DGAT1 in complex with an oleoyl-CoA substrate. Each DGAT1 protomer has nine transmembrane helices, eight of which form a conserved structural fold that we name the MBOAT fold. The MBOAT fold in DGAT1 forms a hollow chamber in the membrane that encloses highly conserved catalytic residues. The chamber has separate entrances for each of the two substrates, fatty acyl-CoA and diacylglycerol. DGAT1 can exist as either a homodimer or a homotetramer and the two forms have similar enzymatic activity. The N terminus of DGAT1 interacts with the neighbouring protomer and these interactions are required for enzymatic activity.

Automated Design of Efficient and Functionally Diverse Enzyme Repertoires.

Substantial improvements in enzyme activity demand multiple mutations at spatially proximal positions in the active site. Such mutations, however, often exhibit unpredictable epistatic (non-additive) effects on activity. Here we describe FuncLib, an automated method for designing multipoint mutations at enzyme active sites using phylogenetic analysis and Rosetta design calculations. We applied FuncLib to two unrelated enzymes, a phosphotriesterase and an acetyl-CoA synthetase. All designs were active, and most showed activity profiles that significantly differed from the wild-type and from one another. Several dozen designs with only 3-6 active-site mutations exhibited 10- to 4,000-fold higher efficiencies with a range of alternative substrates, including hydrolysis of the toxic organophosphate nerve agents soman and cyclosarin and synthesis of butyryl-CoA. FuncLib is implemented as a web server (http://FuncLib.weizmann.ac.il); it circumvents iterative, high-throughput experimental screens and opens the way to designing highly efficient and diverse catalytic repertoires.

Identification of a 1-acyl-glycerol-3-phosphate acyltransferase from Mycobacterium tuberculosis, a key enzyme involved in triacylglycerol biosynthesis.

Latent tuberculosis, caused by dormant Mycobacterium tuberculosis (Mtb), poses a threat to global health through the incubation of undiagnosed infections within the community. Dormant Mtb, which is phenotypically tolerant to antibiotics, accumulates triacylglycerol (TAG) utilizing fatty acids obtained from macrophage lipid droplets. TAG is vital to mycobacteria, serving as a cell envelope component and energy reservoir during latency. TAG synthesis occurs by sequential acylation of glycerol-3-phosphate, wherein the second acylation step is catalyzed by acylglycerol-3-phosphate acyltransferase (AGPAT), resulting in the production of phosphatidic acid (PA), a precursor for the synthesis of TAG and various phospholipids. Here, we have characterized a putative acyltransferase of Mtb encoded by Rv3816c. We found that Rv3816c has all four characteristic motifs of AGPAT, exists as a membrane-bound enzyme, and functions as 1-acylglycerol-3-phosphate acyltransferase. The enzyme could transfer the acyl group to acylglycerol-3-phosphate (LPA) from monounsaturated fatty acyl-coenzyme A of chain length 16 or 18 to produce PA. Complementation of Escherichia coli PlsC mutant in vivo by Rv3816c confirmed that it functions as AGPAT. Its active site mutants, H43A and D48A, were incapable of transferring the acyl group to LPA in vitro and were not able to rescue the growth defect of E. coli PlsC mutant in vivo. Identifying Rv3816c as AGPAT and comparing its properties with other AGPAT homologs is not only a step toward understanding the TAG biosynthesis in mycobacteria but has the potential to explore it as a drug target.

Complete biosynthesis of cannabinoids and their unnatural analogues in yeast.

Cannabis sativa L. has been cultivated and used around the globe for its medicinal properties for millennia1. Some cannabinoids, the hallmark constituents of Cannabis, and their analogues have been investigated extensively for their potential medical applications2. Certain cannabinoid formulations have been approved as prescription drugs in several countries for the treatment of a range of human ailments3. However, the study and medicinal use of cannabinoids has been hampered by the legal scheduling of Cannabis, the low in planta abundances of nearly all of the dozens of known cannabinoids4, and their structural complexity, which limits bulk chemical synthesis. Here we report the complete biosynthesis of the major cannabinoids cannabigerolic acid, Δ9-tetrahydrocannabinolic acid, cannabidiolic acid, Δ9-tetrahydrocannabivarinic acid and cannabidivarinic acid in Saccharomyces cerevisiae, from the simple sugar galactose. To accomplish this, we engineered the native mevalonate pathway to provide a high flux of geranyl pyrophosphate and introduced a heterologous, multi-organism-derived hexanoyl-CoA biosynthetic pathway5. We also introduced the Cannabis genes that encode the enzymes involved in the biosynthesis of olivetolic acid6, as well as the gene for a previously undiscovered enzyme with geranylpyrophosphate:olivetolate geranyltransferase activity and the genes for corresponding cannabinoid synthases7,8. Furthermore, we established a biosynthetic approach that harnessed the promiscuity of several pathway genes to produce cannabinoid analogues. Feeding different fatty acids to our engineered strains yielded cannabinoid analogues with modifications in the part of the molecule that is known to alter receptor binding affinity and potency9. We also demonstrated that our biological system could be complemented by simple synthetic chemistry to further expand the accessible chemical space. Our work presents a platform for the production of natural and unnatural cannabinoids that will allow for more rigorous study of these compounds and could be used in the development of treatments for a variety of human health problems.

Chromodomain Protein CDYL Acts as a Crotonyl-CoA Hydratase to Regulate Histone Crotonylation and Spermatogenesis.

Lysine crotonylation (Kcr) is a newly identified histone modification that is associated with active transcription in mammalian cells. Here we report that the chromodomain Y-like transcription corepressor CDYL negatively regulates histone Kcr by acting as a crotonyl-CoA hydratase to convert crotonyl-CoA to ß-hydroxybutyryl-CoA. We showed that the negative regulation of histone Kcr by CDYL is intrinsically linked to its transcription repression activity and functionally implemented in the reactivation of sex chromosome-linked genes in round spermatids and genome-wide histone replacement in elongating spermatids. Significantly, Cdyl transgenic mice manifest dysregulation of histone Kcr and reduction of male fertility with a decreased epididymal sperm count and sperm cell motility. Our study uncovers a biochemical pathway in the regulation of histone Kcr and implicates CDYL-regulated histone Kcr in spermatogenesis, adding to the understanding of the physiology of male reproduction and the mechanism of the spermatogenic failure in AZFc (Azoospermia Factor c)-deleted infertile men.

Bivalent recognition of fatty acyl-CoA by a human integral membrane palmitoyltransferase.

S-acylation, also known as palmitoylation, is the most abundant form of protein lipidation in humans. This reversible posttranslational modification, which targets thousands of proteins, is catalyzed by 23 members of the DHHC family of integral membrane enzymes. DHHC enzymes use fatty acyl-CoA as the ubiquitous fatty acyl donor and become autoacylated at a catalytic cysteine; this intermediate subsequently transfers the fatty acyl group to a cysteine in the target protein. Protein S-acylation intersects with almost all areas of human physiology, and several DHHC enzymes are considered as possible therapeutic targets against diseases such as cancer. These efforts would greatly benefit from a detailed understanding of the molecular basis for this crucial enzymatic reaction. Here, we combine X-ray crystallography with all-atom molecular dynamics simulations to elucidate the structure of the precatalytic complex of human DHHC20 in complex with palmitoyl CoA. The resulting structure reveals that the fatty acyl chain inserts into a hydrophobic pocket within the transmembrane spanning region of the protein, whereas the CoA headgroup is recognized by the cytosolic domain through polar and ionic interactions. Biochemical experiments corroborate the predictions from our structural model. We show, using both computational and experimental analyses, that palmitoyl CoA acts as a bivalent ligand where the interaction of the DHHC enzyme with both the fatty acyl chain and the CoA headgroup is important for catalytic chemistry to proceed. This bivalency explains how, in the presence of high concentrations of free CoA under physiological conditions, DHHC enzymes can efficiently use palmitoyl CoA as a substrate for autoacylation.

Long-chain fatty acyl coenzyme A inhibits NME1/2 and regulates cancer metastasis.

SignificanceThe study provided a long-sought molecular mechanism that could explain the link between fatty acid metabolism and cancer metastasis. Further understanding may lead to new strategies to inhibit cancer metastasis. The chemical proteomic approach developed here will be useful for discovering other regulatory mechanisms of protein function by small molecule metabolites.

Succinyl-CoA-based energy metabolism dysfunction in chronic heart failure.

Heart failure (HF) is a leading cause of death and repeated hospitalizations and often involves cardiac mitochondrial dysfunction. However, the underlying mechanisms largely remain elusive. Here, using a mouse model in which myocardial infarction (MI) was induced by coronary artery ligation, we show the metabolic basis of mitochondrial dysfunction in chronic HF. Four weeks after ligation, MI mice showed a significant decrease in myocardial succinyl-CoA levels, and this decrease impaired the mitochondrial oxidative phosphorylation (OXPHOS) capacity. Heme synthesis and ketolysis, and protein levels of several enzymes consuming succinyl-CoA in these events, were increased in MI mice, while enzymes synthesizing succinyl-CoA from α-ketoglutarate and glutamate were also increased. Furthermore, the ADP-specific subunit of succinyl-CoA synthase was reduced, while its GDP-specific subunit was almost unchanged. Administration of 5-aminolevulinic acid, an intermediate in the pathway from succinyl-CoA to heme synthesis, appreciably restored succinyl-CoA levels and OXPHOS capacity and prevented HF progression in MI mice. Previous reports also suggested the presence of succinyl-CoA metabolism abnormalities in cardiac muscles of HF patients. Our results identified that changes in succinyl-CoA usage in different metabolisms of the mitochondrial energy production system is characteristic to chronic HF, and although similar alterations are known to occur in healthy conditions, such as during strenuous exercise, they may often occur irreversibly in chronic HF leading to a decrease in succinyl-CoA. Consequently, nutritional interventions compensating the succinyl-CoA consumption are expected to be promising strategies to treat HF.

MadR mediates acyl CoA-dependent regulation of mycolic acid desaturation in mycobacteria.

Mycobacterium tuberculosis has a lipid-rich cell envelope that is remodeled throughout infection to enable adaptation within the host. Few transcriptional regulators have been characterized that coordinate synthesis of mycolic acids, the major cell wall lipids of mycobacteria. Here, we show that the mycolic acid desaturase regulator (MadR), a transcriptional repressor of the mycolate desaturase genes desA1 and desA2, controls mycolic acid desaturation and biosynthesis in response to cell envelope stress. A madR-null mutant of M. smegmatis exhibited traits of an impaired cell wall with an altered outer mycomembrane, accumulation of a desaturated α-mycolate, susceptibility to antimycobacterials, and cell surface disruption. Transcriptomic profiling showed that enriched lipid metabolism genes that were significantly down-regulated upon madR deletion included acyl-coenzyme A (aceyl-CoA) dehydrogenases, implicating it in the indirect control of ß-oxidation pathways. Electromobility shift assays and binding affinities suggest a unique acyl-CoA pool-sensing mechanism, whereby MadR is able to bind a range of acyl-CoAs, including those with unsaturated as well as saturated acyl chains. MadR repression of desA1/desA2 is relieved upon binding of saturated acyl-CoAs of chain length C16 to C24, while no impact is observed upon binding of shorter chain and unsaturated acyl-CoAs. We propose this mechanism of regulation as distinct to other mycolic acid and fatty acid synthesis regulators and place MadR as the key regulatory checkpoint that coordinates mycolic acid remodeling during infection in response to host-derived cell surface perturbation.

NUDT7 regulates total hepatic CoA levels and the composition of the intestinal bile acid pool in male mice fed a Western diet.

Nudix hydrolase 7 (NUDT7) is an enzyme that hydrolyzes CoA species, is highly expressed in the liver, and resides in the peroxisomes. Peroxisomes are organelles where the preferential oxidation of dicarboxylic fatty acids occurs and where the hepatic synthesis of the primary bile acids cholic acid and chenodeoxycholic acid is completed. We previously showed that liver-specific overexpression of NUDT7 affects peroxisomal lipid metabolism but does not prevent the increase in total liver CoA levels that occurs during fasting. We generated Nudt7-/- mice to further characterize the role that peroxisomal (acyl-)CoA degradation plays in the modulation of the size and composition of the acyl-CoA pool and in the regulation of hepatic lipid metabolism. Here, we show that deletion of Nudt7 alters the composition of the hepatic acyl-CoA pool in mice fed a low-fat diet, but only in males fed a Western diet does the lack of NUDT7 activity increase total liver CoA levels. This effect is driven by the male-specific accumulation of medium-chain dicarboxylic acyl-CoAs, which are produced from the ß-oxidation of dicarboxylic fatty acids. We also show that, under conditions of elevated synthesis of chenodeoxycholic acid derivatives, Nudt7 deletion promotes the production of tauromuricholic acid, decreasing the hydrophobicity index of the intestinal bile acid pool and increasing fecal cholesterol excretion in male mice. These findings reveal that NUDT7-mediated hydrolysis of acyl-CoA pathway intermediates in liver peroxisomes contributes to the regulation of dicarboxylic fatty acid metabolism and the composition of the bile acid pool.

Clinical, pathological and genetic features and follow-up of 110 patients with late-onset MADD: a single-center retrospective study.

To observe a long-term prognosis in late-onset multiple acyl-coenzyme-A dehydrogenation deficiency (MADD) patients and to determine whether riboflavin should be administrated in the long-term and high-dosage manner, we studied the clinical, pathological and genetic features of 110 patients with late-onset MADD in a single neuromuscular center. The plasma riboflavin levels and a long-term follow-up study were performed. We showed that fluctuating proximal muscle weakness, exercise intolerance and dramatic responsiveness to riboflavin treatment were essential clinical features for all 110 MADD patients. Among them, we identified 106 cases with ETFDH variants, 1 case with FLAD1 variants and 3 cases without causal variants. On muscle pathology, fibers with cracks, atypical ragged red fibers (aRRFs) and diffuse decrease of SDH activity were the distinctive features of these MADD patients. The plasma riboflavin levels before treatment were significantly decreased in these patients as compared to healthy controls. Among 48 MADD patients with a follow-up of 6.1 years on average, 31 patients were free of muscle weakness recurrence, while 17 patients had episodes of slight muscle weakness upon riboflavin withdrawal, but recovered after retaking a small-dose of riboflavin for a short-term. Multivariate Cox regression analysis showed vegetarian diet and masseter weakness were independent risk factors for muscle weakness recurrence. In conclusion, fibers with cracks, aRRFs and diffuse decreased SDH activity could distinguish MADD from other genotypes of lipid storage myopathy. For late-onset MADD, increased fatty acid oxidation and reduced riboflavin levels can induce episodes of muscle symptoms, which can be treated by short-term and small-dose of riboflavin therapy.