Discovery of a metagenome-derived enzyme that produces branched-chain acyl-(acyl-carrier-protein)s from branched-chain α-keto acids.
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ABSTRACT:
SUBMITTER: Craig JW
PROVIDER: S-EPMC3495001 | biostudies-literature | 2011 Aug
REPOSITORIES: biostudies-literature
Publications
Discovery of a metagenome-derived enzyme that produces branched-chain acyl-(acyl-carrier-protein)s from branched-chain α-keto acids.
Craig Jeffrey W JW Brady Sean F SF
Chembiochem : a European journal of chemical biology 20110628 12
Similar Datasets
Project description:Branched-chain amino acid (BCAA) metabolism is linked to glucose homeostasis, but the underlying signaling mechanisms are unclear. We find that gluconeogenesis is reduced in mice deficient of Ppm1k, a positive regulator of BCAA catabolism, which protects against obesity-induced glucose intolerance. Accumulation of branched-chain keto acids (BCKAs) inhibits glucose production in hepatocytes. BCKAs suppress liver mitochondrial pyruvate carrier (MPC) activity and pyruvate-supported respiration. Pyruvate-supported gluconeogenesis is selectively suppressed in Ppm1k-deficient mice and can be restored with pharmacological activation of BCKA catabolism by BT2. Finally, hepatocytes lack branched-chain aminotransferase that alleviates BCKA accumulation via reversible conversion between BCAAs and BCKAs. This renders liver MPC most susceptible to circulating BCKA levels hence a sensor of BCAA catabolism.
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Project description:Compound lipids comprise a diverse group of metabolites present in living systems, and metabolic- and environmentally-driven structural distinctions across this family are increasingly linked to biological function. However, methods for deconvoluting these often isobaric lipid species are lacking or require specialized instrumentation. Notably, acyl-chain diversity within cells may be influenced by nutritional states, metabolic dysregulation, or genetic alterations. Therefore, a reliable, validated method of quantifying structurally similar even-, odd-, and branched-chain acyl groups within intact compound lipids will be invaluable for gaining molecular insights into their biological functions. Here we demonstrate the chromatographic resolution of isobaric lipids containing distinct combinations of straight-chain and branched-chain acyl groups via ultra-high-pressure liquid chromatography (UHPLC)-mass spectrometry (MS) using a C30 liquid chromatography column. Using metabolically engineered adipocytes lacking branched-keto acid dehydrogenase A (Bckdha), we validate this approach through a combination of fatty acid supplementation and metabolic tracing using monomethyl branched-chain fatty acids and valine. We observe the resolution of numerous isobaric triacylglycerols and other compound lipids, demonstrating the resolving utility of this method. This approach adds to the toolbox for laboratories to quantify and characterize acyl chain diversity across the lipidome.
| S-EPMC11621494 | biostudies-literature
Project description:Leptomeningeal disease (LMD) occurs when tumors seed into the leptomeningeal space and cerebrospinal fluid (CSF), leading to severe neurological deterioration and poor survival outcomes. We utilized comprehensive multi-omics analyses of CSF from patients with lymphoma LMD to demonstrate an immunosuppressive cellular microenvironment and identified dysregulations in proteins and lipids indicating neurodegenerative processes. Strikingly, we found a significant accumulation of toxic branched-chain keto acids (BCKA) in the CSF of patients with LMD. The BCKA accumulation was found to be a pan-cancer occurrence, evident in lymphoma, breast cancer, and melanoma LMD patients. Functionally, BCKA disrupted the viability and function of endogenous T lymphocytes, chimeric antigen receptor (CAR) T cells, neurons, and meningeal cells. Treatment of LMD mice with BCKA-reducing sodium phenylbutyrate significantly improved neurological function, survival outcomes, and efficacy of anti-CD19 CAR T cell therapy. This is the first report of BCKA accumulation in LMD and provides preclinical evidence that targeting these toxic metabolites improves outcomes.
| S-EPMC10769272 | biostudies-literature
Project description:Branched chain amino acids (BCAAs), leucine, isoleucine and valine, are essential amino acids widely studied for their crucial role in the regulation of protein synthesis mainly through the activation of the mTOR signaling pathway and their emerging recognition as players in the regulation of various physiological and metabolic processes, such as glucose homeostasis. BCAA supplementation is primarily used as a beneficial nutritional intervention in chronic liver and kidney disease as well as in muscle wasting disorders. However, downregulated/upregulated plasma BCAAs and their defective catabolism in various tissues, mainly due to altered enzymatic activity of the first two enzymes in their catabolic pathway, BCAA aminotransferase (BCAT) and branched-chain α-keto acid dehydrogenase (BCKD), have been investigated in many nutritional and disease states. The current review focused on the underlying mechanisms of altered BCAA catabolism and its contribution to the pathogenesis of a numerous pathological conditions such as diabetes, heart failure and cancer. In addition, we summarize findings that indicate that the recovery of the dysregulated BCAA catabolism may be associated with an improved outcome and the prevention of serious disease complications.
| S-EPMC8999875 | biostudies-literature
Project description:ObjectivesInsulin resistance (IR) in adolescents with obesity is associated with a sex-dependent metabolic 'signature' comprising the branched-chain amino acids (BCAAs), glutamate/glutamine, C3/C5 acylcarnitines and uric acid. Here, we compared the levels of branched-chain α-keto acids (BCKAs) and glutamate/glutamine, which are the byproducts of BCAA catabolism and uric acid among adolescents with obesity prior to and following a 6-month lifestyle-intervention program.MethodsFasting plasma samples from 33 adolescents with obesity (16 males, 17 females, aged 12-18 year) were analysed by flow-injection tandem MS and LC-MS/MS. Multiple linear regression models were used to correlate changes in BCKAs, glutamate/glutamine and uric acid with changes in weight and insulin sensitivity as assessed by HOMA-IR, adiponectin and the ratio of triglyceride (TG) to HDL. In predictive models, BCKAs, glutamate/glutamine and uric acid at baseline were used as explanatory variables.ResultsBaseline BCKAs, glutamate/glutamine and uric acid were higher in males than females despite comparable BMI-metrics. Following lifestyle-intervention, α-keto-β-methylvalerate (α-KMV, a metabolic by product of isoleucine) decreased in males but not in females. The ratio of BCKA/BCAA trended lower in males. In the cohort as a whole, BCKAs correlated positively with the ratio of TG to HDL at baseline and HOMA-IR at 6-month-follow-up. Glutamate/glutamine was positively associated with HOMA-IR at baseline and 6-month-follow-up. A reduction in BCKAs was associated with an increase in adiponectin, and those with higher BCKAs at baseline had higher adiponectin levels at 6-month-follow-up. Interestingly those adolescents with higher uric acid levels at baseline had greater reduction in weight.ConclusionsBCKAs and glutamate/glutamine may serve as biomarkers of IR in adolescents with obesity, and uric acid might serve as a predictor of weight loss in response to lifestyle-intervention. Differential regulation of BCAA catabolism in adolescent males and females implicates critical roles for sex steroids in metabolic homeostasis.
| S-EPMC9836245 | biostudies-literature
Project description:Triheptanoin (triheptanoylglycerol) has shown value as anaplerotic therapy for patients with long chain fatty acid oxidation disorders but is contraindicated in medium-chain acyl-CoA dehydrogenase (MCAD) deficiency. In search for anaplerotic therapy for patients with MCAD deficiency, fibroblasts from three patients homozygous for the most common mutation, ACADMG985A/G985A, were treated with fatty acids hypothesized not to require MCAD for their metabolism, including heptanoic (C7; the active component of triheptanoin), 2,6-dimethylheptanoic (dMC7), 6-amino-2,4-dimethylheptanoic (AdMC7), or 4,8-dimethylnonanoic (dMC9) acids. Their effectiveness as anaplerotic fatty acids was assessed in live cells by monitoring changes in cellular oxygen consumption rate (OCR) and mitochondrial protein lysine succinylation, which reflects cellular succinyl-CoA levels, using immunofluorescence (IF) staining. Krebs cycle intermediates were also quantitated in these cells using targeted metabolomics. The four fatty acids induced positive changes in OCR parameters, consistent with their oxidative catalysis and utilization. Increases in cellular IF staining of succinylated lysines were observed, indicating that the fatty acids were effective sources of succinyl-CoA in the absence of media glucose, pyruvate, and lipids. The ability of MCAD deficient cells to metabolize C7 was confirmed by the ability of extracts to enzymatically utilize C7-CoA as substrate but not C8-CoA. To evaluate C7 therapeutic potential in vivo, Acadm-/- mice were treated with triheptanoin for seven days. Dose dependent increase in plasma levels of heptanoyl-, valeryl-, and propionylcarnitine indicated efficient metabolism of the medication. The pattern of the acylcarnitine profile paralleled resolution of liver pathology including reversing hepatic steatosis, increasing hepatic glycogen content, and increasing hepatocyte protein succinylation, all indicating improved energy homeostasis in the treated mice. These results provide the impetus to evaluate triheptanoin and the medium branched chain fatty acids as potential therapeutic agents for patients with MCAD deficiency.
| S-EPMC10840664 | biostudies-literature
Project description:In some microorganisms, polyunsaturated fatty acids (PUFAs) are biosynthesized by PUFA synthases characterized by tandem acyl carrier proteins (ACPs) in subunit A. These ACPs were previously shown to be important for PUFA productivity. In this study, we examined their function in more detail. PUFA productivities increased depending on the number of ACPs without profile changes in each subunit A of eukaryotic and prokaryotic PUFA synthases. We also constructed derivative enzymes from subunit A with 5 × ACPs. Enzymes possessing one inactive ACP at any position produced ~30% PUFAs compared with the parental enzyme but unexpectedly had ~250% productivity compared with subunit A with 4 × ACPs. Enzymes constructed by replacing the 3rd ACP with an inactive ACP from another subunit A or ACP-unrelated sequences produced ~100% and ~3% PUFAs compared with the parental 3rd ACP-inactive enzyme, respectively. These results suggest that both the structure and number of ACP domains are important for PUFA productivity.
| S-EPMC5067506 | biostudies-literature
Project description:Deficient branched-chain amino acids (BCAAs) are implicated in cognitive dysfunction after traumatic brain injury (TBI). The mechanism remains unknown. BCAAs are catabolized by neuron-specific cytosolic and astrocyte-specific mitochondrial branched-chain aminotransferases (BCATc, BCATm) to generate glutamate and branched-chain keto-acids (BCKAs) that are metabolized by the mitochondrial branched-chain keto-acid dehydrogenase (BCKD) whose activity is regulated by its phosphorylation state. BCKD phosphorylation by BCKD kinase (BCKDK) inactivates BCKD and cause neurocognitive dysfunction, whereas dephosphorylation by specific phosphatase restores BCKD activity. Real-time polymerase chain reaction showed rapidly and significantly decreased BCATc messenger RNA (mRNA) levels, but significantly increased BCATm mRNA level post-CCI (controlled cortical impact). BCKD and BCKDK mRNA decreased significantly immediately after CCI-induced TBI (CCI) in the rat. Phosphorylated BCKD proteins (pBCKD) increased significantly in the ipsilateral-CCI hemisphere. Immunohistochemistry revealed significantly increased pBCKD proteins in ipsilateral astrocytes post-CCI. BCKD protein expression is higher in primarily cultured cortical neurons than in astrocytes, whereas pBCKD protein level is higher in astrocytes than in cortical neurons. Transforming growth factor beta treatment (10 μg/mL for 48 h) significantly increased pBCKD protein expression in astrocytes, whereas glutamate treatment (25 μM for 24 h) significantly decreased pBCKD protein in neurons. Because increased pBCKD would lead to increased BCKA accumulation, BCKA-mediated astrocyte activation, cell death, and cognitive dysfunction as found in maple syrup urine disease; thus, TBI may potentially induce cognitive deficit through diverting BCAA from glutamate production in neurons to BCKA production in astrocytes through the pBCKD-dependent mechanism.
| S-EPMC6196747 | biostudies-literature
Project description:Genes encoding a branched-chain alpha-keto acid dehydrogenase from Enterococcus faecalis 10C1, E1alpha (bkdA), E1beta (bkdB), E2 (bkdC), and E3 (bkdD), were found to reside in the gene cluster ptb-buk-bkdDABC. The predicted products of ptb and buk exhibited significant homology to the phosphotransbutyrylase and butyrate kinase, respectively, from Clostridium acetobutylicum. Activity and redox properties of the purified recombinant enzyme encoded by bkdD indicate that E. faecalis has a lipoamide dehydrogenase that is distinct from the lipoamide dehydrogenase associated with the pyruvate dehydrogenase complex. Specific activity of the ptb gene product expressed in Escherichia coli was highest with the substrates valeryl-coenzyme A (CoA), isovaleryl-CoA, and isobutyryl-CoA. In cultures, a stoichiometric conversion of alpha-ketoisocaproate to isovalerate was observed, with a concomitant increase in biomass. We propose that alpha-ketoisocaproate is converted via the BKDH complex to isovaleryl-CoA and subsequently converted into isovalerate via the combined actions of the ptb and buk gene products with the concomitant phosphorylation of ADP. In contrast, an E. faecalis bkd mutant constructed by disruption of the bkdA gene did not benefit from having alpha-ketoisocaproate in the growth medium, and conversion to isovalerate was less than 2% of the wild-type conversion. It is concluded that the bkd gene cluster encodes the enzymes that constitute a catabolic pathway for branched-chain alpha-keto acids that was previously unidentified in E. faecalis.
| S-EPMC94053 | biostudies-literature
Project description:Branched chain α-keto acids (BCKAs) are endogenous metabolites of branched-chain amino acids (BCAAs). BCAA and BCKA are significantly elevated in pathologically stressed heart and contribute to chronic pathological remodeling and dysfunction. However, their direct impact on acute cardiac injury is unknown. Here, we demonstrated that elevated BCKAs significantly attenuated ischemia-reperfusion (I/R) injury and preserved post I/R function in isolated mouse hearts. BCKAs protected cardiomyocytes from oxidative stress-induced cell death in vitro. Mechanistically, BCKA protected oxidative stress induced cell death by inhibiting necrosis without affecting apoptosis or autophagy. Furthermore, BCKAs, but not BCAAs, protected mitochondria and energy production from oxidative injury. Finally, administration of BCKAs during reperfusion was sufficient to significantly attenuate cardiac I/R injury. These findings uncover an unexpected role of BCAA metabolites in cardioprotection against acute ischemia/reperfusion injury, and demonstrate the potential use of BCKA treatment to preserve ischemic tissue during reperfusion.
| S-EPMC5174097 | biostudies-literature