ABSTRACT
Neuraminic acid synthases are an important yet underexplored group of enzymes. Thus, in this research, we performed a detailed kinetic and stability analysis and a comparison of previously known neuraminic acid synthase from Neisseria meningitidis, and a novel enzyme, PNH5, obtained from a metagenomic library. A systematic analysis revealed a high level of similarity of PNH5 to other known neuraminic acid synthases, except for its pH optimum, which was found to be at 5.5 for the novel enzyme. This is the first reported enzyme from this family that prefers an acidic pH value. The effect of different metal cofactors on enzyme activity, i.e. Co2+, Mn2+ and Mg2+, was studied systematically. The kinetics of neuraminic acid synthesis was completely elucidated, and an appropriate kinetic model was proposed. Enzyme stability study revealed that the purified enzyme exhibits changes in its structure during time as observed by differential light scattering, which cause a drop in its activity and protein concentration. The operational enzyme stability for the neuraminic acid synthase from N. meningitidis is excellent, where no activity drop was observed during the batch reactor experiments. In the case of PNH5, some activity drop was observed at higher concentration of substrates. The obtained results present a solid platform for the future application of these enzymes in the synthesis of sialic acids. KEY POINTS: ⢠A novel neuraminic acid synthase was characterized. ⢠The effect of cofactors on NeuS activity was elucidated. ⢠Kinetic and stability characterization of two neuraminic acid synthases was performed.
Subject(s)
Enzyme Stability , Neisseria meningitidis , Kinetics , Hydrogen-Ion Concentration , Neisseria meningitidis/enzymology , Neisseria meningitidis/genetics , Oxo-Acid-Lyases/metabolism , Oxo-Acid-Lyases/genetics , Oxo-Acid-Lyases/chemistry , Coenzymes/metabolismABSTRACT
Kainoid synthases are key enzymes in the biosynthesis of kainoids. Kainoids, as represented by DA and KA, are a class of naturally occurring non-protein amino acids with strong neurotransmitter activity in the mammalian central nervous system. Marine algae kainoid synthases include PnDabC from diatoms, which synthesizes domoic acid (DA), and DsKabC and GfKabC from red algae, which synthesize kainic acid (KA). Elucidation of the catalytic mechanism of kainoid synthases is of great significance for the rational design of better biocatalysts to promote the industrial production of kainoids for use in new drugs. Through modeling, molecular docking, and molecular dynamics simulations, we investigated the conformational dynamics of kainoid synthases. We found that the kainoid synthase complexes showed different stability in the simulation, and the binding and catalytic processes showed significant conformational transformations of kainoid synthase. The residues involved in specific interactions with the substrate contributed to the binding energy throughout the simulation process. Binding energy, the relaxed active pocket, electrostatic potential energy of the active pocket, the number and rotation of aromatic residues interacting with substrates during catalysis, and the number and frequency of hydrogen bonds between the individual functional groups revealed the structure-activity relationships and affected the degree of promiscuity of kainoid synthases. Our research enriches the understanding of the conformational dynamics of kainoid synthases and has potential guiding significance for their rational design.
Subject(s)
Diatoms , Kainic Acid , Molecular Docking Simulation , Molecular Dynamics Simulation , Structure-Activity Relationship , Kainic Acid/analogs & derivatives , Diatoms/enzymology , Rhodophyta/enzymology , Oxo-Acid-Lyases/chemistry , Oxo-Acid-Lyases/metabolism , Hydrogen BondingABSTRACT
Itaconate is a key anti-inflammatory/antibacterial metabolite in pathogen-macrophage interactions that induces adaptive changes in Pseudomonas aeruginosa-exposed airways. However, the impact and mechanisms underlying itaconate metabolism remain unclear. Our study reveals that itaconate significantly upregulates the expression of pyoverdine in P. aeruginosa and enhances its tolerance to tobramycin. Notably, the enzymes responsible for efficient itaconate metabolism, PaIch and PaCcl, play crucial roles in both utilizing itaconate and clearing its toxic metabolic intermediates. By using protein crystallography and molecular dynamics simulations analyses, we have elucidated the unique catalytic center and substrate-binding pocket of PaIch, which contribute to its highly efficient catalysis. Meanwhile, analysis of PaCcl has revealed how interactions between domains regulate the conformational changes of the active sites and binding pockets, influencing the catalytic process. Overall, our research uncovers the significance and mechanisms of PaIch and PaCcl in the efficient metabolism of itaconate by P. aeruginosa.
Subject(s)
Bacterial Proteins , Catalytic Domain , Oxo-Acid-Lyases , Pseudomonas aeruginosa , Succinates , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Binding Sites , Crystallography, X-Ray , Hydro-Lyases/metabolism , Hydro-Lyases/chemistry , Hydro-Lyases/genetics , Molecular Dynamics Simulation , Protein Binding , Pseudomonas aeruginosa/metabolism , Pseudomonas aeruginosa/enzymology , Substrate Specificity , Succinates/metabolism , Succinates/chemistry , Oxo-Acid-Lyases/chemistryABSTRACT
Three new formyl phloroglucinol meroterpenoids, eumaidials A-C (1-3), were isolated from the leaves of Eucalyptus globulus subsp. maidenii, along with ten known analogues (4-13). Their chemical structures were determined by various spectral data and electronic circular dichroism calculations. Eumaidial A (1) is the first ß-caryophyllene-based formyl phloroglucinol meroterpenoids from the genus Eucalyptus. Compounds 1-4 and 10 exhibited ATP-citrate lyase inhibitory activities, and compounds 2 and 3 suppressed the hepatocyte lipogenesis.
Subject(s)
Eucalyptus , Multienzyme Complexes , Oxo-Acid-Lyases , Molecular Structure , Eucalyptus/chemistry , Phloroglucinol/pharmacology , Phloroglucinol/chemistry , Plant Leaves/chemistry , Adenosine TriphosphateABSTRACT
Biallelic genetic variants in N-acetylneuraminic acid synthase (NANS), a critical enzyme in endogenous sialic acid biosynthesis, are clinically associated with neurodevelopmental disorders. However, the mechanism underlying the neuropathological consequences has remained elusive. Here, we found that NANS mutation resulted in the absence of both sialic acid and protein polysialylation in the cortical organoids and notably reduced the proliferation and expansion of neural progenitors. NANS mutation dysregulated neural migration and differentiation, disturbed synapse formation, and weakened neuronal activity. Single-cell RNA sequencing revealed that NANS loss of function markedly altered transcriptional programs involved in neuronal differentiation and ribosomal biogenesis in various neuronal cell types. Similarly, Nans heterozygous mice exhibited impaired cortical neurogenesis and neurobehavioral deficits. Collectively, our findings reveal a crucial role of NANS-mediated endogenous sialic acid biosynthesis in regulating multiple features of human cortical development, thus linking NANS mutation with its clinically relevant neurodevelopmental disorders.
Subject(s)
N-Acetylneuraminic Acid , Oxo-Acid-Lyases , Humans , Mice , Animals , N-Acetylneuraminic Acid/metabolism , Oxo-Acid-Lyases/genetics , Organoids/metabolism , Mutation , Neurogenesis/geneticsABSTRACT
Bempedoic acid is an adenosine triphosphate citrate lyase inhibitor that lowers low-density lipoprotein cholesterol by inhibiting cholesterol synthesis and upregulating hepatic low-density lipoprotein receptor expression. After oral dosing, bempedoic acid was readily absorbed, attaining maximum concentrations with a median time of 3.5 hours, and may be taken without regard to food. Steady-state oral pharmacokinetics in healthy adults receiving bempedoic acid at the approved 180 mg/day dose were characterized by mean maximum concentration of 20.6 µg/mL, area under the concentration-time curve over 24 hours of 289 µg·h/mL, and elimination half-life of 21.1 hours. Multiple-dose pharmacokinetics were linear at bempedoic acid doses of 120-220 mg/day. Circulating concentrations of the active metabolite ESP15228 were 18.0% of bempedoic acid concentrations on average. Comparisons of bempedoic acid 180 mg/day pharmacokinetics after single and multiple dosing revealed no clinically meaningful differences between Japanese, Chinese, and Western subjects. Mean estimates of bempedoic acid elimination half-life in Japanese (25.2 hours) and Chinese (20.0 hours) subjects were comparable to Western subjects (23.9 hours) following 14 days of once-daily dosing. Bempedoic acid was generally safe and well tolerated up to a dose of 220 mg/day across the study populations described herein.
Subject(s)
East Asian People , Hypolipidemic Agents , Oxo-Acid-Lyases , Adult , Humans , Adenosine Triphosphate/antagonists & inhibitors , Cholesterol, LDL , Oxo-Acid-Lyases/administration & dosage , Oxo-Acid-Lyases/adverse effects , Oxo-Acid-Lyases/antagonists & inhibitors , Hypolipidemic Agents/adverse effects , Hypolipidemic Agents/pharmacokinetics , Administration, OralABSTRACT
PURPOSE: The aim of our study is to describe the genetic features and correlation between the genotype and phenotype of Chinese patients with primary hyperoxaluria type 3 (PH3). METHODS: The genetic and clinical data of PH3 patients in our cohort were collected and analyzed retrospectively. All published studies of Chinese PH3 populations between January 2010 and November 2022 were searched and enrolled based on inclusive standards. RESULTS: A total of 60 Chinese PH3 patients (21 cases from our cohort and 39 cases from previous studies) were included. The mean age of onset was 1.62 ± 1.35 (range 0.4-7) years. A total of 29 different variants in the HOGA1 gene were found. The mutations were most commonly clustered in exons 1, 6, and 7. Among the genotypes, exon 6 skipping (c.834G > A and c.834_834 + 1GG > TT mutations) was the most common, followed by c.769 T > G; the allele frequencies (AFs) were 48.76% and 12.40%, respectively. Patients homozygous for exon 6 skipping exhibited a median age of onset of 0.67 (0.58-1) years, which was significantly lower than that observed among heterozygotes and nonexon 6 skipping patients (p = 0.021). A total of 22.5% (9/40) of PH3 patients had a decreased estimated glomerular filtration rate, and one patient with homozygous exon 6 skipping developed end-stage renal disease. CONCLUSIONS: A hotspot mutation, potential hotspot mutation and genotype-phenotype correlation were found in Chinese PH3 patients. This study expands the mutational spectrum and contributes to the understanding of genotypic profiles of PH3, which may provide a potential diagnostic and therapeutic target.
Subject(s)
Hyperoxaluria, Primary , Oxo-Acid-Lyases , Humans , East Asian People , Genotype , Hyperoxaluria, Primary/genetics , Mutation , Phenotype , Retrospective Studies , Oxo-Acid-Lyases/geneticsABSTRACT
In an era of increased accessibility to genetic testing, nephrologists may be able to better understand pathophysiologic mechanisms by which their patients develop specific conditions. In this study, we describe clinical and genetic findings of two patients with kidney cysts, who were found to have variants in HOGA1, a mitochondrial 4-hydroxy-2-oxoglutarate aldolase enzyme associated with primary hyperoxaluria type 3 and the development of oxalate-containing kidney stones. We describe possible mechanisms by which mutations in this enzyme could result in the kidney cyst formation seen in our two patients. We propose that patients with mutations in HOGA1 are predisposed to crystal or stone deposition, tubule dilation, and inflammasome activation, which can result in kidney cyst formation.
Subject(s)
Cysts , Hyperoxaluria, Primary , Kidney Calculi , Oxo-Acid-Lyases , Humans , Hyperoxaluria, Primary/genetics , Kidney , Oxo-Acid-Lyases/chemistry , Oxo-Acid-Lyases/geneticsABSTRACT
Aminodeoxychorismate lyase (ADCL) is a kind of pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes the conversion of 4-amino-4-deoxychorismate (ADC) to p-aminobenzoate (PABA), which is a key step for the biosynthesis of folate. To illuminate the reaction details at the atomistic level, an enzyme-substrate reactant model has been constructed, and QM/MM calculations have been performed. Our calculation results reveal that the overall catalytic cycle contains 11 elementary steps, which can be described by three stages, including the transamination reaction of PLP, the release of pyruvate and aromatization of ADC, and the recovery to the initial aldimine. During the reaction, a series of intramolecular proton transfer are involved, which are the key for the C-N bond formation and cleavage as well as the aromatization of the ADC ring. In addition to forming the Schiff base with the pocket residue Lys251 and substrate in the internal aldimine and the external aldimine, respectively, the coenzyme PLP also plays a critical role in the intramolecular proton transfer by employing its hydroxyl oxygen anion and phosphate group. These findings may provide useful information for further understanding the catalytic mechanism of other PLP-dependent enzymes.
Subject(s)
Oxo-Acid-Lyases , Protons , Pyridoxal Phosphate/chemistry , Oxo-Acid-Lyases/chemistry , PhosphatesABSTRACT
BACKGROUND: Metabolic disorder is an essential characteristic of tumor development. Ketogenesis is a heterogeneous factor in multiple cancers, but the effect of ketogenesis on hepatocellular carcinoma (HCC) is elusive. METHODS: We aimed to explain the role of ketogenesis-related hydroxy-methyl-glutaryl-CoA lyase (HMGCL) on HCC suppression. Expression pattern of HMGCL in HCC specimens was evaluated by immunohistochemistry (IHC). HMGCL was depleted or overexpressed in HCC cells to investigate the functions of HMGCL in vitro and in vivo. The anti-tumor function of HMGCL was studied in subcutaneous xenograft and Trp53Δhep/Δhep; c-Myc-driven HCC mouse models. The mechanism of HMGCL-mediated tumor suppression was studied by IHC, western blot (WB) and Cut & Tag. RESULTS: HMGCL depletion promoted HCC proliferation and metastasis, whereas its overexpression reversed this trend. As HMGCL catalyzes ß-hydroxy-butyric acid (ß-OHB) production, we discovered that HMGCL increased acetylation at histone H3K9, which further promoted the transcription of dipeptidyl peptidase 4 (DPP4), a key protein maintains intracellular lipid peroxidation and iron accumulation, leading to HCC cells vulnerability to erastin- and sorafenib-induced ferroptosis. CONCLUSION: Our study identified a critical role of HMGCL on HCC suppression, of which HMGCL regulated H3K9 acetylation through ß-OHB and modulating the expression of DPP4 in a dose-dependent manner, which led to ferroptosis in HCC cells.
Subject(s)
Carcinoma, Hepatocellular , Dipeptidyl Peptidase 4 , Ferroptosis , Liver Neoplasms , Oxo-Acid-Lyases , Animals , Humans , Mice , 3-Hydroxybutyric Acid/metabolism , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Dipeptidyl Peptidase 4/genetics , Dipeptidyl Peptidase 4/metabolism , Ferroptosis/genetics , Ferroptosis/physiology , Histones/metabolism , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Lyases/genetics , Lyases/metabolism , Oxo-Acid-Lyases/genetics , Oxo-Acid-Lyases/metabolismABSTRACT
Primary hyperoxalurias (PH) are a group of rare heterogeneous disorders characterized by deficiencies in glyoxylate metabolism. To date, three genes have been identified to cause three types of PH (I, II, and III). The HOGA1 gene caused type III in around 10% of the PH cases. Disease-associated pathogenic variants have been reported from several populations and a comprehensive spectrum of these mutations and genotype-phenotype correlation has never been presented. In this study, we describe new cases of the HOGA1 gene pathogenic variants identified in our population. We report the first case of ESKD with successful kidney transplantation with 5 years of follow-up. Furthermore, a comprehensive overview of PH type III associated HOGA1 gene variants was carried out. Compiling the data from the literature, we reviewed 57 distinct HOGA1 gene pathogenic variants in 175 patients worldwide. The majority of reported variants are missense variants that predicted a loss of function mechanism as the underlying pathology. There has been evidence of the presence of founder mutations in several populations like Europeans, Ashkenazi Jews, Arab, and Chinese populations. No significant genotype-phenotype correlation was identified concerning the ages of onset of the disease and biochemical and metabolic parameters. Nephrocalcinosis was rare in patients with disease-associated variants. Most of the patients were presented with urolithiasis early in life; only five cases reported disease progression after the second decade of life. The establishment of impairment of renal function in 8% of all the reported cases makes this type a relatively severe form of primary hyperoxaluria, not a benign etiology as suggested previously.
Subject(s)
Hyperoxaluria, Primary , Oxo-Acid-Lyases , Humans , Hyperoxaluria, Primary/diagnosis , Hyperoxaluria, Primary/genetics , Hyperoxaluria, Primary/metabolism , Mutation , Oxo-Acid-Lyases/genetics , Oxo-Acid-Lyases/metabolismABSTRACT
Succinyl-CoA synthetase (SCS) catalyzes a three-step reaction in the citric acid cycle with succinyl-phosphate proposed as a catalytic intermediate. However, there are no structural data to show the binding of succinyl-phosphate to SCS. Recently, the catalytic mechanism underlying acetyl-CoA production by ATP-citrate lyase (ACLY) has been debated. The enzyme belongs to the family of acyl-CoA synthetases (nucleoside diphosphate-forming) for which SCS is the prototype. It was postulated that the amino-terminal portion catalyzes the full reaction and the carboxy-terminal portion plays only an allosteric role. This interpretation was based on the partial loss of the catalytic activity of ACLY when Glu599 was mutated to Gln or Ala, and on the interpretation that the phospho-citryl-CoA intermediate was trapped in the 2.85â Å resolution structure from cryogenic electron microscopy (cryo-EM). To better resolve the structure of the intermediate bound to the E599Q mutant, the equivalent mutation, E105αQ, was made in human GTP-specific SCS. The structure of the E105αQ mutant shows succinyl-phosphate bound to the enzyme at 1.58â Å resolution when the mutant, after phosphorylation in solution by Mg2+-ATP, was crystallized in the presence of magnesium ions, succinate and desulfo-CoA. The E105αQ mutant is still active but has a specific activity that is 120-fold less than that of the wild-type enzyme, with apparent Michaelis constants for succinate and CoA that are 50-fold and 11-fold higher, respectively. Based on this high-resolution structure, the cryo-EM maps of the E599Q ACLY complex reported previously should have revealed the binding of citryl-phosphate and CoA and not phospho-citryl-CoA.
Subject(s)
ATP Citrate (pro-S)-Lyase , Succinate-CoA Ligases , ATP Citrate (pro-S)-Lyase/chemistry , ATP Citrate (pro-S)-Lyase/genetics , ATP Citrate (pro-S)-Lyase/metabolism , Acetyl Coenzyme A , Acyl Coenzyme A , Adenosine Triphosphate/metabolism , Crystallography, X-Ray , Diphosphates , Guanosine Triphosphate/metabolism , Humans , Magnesium , Multienzyme Complexes , Nucleosides , Oxo-Acid-Lyases , Succinate-CoA Ligases/chemistry , Succinates , Succinic Acid/metabolismABSTRACT
BACKGROUND: ATP-citrate lyase (ACL) plays a pivotal role in histone acetylation and aerobic glycolysis. In plant, ACL is a heteromeric enzyme composed of ACLA (45 kD) and ACLB (65 kD). So far, the function of ACL genes in cotton still remains unknown. RESULTS: Here, we identified three ACLA homologous sequences and two ACLB homologous in each genome/sub-genome of cotton species. Silencing ACLB in cotton led to cell death at newly-grown leaves and stem apexes. Simultaneously, in ACLB-silenced plants, transcription factors related to senescence including SGR, WRKY23 and Osl57 were observed to be activated. Further investigation showed that excessive H2O2 was accumulated, salicylic acid-dependent defense response and pathogenesis-related gene expressions were evidently enhanced in ACLB-silenced plants, implying that knockdown of ACLB genes leads to hypersensitive response-like cell death in cotton seedlings. However, as noted, serious cell death happened in newly-grown leaves and stem apexes in ACLB-silenced plants, which led to the failure of subsequent fungal pathogenicity assays. To confirm the role of ACLB gene in regulating plant immune response, the dicotyledonous model plant Arabidopsis was selected for functional verification of ACLB gene. Our results indicate the resistance to Verticillium dahliae infection in the Arabidopsis mutant aclb-2 were enhanced without causing strong cell death. Ectopic expression of GausACLB-2 in Arabidopsis weakened its resistance to V. dahliae either in Col-0 or in aclb-2 background, in which the expression level of ACLB is negatively correlated with the resistance to V. dahliae. CONCLUSIONS: These results indicate that ACLB has a new function in negatively affecting the induction of plant defense response and cell death in cotton, which provides theoretical guidance for developing cotton varieties with resistance against Verticillium wilt.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Verticillium , ATP Citrate (pro-S)-Lyase/metabolism , Adenosine Triphosphate , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Cell Death , Histones , Hydrogen Peroxide/metabolism , Multienzyme Complexes , Oxo-Acid-Lyases , Plant Diseases/genetics , Plant Diseases/microbiology , Salicylic Acid/metabolism , Transcription Factors/metabolism , Verticillium/physiologyABSTRACT
AIM: The present study aimed mainly to demonstrate the metabolic effects of lycopene (LYC) or atorvastatin (ATOR) in diabetic hyperlipidemic rat model. MAIN METHODS: Rats were randomly classified into four groups; the first was fed normal chow diet (NC) while the other three groups received streptozotocin (STZ) along with CCT-diet. The second group received no treatment (diabetic hyperlipidemic control, DHC), the third one received ATOR (50 mg/kg/day) while the fourth one received LYC (20 mg/kg/day). Serum and tissue samples were collected for biochemical and histological evaluations. KEY FINDINGS: DHC rats demonstrated significant hyperglycemia, dyslipidemia, increased hepatic fatty acid synthetase (FAS), malondialdehyde (MDA), tumor necrosis factor- alpha (TNF-α), 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase and ATP citrate lyase (ACLY). However, hepatic reduced glutathione (GSH) and phosphorylated form of AMP-activated protein kinase (AMPK-P) activities showed significant decreases. ATOR or LYC administration induced hypoglycemic and hypolipidemic effects; decreased hepatic levels of MDA, TNF-α, HMG-CoA reductase, ACLY and FAS along with GSH and AMPK-P increases. Histopathological findings showed clear correlation with the biomarkers results. SIGNIFICANCE: LYC demonstrated favorable significant effects regarding the biomarkers studied as compared to ATOR and may be expressed as a potent therapeutic agent of natural origin for hyperlipidemia complications either alone or in combination with other hypolipidemic drugs.
Subject(s)
Diabetes Mellitus , Hyperlipidemias , AMP-Activated Protein Kinases , ATP Citrate (pro-S)-Lyase/metabolism , Adenosine Triphosphate , Animals , Atorvastatin/therapeutic use , Biomarkers , Coenzyme A , Fatty Acid Synthases , Glutathione , Hyperlipidemias/complications , Hyperlipidemias/drug therapy , Hypoglycemic Agents/therapeutic use , Hypolipidemic Agents/therapeutic use , Lycopene , Malondialdehyde , Multienzyme Complexes , Oxo-Acid-Lyases , Rats , Streptozocin , Tumor Necrosis Factor-alphaABSTRACT
Aldolases are enzymes that reversibly catalyze the cleavage of carbon-carbon bonds. Here we describe a recombinant sialic acid aldolase originating from the freshwater snail Biomphalaria glabrata (sNPL), and compare its substrate spectrum with a sialic acid aldolase originating from chicken (chNPL). In contrast to vertebrate animals which can synthesize, degrade, and incorporate sialic acids on glycoconjugate ubiquitously, snails (as all mollusks) cannot synthesize sialic acids endogenously, and therefore the biological function and substrate scope of sNPL ought to differ significantly from vertebrate sialic aldolases such as chNPL. sNPL was active towards a series of sialic acid derivatives but was in contrast to chNPL unable to catalyze the cleavage of N-acetylneuraminic acid into N-acetylmannosamine and pyruvate. Interestingly, chNPL and sNPL showed contrasting C4(R)/(S) diastereoselectivity towards the substrates d-mannose and d-galactose in the presence of pyruvate. In addition, sNPL was able to synthesize a series of 4-hydroxy-2-oxoates using the corresponding aliphatic aldehyde substrates in the presence of pyruvate, which could be not achieved by chNPL.
Subject(s)
Aldehyde-Lyases , Aldehydes , Aldehyde-Lyases/metabolism , Aldehydes/metabolism , Animals , Carbon , Fructose-Bisphosphate Aldolase/metabolism , N-Acetylneuraminic Acid , Oxo-Acid-Lyases , Pyruvic Acid , Sialic Acids , Snails/metabolism , Substrate SpecificityABSTRACT
N-acetylneuraminic acid (sialic acid) is an abundantly found carbohydrate moiety covering the surface of all vertebrate cells and secreted glycoproteins. The human N-acetylneuraminate pyruvate lyase (NPL) interconverts sialic acid to N-acetylmannosamine and pyruvate, and mutations of the NPL gene were found to cause sialuria and impair the functionality of muscles. Here we report the soluble and functional expression of human NPL in Escherichia coli, which allowed us to study the biochemical properties of two clinically relevant NLP mutations (Asn45Asp and Arg63Cys). The Asn45Asp mutant variant was enzymatically active, but had lower expression levels and showed reduced stability when compared to the wild-type NPL variant. Expression trials of the Arg63Cys mutant did not yield any recombinant protein and consequently, no enzymatic activity was detected. The locations of these clinically relevant amino acid substitutions are also discussed by using a human NPL homology model.
Subject(s)
Lyases , Oxo-Acid-Lyases , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , N-Acetylneuraminic Acid/chemistry , Oxo-Acid-Lyases/chemistry , Oxo-Acid-Lyases/genetics , Oxo-Acid-Lyases/metabolism , PyruvatesABSTRACT
INTRODUCTION: ATP citrate lyase (ACLY) is a key enzyme in cellular metabolism, being the main source of acetyl-Coenzyme A, an important precursor for fatty acid, cholesterol, and isoprenoid biosynthesis, and it is also involved in protein acetylation. Its expression changes are related to hyperlipidemia and cardiovascular diseases. Other studies have shown that ACLY is closely related to the occurrence of cancer: the increase in lipid synthesis provides the necessary building blocks for cell growth and division. Therefore, finding effective ACLY inhibitors has very important application prospects for lipid-related pathologies and cancer. AREAS COVERED: This review covers patents concerning ACLY inhibitors and alternative strategies to modulate ACLY activity, with their potential therapeutic applications. EXPERT OPINION: In recent years, ACLY as a drug target has become a hot spot in the research of innovative drugs for disorders of glucose and lipid metabolism. Many types of small-molecule ACLY inhibitors have been discovered, but few ACLY inhibitors proved to be highly effective in vitro and in vivo, since their main limitations were low cell penetration and low affinity to ACLY. The search for new effective ACLY inhibitors is of great significance and has broad application prospects for the treatment of hyperlipidemia and cancer.
Subject(s)
Neoplasms , Patents as Topic , ATP Citrate (pro-S)-Lyase/metabolism , Adenosine Triphosphate , Fatty Acids , Humans , Multienzyme Complexes , Neoplasms/drug therapy , Neoplasms/pathology , Oxo-Acid-LyasesABSTRACT
Pancreatic ductal adenocarcinoma (PDA) tumor cells are deprived of oxygen and nutrients and therefore must adapt their metabolism to ensure proliferation. In some physiological states, cells rely on ketone bodies to satisfy their metabolic needs, especially during nutrient stress. Here, we show that PDA cells can activate ketone body metabolism and that ß-hydroxybutyrate (ßOHB) is an alternative cell-intrinsic or systemic fuel that can promote PDA growth and progression. PDA cells activate enzymes required for ketogenesis, utilizing various nutrients as carbon sources for ketone body formation. By assessing metabolic gene expression from spontaneously arising PDA tumors in mice, we find HMG-CoA lyase (HMGCL), involved in ketogenesis, to be among the most deregulated metabolic enzymes in PDA compared to normal pancreas. In vitro depletion of HMGCL impedes migration, tumor cell invasiveness, and anchorage-independent tumor sphere compaction. Moreover, disrupting HMGCL drastically decreases PDA tumor growth in vivo, while ßOHB stimulates metastatic dissemination to the liver. These findings suggest that ßOHB increases PDA aggressiveness and identify HMGCL and ketogenesis as metabolic targets for limiting PDA progression.
Subject(s)
Ketone Bodies , Pancreatic Neoplasms , 3-Hydroxybutyric Acid/metabolism , Animals , Ketone Bodies/metabolism , Mice , Oxo-Acid-Lyases , Pancreas/metabolismABSTRACT
Sialic acid and its catabolism are involved in bacterial pathogenicity. N-acetylneuraminate lyase (NAL), which catalyzes the reversible aldol cleavage of sialic acid to form N-acetyl-D-mannosamine in the first step of sialic acid degradation, has been recently investigated to elucidate whether NAL enhances bacterial virulence; however, the role of NAL in bacterial pathogenicity remains unclear. In the present study, we demonstrated that the existence of two enzymes in Edwardsiella piscicida, referred to as dihydrodipicolinate synthase (DHDPS) and NAL, induced the cleavage/condensation activity toward sialic acids such as N-acetylneuraminic acid, N-glycolylneuraminic acid and 3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid. NAL enhanced cellular infection in vitro and suppressed the survival rate in zebrafish larvae in bath-infection in vivo, whereas DHDPS did not. Furthermore, NAL strongly activated the expression of E. piscicida phenotypes such as biofilm formation and motility, whereas DHDPS did not. Besides, the gene expression level of nanK, nanE, and glmU were up-regulated in the NAL-overexpressing strain, along with an increase in the total amount of N-acetylglucosamine.
Subject(s)
N-Acetylneuraminic Acid , Zebrafish , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biofilms , Edwardsiella , N-Acetylneuraminic Acid/metabolism , Oxo-Acid-LyasesABSTRACT
The ethanolic extracts of the dried flower buds of two Caprifoliaceae plants, Lonicera japonica and Abelia × grandiflora, showed considerable inhibitory activities against adenosine triphosphate (ATP)-citrate lyase (ACL), a new promising drug target for the treatment of metabolic disorders. Bioassay-guided purification in conjunction with HPLC-PDA profiling led to the isolation and characterization of thirty-five (1-35) and fourteen (1'-14') structurally diverse compounds from the above two plant extracts, respectively. Compounds 1-9 and 1'-6' are previously undescribed glycosides. Their structures were elucidated on the basis of spectroscopic data, electronic circular dichroism (ECD), and single crystal X-ray diffraction analyses. In particular, lonicejaposide A (1) has an unprecedented skeleton generated through the coupling of C-7 in secologanin with C-2'' in phenylacetaldehyde via an aldol condensation. Abeliflorosides A (1') and B (2') are hitherto unknown glycosides of triterpene and bisiridoid conjugates constructed through the formation of a 1,3-dioxane moiety. All the isolates were evaluated for their inhibitory activities against ACL. Compounds 9, 25-28, 31, 1', 2', and 14' displayed significant inhibitory effects, with IC50 values ranging from 0.1 to 14.2 µM. The interactions of selected compounds possessing different structure features (e.g., 9, 25, 31, and 2') with ACL were thereafter performed by employing molecular docking studies. In addition, compound 2', the most complex triterpene-bisiridoid conjugate glycoside reported herein, also inhibited acetyl-CoA carboxylase 1 (ACC1), with an IC50 value of 7.9 µM. The dried material of the flower buds of L. japonica (honeysuckle) is a well-known traditional oriental medicine (i.e., Flos Lonicerae Japonicae, FLJ) and has long been used in large quantities. The above findings not only provide new insights for the development of multipurpose utilization of FLJ in healthcare community, but also provide profitable clues indicating that the flower buds of A. × grandiflora might be a potential alternative to FLJ in the traditional Chinese medicine market.