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1.
Eur J Pharmacol ; 954: 175849, 2023 Sep 05.
Article in English | MEDLINE | ID: mdl-37331684

ABSTRACT

Short-chain acyl-CoA dehydrogenase (SCAD), the rate-limiting enzyme for fatty acid ß-oxidation, has a negative regulatory effect on pathological cardiac hypertrophy and fibrosis. FAD, a coenzyme of SCAD, participates in the electron transfer of SCAD-catalyzed fatty acid ß-oxidation, which plays a crucial role in maintaining the balance of myocardial energy metabolism. Insufficient riboflavin intake can lead to symptoms similar to short-chain acyl-CoA dehydrogenase (SCAD) deficiency or flavin adenine dinucleotide (FAD) gene abnormality, which can be alleviated by riboflavin supplementation. However, whether riboflavin can inhibit pathological cardiac hypertrophy and fibrosis remains unclear. Therefore, we observed the effect of riboflavin on pathological cardiac hypertrophy and fibrosis. In vitro experiments, riboflavin increased SCAD expression and the content of ATP, decreased the free fatty acids content and improved PE-induced cardiomyocytes hypertrophy and AngⅡ-induced cardiac fibroblasts proliferation by increasing the content of FAD, which were attenuated by knocking down the expression of SCAD using small interfering RNA. In vivo experiments, riboflavin significantly increased the expression of SCAD and the energy metabolism of the heart to improve TAC induced pathological myocardial hypertrophy and fibrosis in mice. The results demonstrate that riboflavin improves pathological cardiac hypertrophy and fibrosis by increasing the content of FAD to activate SCAD, which may be a new strategy for treating pathological cardiac hypertrophy and fibrosis.


Subject(s)
Butyryl-CoA Dehydrogenase , Flavin-Adenine Dinucleotide , Animals , Mice , Butyryl-CoA Dehydrogenase/genetics , Butyryl-CoA Dehydrogenase/metabolism , Flavin-Adenine Dinucleotide/pharmacology , Riboflavin/pharmacology , Cardiomegaly/pathology , Fatty Acids, Nonesterified , Fibrosis
2.
J Hypertens ; 41(5): 775-793, 2023 05 01.
Article in English | MEDLINE | ID: mdl-36883465

ABSTRACT

OBJECTIVES: Short-chain acyl-CoA dehydrogenase (SCAD), a key enzyme in the fatty acid oxidation process, is not only involved in ATP synthesis but also regulates the production of mitochondrial reactive oxygen species (ROS) and nitric oxide synthesis. The purpose of this study was to investigate the possible role of SCAD in hypertension-associated vascular remodelling. METHODS: In-vivo experiments were performed on spontaneously hypertensive rats (SHRs, ages of 4 weeks to 20 months) and SCAD knockout mice. The aorta sections of hypertensive patients were used for measurement of SCAD expression. In-vitro experiments with t-butylhydroperoxide (tBHP), SCAD siRNA, adenovirus-SCAD (MOI 90) or shear stress (4, 15 dynes/cm 2 ) were performed using human umbilical vein endothelial cells (HUVECs). RESULTS: Compared with age-matched Wistar rats, aortic SCAD expression decreased gradually in SHRs with age. In addition, aerobic exercise training for 8 weeks could significantly increase SCAD expression and enzyme activity in the aortas of SHRs while decreasing vascular remodelling in SHRs. SCAD knockout mice also exhibited aggravated vascular remodelling and cardiovascular dysfunction. Likewise, SCAD expression was also decreased in tBHP-induced endothelial cell apoptosis models and the aortas of hypertensive patients. SCAD siRNA caused HUVEC apoptosis in vitro , whereas adenovirus-mediated SCAD overexpression (Ad-SCAD) protected against HUVEC apoptosis. Furthermore, SCAD expression was decreased in HUVECs exposed to low shear stress (4 dynes/cm 2 ) and increased in HUVECs exposed to 15 dynes/cm 2 compared with those under static conditions. CONCLUSION: SCAD is a negative regulator of vascular remodelling and may represent a novel therapeutic target for vascular remodelling.


Subject(s)
Butyryl-CoA Dehydrogenase , Hypertension , Rats , Animals , Mice , Humans , Infant, Newborn , Butyryl-CoA Dehydrogenase/genetics , Butyryl-CoA Dehydrogenase/metabolism , Vascular Remodeling , Rats, Inbred SHR , Rats, Wistar , Human Umbilical Vein Endothelial Cells/metabolism , RNA, Small Interfering/metabolism , Mice, Knockout
4.
Int J Med Sci ; 18(16): 3631-3643, 2021.
Article in English | MEDLINE | ID: mdl-34790035

ABSTRACT

Background: Acyl-CoA dehydrogenase short-chain (ACADS) is a crucial enzyme in the fatty acid metabolism pathway located in mitochondria. However, the expression level and prognostic value of ACADS in colorectal cancer (CRC) remain unclear. Methods: The mRNA and protein expression data of ACADS was obtained from The Cancer Genome Atlas (TCGA), Clinical Proteomic Tumor Analysis Consortium (CPTAC), and Oncomine. Prognostic values of ACADS were calculated using Kaplan-Meier survival analysis. Correlations between ACADS and immune infiltration were estimated using TIMER, CIBERSORT, EPIC, quanTIseq, and xCell. The UALCAN and MEXPRESS databases were utilized for Methylation analysis. The co-expression analysis based on mRNA expression and interaction network of ACADS were performed via several online tools. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis on ACADS co-expressed genes were performed using the Metascape. Results: The expression analysis demonstrated that ACADS was down-regulated in CRC tissues compared with paired normal tissue. Expression of ACADS was found to be significantly associated with clinical cancer stages and the consensus molecular subgroups (CMS) constituent ratio in CRC patients. Besides, lower ACADS expression was found to predict poor prognosis and be significantly associated with common immune checkpoint genes and MMR genes in CRC. ACADS expression levels were positively related to B cells, CD4+ T cells, CD8+ T cells, M1 macrophages, neutrophils, and Tregs, while negatively correlated with M0 macrophages, M2 macrophages. The methylation level of ACADS in normal tissues was significantly higher than that in tumor tissues, and several methylation sites were identified. The enrichment analysis suggested the co-expressed genes mainly enriched in cell mitochondrial metabolism. Conclusions: The present study provided multilevel evidences for expression of ACADS in CRC and the function of ACADS in prognostic prediction, immune infiltration, and methylation. ACADS might have the potential as the novel biomarker and therapeutic target in CRC patients.


Subject(s)
Butyryl-CoA Dehydrogenase/genetics , Butyryl-CoA Dehydrogenase/metabolism , Colorectal Neoplasms/diagnosis , Adenocarcinoma/diagnosis , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Adenocarcinoma/mortality , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Carcinoma/diagnosis , Carcinoma/genetics , Carcinoma/metabolism , Carcinoma/mortality , Cell Line, Tumor , Chemotaxis, Leukocyte/genetics , Chemotaxis, Leukocyte/immunology , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/mortality , Fatty Acids/metabolism , Gene Expression Profiling , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Humans , Lipid Metabolism/genetics , Predictive Value of Tests , Prognosis , Proteomics , Survival Analysis , Tumor Microenvironment/genetics , Tumor Microenvironment/immunology
5.
Biochem Pharmacol ; 178: 114100, 2020 08.
Article in English | MEDLINE | ID: mdl-32540485

ABSTRACT

Short-chain acyl-CoA dehydrogenase (SCAD), the rate-limiting enzyme for fatty acid ß-oxidation, has a negative regulatory effect on pathological cardiac hypertrophy and fibrosis. Furthermore, flavin adenine dinucleotide (FAD) can enhance the expression and enzyme activity of SCAD. However, whether FAD can inhibit pathological cardiac hypertrophy and fibrosis remains unclear. Therefore, we observed the effect of FAD on pathological cardiac hypertrophy and fibrosis. FAD significantly inhibited PE-induced cardiomyocyte hypertrophy and AngII-induced cardiac fibroblast proliferation. In addition, FAD ameliorated pathological cardiac hypertrophy and fibrosis in SHR. FAD significantly increased the expression and enzyme activity of SCAD. Meanwhile, ATP content was increased, the content of free fatty acids and reactive oxygen species were decreased by FAD in vivo and in vitro. In addition, molecular dynamics simulations were also used to provide insights into the structural stability and dynamic behavior of SCAD. The results demonstrated that FAD may play an important structural role on the SCAD dimer stability and maintenance of substrate catalytic pocket to increase the expression and enzyme activity of SCAD. In conclusion, FAD can inhibit pathological cardiac hypertrophy and fibrosis through activating SCAD, which may be a novel effective treatment for pathological cardiac hypertrophy and fibrosis, thus prevent them from developing into heart failure.


Subject(s)
Butyryl-CoA Dehydrogenase/genetics , Cardiomegaly/prevention & control , Cardiotonic Agents/pharmacology , Fibroblasts/drug effects , Flavin-Adenine Dinucleotide/pharmacology , Gene Expression Regulation/drug effects , Adenosine Triphosphate/biosynthesis , Animals , Binding Sites , Butyryl-CoA Dehydrogenase/metabolism , Cardiomegaly/enzymology , Cardiomegaly/genetics , Cardiomegaly/pathology , Cell Proliferation/drug effects , Energy Metabolism/drug effects , Energy Metabolism/genetics , Enzyme Stability , Fatty Acids, Nonesterified/antagonists & inhibitors , Fatty Acids, Nonesterified/metabolism , Fibroblasts/metabolism , Fibroblasts/pathology , Fibrosis , Heart Failure/prevention & control , Male , Molecular Dynamics Simulation , Myocardium/enzymology , Myocardium/pathology , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Multimerization , Rats , Rats, Inbred SHR , Rats, Wistar , Reactive Oxygen Species/antagonists & inhibitors , Reactive Oxygen Species/metabolism
6.
Fungal Genet Biol ; 127: 23-34, 2019 06.
Article in English | MEDLINE | ID: mdl-30822500

ABSTRACT

Short-chain acyl-CoA dehydrogenase (Scad) mediated ß-oxidation serves as the fastest route for generating essential energies required to support the survival of organisms under stress or starvation. In this study, we identified three putative SCAD genes in the genome of the globally destructive rice blast pathogen Magnaporthe oryzae, named as MoSCAD1, MoSCAD2, and MoSCAD3. To elucidate their function, we deployed targeted gene deletion strategy to investigate individual and the combined influence of MoSCAD genes on growth, stress tolerance, conidiation and pathogenicity of the rice blast fungus. First, localization and co-localization results obtained from this study showed that MoScad1 localizes to the endoplasmic reticulum (ER), MoScad2 localizes exclusively to the mitochondria while MoScad3 partially localizes to the mitochondria and peroxisome at all developmental stages of M. oryzae. Results obtained from this investigation showed that the deletion of MoSCAD1 and MoSCAD2 caused a minimal but significant reduction in the growth of ΔMoscad1 and ΔMoscad2 strains, while, growth characteristics exhibited by the ΔMoscad3 strain was similar to the wild-type strain. Furthermore, we observed that deletion of MoSCAD2 resulted in drastic reduction in conidiation, delayed conidia germination, triggered the development of abnormal appressorium and suppressed host penetration and colonization efficiencies of the ΔMoscad1 strain. This study provides first material evidence confirming the possible existence of ER ß-oxidation pathway in M. oryzae. We also infer that mitochondria ß-oxidation rather than peroxisomal and ER ß-oxidation play an essential role in the vegetative growth, conidiation, appressorial morphogenesis and progression of pathogenesis in M. oryzae.


Subject(s)
Butyryl-CoA Dehydrogenase/genetics , Fungal Proteins/genetics , Magnaporthe/genetics , Magnaporthe/pathogenicity , Spores, Fungal/growth & development , Endoplasmic Reticulum , Free Radicals/metabolism , Gene Deletion , Gene Expression Regulation, Fungal , Magnaporthe/enzymology , Mitochondria/metabolism , Oryza/microbiology , Oxidation-Reduction , Peroxisomes/metabolism , Plant Diseases/microbiology , Spores, Fungal/genetics
7.
BMC Med Genet ; 19(1): 64, 2018 04 20.
Article in English | MEDLINE | ID: mdl-29678161

ABSTRACT

BACKGROUND: Short-chain acyl-CoA dehydrogenase deficiency (SCADD) represents a rare autosomal recessive inborn metabolic disorder of mitochondrial ß-oxidation of monocarboxylic acids. Clinical symptoms can vary from a severe life-threatening condition to an asymptomatic state, reported in the majority of cases. Since the expansion of newborn screenings, more than three hundred probands were admitted for molecular-genetic analysis, most selected because of elevated values of C4-acylcarnitine detected in newborn screenings in Slovakia. Searching for the principal genomic changes led us to the selection of sixty-two patients in whom the presence of sequence variants in the ACADS gene was analysed and correlated with the available biochemical and clinical data. METHODS: Biochemical and molecular genetic tests were performed. Acylcarnitine profiles focused on an elevated level of C4-acylcarnitine, which was analysed via tandem mass spectrometry. Urinary organic acids, specifically a quantity of ethylmalonic acid, were determined by gas chromatography/mass spectrometry. The entire coding region of the ACADS gene was sequenced. A low-cost restriction fragment length polymorphism of PCR amplified fragments analysis (PCR-RFLP) of pathogenic variants was introduced and implemented for the molecular-genetic algorithm appropriate for the Slovak population. RESULTS: Our molecular genetic study was performed on sixty-two patients with a pathological biochemical pattern related to short-chain acyl-CoA dehydrogenase deficiency. In this cohort, we discovered a high occurrence of two rare pathogenic variants-the deletion c.310_312delGAG and the substitution c.1138C>T, with allelic frequencies of 64% and 31%, respectively. Up to 86% of investigated individuals belong to the Roma ethnic group. CONCLUSIONS: Analogous to other countries, SCADD is not included in the newborn screening programme. Based on the exceeded levels of the specific biomarker C4-acylcarnitine as well as ethylmalonic acid, we revealed a high prevalence of short-chain acyl-CoA dehydrogenase deficiency cases, confirmed by the findings of two rare pathogenic variants. A deletion c.310_312delGAG and c.1138C > T substitution in the ACADS gene appear with a high frequency in the Roma ethnic group of Slovakia. Due to the uncertainty of the pathogenicity and clinical consequences, it is important to follow up the morbidity and mortality in these patients over time and evaluate SCADD in relation to clinical outcomes and preventive healthcare recommendations.


Subject(s)
Acyl-CoA Dehydrogenase/deficiency , Butyryl-CoA Dehydrogenase/genetics , Carnitine/analogs & derivatives , Ethnicity/genetics , Lipid Metabolism, Inborn Errors/genetics , Mutation , Acyl-CoA Dehydrogenase/genetics , Acyl-CoA Dehydrogenase/metabolism , Carnitine/metabolism , Female , Gene Frequency , Genetic Testing , Humans , Infant, Newborn , Lipid Metabolism, Inborn Errors/ethnology , Lipid Metabolism, Inborn Errors/metabolism , Male , Neonatal Screening/methods , Polymorphism, Restriction Fragment Length , Sequence Analysis, DNA , Slovakia/ethnology
8.
FEBS Lett ; 591(12): 1785-1797, 2017 06.
Article in English | MEDLINE | ID: mdl-28524228

ABSTRACT

To investigate the function of the pa4079 gene from the opportunistic pathogen Pseudomonas aeruginosa PAO1, we determined its crystal structure and confirmed it to be a NAD(P)-dependent short-chain dehydrogenase/reductase. Structural similarity and activity for a broad range of substrates indicate that PA4079 functions as a carbonyl reductase. Comparison of apo- and holo-PA4079 shows that NADP stabilizes the active site specificity loop, and small molecule binding induces rotation of the Tyr183 side chain by approximately 90° out of the active site. Quantitative real-time PCR results show that pa4079 maintains high expression levels during antibiotic exposure. This work provides a starting point for understanding substrate recognition and selectivity by PA4079, as well as its possible reduction of antimicrobial drugs. DATABASE: Structural data are available in the Protein Data Bank (PDB) under the following accession numbers: apo PA4079 (condition I), 5WQM; apo PA4079 (condition II), 5WQN; PA4079 + NADP (condition I), 5WQO; PA4079 + NADP (condition II), 5WQP.


Subject(s)
Aldehyde Reductase/metabolism , Bacterial Proteins/metabolism , Butyryl-CoA Dehydrogenase/metabolism , Models, Molecular , NADP/metabolism , Pseudomonas aeruginosa/metabolism , Aldehyde Reductase/chemistry , Aldehyde Reductase/genetics , Aldo-Keto Reductases , Amino Acid Sequence , Amino Acid Substitution , Anti-Bacterial Agents/pharmacology , Apoenzymes/chemistry , Apoenzymes/genetics , Apoenzymes/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Binding Sites , Butyryl-CoA Dehydrogenase/chemistry , Butyryl-CoA Dehydrogenase/genetics , Catalytic Domain , Conserved Sequence , Crystallography, X-Ray , Enzyme Stability , Gene Expression Regulation, Bacterial/drug effects , Holoenzymes/chemistry , Holoenzymes/genetics , Holoenzymes/metabolism , Ligands , Mutation , NADP/chemistry , Protein Conformation , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/growth & development , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Structural Homology, Protein , Substrate Specificity
9.
Appl Environ Microbiol ; 81(14): 4782-90, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25956767

ABSTRACT

Eubacterium limosum KIST612 is one of the few acetogens that can produce butyrate from carbon monoxide. We have used a genome-guided analysis to delineate the path of butyrate formation, the enzymes involved, and the potential coupling to ATP synthesis. Oxidation of CO is catalyzed by the acetyl-coenzyme A (CoA) synthase/CO dehydrogenase and coupled to the reduction of ferredoxin. Oxidation of reduced ferredoxin is catalyzed by the Rnf complex and Na(+) dependent. Consistent with the finding of a Na(+)-dependent Rnf complex is the presence of a conserved Na(+)-binding motif in the c subunit of the ATP synthase. Butyrate formation is from acetyl-CoA via acetoacetyl-CoA, hydroxybutyryl-CoA, crotonyl-CoA, and butyryl-CoA and is consistent with the finding of a gene cluster that encodes the enzymes for this pathway. The activity of the butyryl-CoA dehydrogenase was demonstrated. Reduction of crotonyl-CoA to butyryl-CoA with NADH as the reductant was coupled to reduction of ferredoxin. We postulate that the butyryl-CoA dehydrogenase uses flavin-based electron bifurcation to reduce ferredoxin, which is consistent with the finding of etfA and etfB genes next to it. The overall ATP yield was calculated and is significantly higher than the one obtained with H2 + CO2. The energetic benefit may be one reason that butyrate is formed only from CO but not from H2 + CO2.


Subject(s)
Butyrates/metabolism , Carbon Monoxide/metabolism , Eubacterium/metabolism , Acyl Coenzyme A/metabolism , Adenosine Triphosphate/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Butyryl-CoA Dehydrogenase/genetics , Butyryl-CoA Dehydrogenase/metabolism , Energy Metabolism , Eubacterium/enzymology , Eubacterium/genetics , Flavins/metabolism , Genomics , Oxidation-Reduction
10.
FEBS J ; 282(16): 3149-60, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25903584

ABSTRACT

Over 50 years ago, it was reported that, in the anaerobic rumen bacterium Megasphaera elsdenii, the reduction of crotonyl-CoA to butyryl-CoA by NADH involved an electron transferring flavoprotein (Etf) as mediator [Baldwin RL, Milligan LP (1964) Biochim Biophys Acta 92, 421-432]. Purification and spectroscopic characterization revealed that this Etf contained 2 FAD, whereas, in the Etfs from aerobic and facultative bacteria, one FAD is replaced by AMP. Recently we detected a similar system in the related anaerobe Acidaminococcus fermentans that differed in the requirement of additional ferredoxin as electron acceptor. The whole process was established as flavin-based electron bifurcation in which the exergonic reduction of crotonyl-CoA by NADH mediated by Etf + butyryl-CoA dehydrogenase (Bcd) was coupled to the endergonic reduction of ferredoxin also by NADH. In the present study, we demonstrate that, under anaerobic conditions, Etf + Bcd from M. elsdenii bifurcate as efficiently as Etf + Bcd from A. fermentans. Under the aerobic conditions used in the study by Baldwin and Milligan and in the presence of catalytic amounts of crotonyl-CoA or butyryl-CoA, however, Etf + Bcd act as NADH oxidase producing superoxide and H2 O2 , whereas ferredoxin is not required. We hypothesize that, during bifurcation, oxygen replaces ferredoxin to yield superoxide. In addition, the formed butyryl-CoA is re-oxidized by a second oxygen molecule to crotonyl-CoA, resulting in a stoichiometry of 2 NADH consumed and 2 H2 O2 formed. As a result of the production of reactive oxygen species, electron bifurcation can be regarded as an Achilles' heel of anaerobes when exposed to air.


Subject(s)
Bacterial Proteins/metabolism , Electron-Transferring Flavoproteins/metabolism , Ferredoxins/metabolism , Megasphaera/metabolism , Acidaminococcus/genetics , Acidaminococcus/metabolism , Anaerobiosis , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Butyryl-CoA Dehydrogenase/chemistry , Butyryl-CoA Dehydrogenase/genetics , Butyryl-CoA Dehydrogenase/metabolism , Electron Transport , Electron-Transferring Flavoproteins/chemistry , Electron-Transferring Flavoproteins/genetics , Megasphaera/genetics , Multienzyme Complexes/chemistry , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , NAD/metabolism , NADH, NADPH Oxidoreductases/chemistry , NADH, NADPH Oxidoreductases/genetics , NADH, NADPH Oxidoreductases/metabolism , Oxidation-Reduction , Oxygen/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spectrophotometry
11.
J Cell Mol Med ; 19(7): 1672-88, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25753319

ABSTRACT

This study was designed to investigate the expression of short-chain acyl-CoA dehydrogenase (SCAD), a key enzyme of fatty acid ß-oxidation, during rat heart development and the difference of SCAD between pathological and physiological cardiac hypertrophy. The expression of SCAD was lowest in the foetal and neonatal heart, which had time-dependent increase during normal heart development. In contrast, a significant decrease in SCAD expression was observed in different ages of spontaneously hypertensive rats (SHR). On the other hand, swim-trained rats developed physiological cardiac hypertrophy, whereas SHR developed pathological cardiac hypertrophy. The two kinds of cardiac hypertrophy exhibited divergent SCAD changes in myocardial fatty acids utilization. In addition, the expression of SCAD was significantly decreased in pathological cardiomyocyte hypertrophy, however, increased in physiological cardiomyocyte hypertrophy. SCAD siRNA treatment triggered the pathological cardiomyocyte hypertrophy, which showed that the down-regulation of SCAD expression may play an important role in pathological cardiac hypertrophy. The changes in peroxisome proliferator-activated receptor α (PPARα) was accordant with that of SCAD. Moreover, the specific PPARα ligand fenofibrate treatment increased the expression of SCAD and inhibited pathological cardiac hypertrophy. Therefore, we speculate that the down-regulated expression of SCAD in pathological cardiac hypertrophy may be responsible for 'the recapitulation of foetal energy metabolism'. The deactivation of PPARα may result in the decrease in SCAD expression in pathological cardiac hypertrophy. Changes in SCAD are different in pathological and physiological cardiac hypertrophy, which may be used as the molecular markers of pathological and physiological cardiac hypertrophy.


Subject(s)
Butyryl-CoA Dehydrogenase/metabolism , Cardiomegaly/enzymology , Heart/growth & development , Animals , Animals, Newborn , Blood Pressure/drug effects , Butyryl-CoA Dehydrogenase/genetics , Cardiomegaly/diagnostic imaging , Cardiomegaly/pathology , Cardiomegaly/physiopathology , Disease Models, Animal , Fatty Acids/metabolism , Fenofibrate/pharmacology , Heart/drug effects , Heart/physiopathology , Heart Ventricles/pathology , Heart Ventricles/physiopathology , Insulin-Like Growth Factor I/pharmacology , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Organ Size/drug effects , Oxidation-Reduction/drug effects , PPAR alpha/metabolism , Phenylephrine/pharmacology , RNA Interference/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Rats, Inbred SHR , Rats, Wistar , Substrate Specificity/drug effects , Systole/drug effects , Time Factors , Ultrasonography
12.
J Ind Microbiol Biotechnol ; 41(9): 1425-33, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25037723

ABSTRACT

Klebsiella pneumoniae synthesize large amounts of L-2,3-butanediol (L-2,3-BD), but the underlying mechanism has been unknown. In this study, we provide the first identification and characterization of an L-2,3-BD dehydrogenase from K. pneumoniae, demonstrating its reductive activities toward diacetyl and acetoin, and oxidative activity toward L-2,3-BD. Optimum pH, temperature, and kinetics determined for reductive and oxidative reactions support the preferential production of 2,3-BD during cell growth. Synthesis of L-2,3-BD was remarkably enhanced by increasing gene dosage, reaching levels that, to the best of our knowledge, are the highest achieved to date.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Butylene Glycols/metabolism , Butyryl-CoA Dehydrogenase/chemistry , Butyryl-CoA Dehydrogenase/metabolism , Klebsiella pneumoniae/enzymology , Acetoin/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Butyryl-CoA Dehydrogenase/genetics , Enzyme Stability , Klebsiella pneumoniae/chemistry , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/metabolism , Molecular Sequence Data , Sequence Alignment
13.
Mol Biosyst ; 10(6): 1246-54, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24554194

ABSTRACT

Data on neurobiological mechanisms underlying mood disorders are elusive; the aetiology of such states is multifactorial, including genetic predisposition and environmental factors. Diagnosis is currently being made only on an interview-based methodology. Biological markers, which could improve the current classification, and in perspective, stratify patients on a biological basis into more homogeneous clinically distinct subgroups, are highly needed. We describe here a comparative proteomic analysis of peripheral lymphocytes from patients affected by acute psychotic bipolar disorder (PBD) (n = 15), major depressive episode (MDE) with no personal or family history of psychosis (n = 11), and a group of demographically matched healthy controls (HC) (n = 15). All patients were evaluated by means of Structured Clinical Interview for DSM-IV-Patient version (SCID-I-P), Positive and Negative Symptoms Scale (PANSS), Young Mania Rating Scale (YMRS), Hamilton Anxiety Rating Scale (HAM-A) and Hamilton Depression Rating Scale (HAM-D-17) questionnaires. Blood lymphocytes were obtained by gradient separation, and 2-DE was carried out on protein extracts. Significant differences in protein patterns among the three groups were observed. Thirty-six protein spots were found to be differentially expressed in patients compared to controls, which collapsed into 25 different proteins after mass spectrometry identification. Twenty-one of these proteins failed to discriminate between PBD and MDE, suggesting common signatures for these disorders. Nevertheless, after the western blot validation only two of the remaining proteins, namely LIM and SH3 domain protein1, and short-chain specific acyl-CoA dehydrogenase mitochondrial protein, resulted in being significantly upregulated in PBD samples suggesting additional mechanisms that could be associated with the psychotic features of bipolar disorder.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Bipolar Disorder/blood , Bipolar Disorder/pathology , Butyryl-CoA Dehydrogenase/metabolism , Cytoskeletal Proteins/metabolism , Depressive Disorder, Major/blood , Depressive Disorder, Major/pathology , LIM Domain Proteins/metabolism , Adaptor Proteins, Signal Transducing/blood , Adaptor Proteins, Signal Transducing/genetics , Adult , Biomarkers/blood , Bipolar Disorder/metabolism , Butyryl-CoA Dehydrogenase/blood , Butyryl-CoA Dehydrogenase/genetics , Case-Control Studies , Cytoskeletal Proteins/blood , Cytoskeletal Proteins/genetics , Depressive Disorder, Major/metabolism , Female , Gene Expression Regulation , Humans , LIM Domain Proteins/blood , LIM Domain Proteins/genetics , Male , Middle Aged , Proteomics
14.
Mol Genet Metab ; 111(3): 360-368, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24485985

ABSTRACT

Short-chain acyl-CoA dehydrogenase (SCAD) deficiency is a rare inherited autosomal recessive disorder with not yet well established mechanisms of disease. In the present study, the mitochondrial proteome of five symptomatic patients homozygous for missense variations in the SCAD gene ACADS was investigated in an extensive large-scale proteomic study to map protein perturbations linked to the disease. Fibroblast cultures of patient cells homozygous for either c.319C>T/p.Arg107Cys (n=2) or c.1138C>T/p.Arg380Trp (n=3) in ACADS, and healthy controls (normal human dermal fibroblasts), were studied. The mitochondrial proteome derived from these cultures was analyzed by label free proteomics using high mass accuracy nanoliquid chromatography tandem mass spectrometry (nanoLC-MS/MS). More than 300 mitochondrial proteins were identified and quantified. Thirteen proteins had significant alteration in protein levels in patients carrying variation c.319C>T in ACADS compared to controls and they belonged to various pathways, such as the antioxidant system and amino acid metabolism. Twenty-two proteins were found significantly altered in patients carrying variation c.1138C>T which included proteins associated with fatty acid ß-oxidation, amino acid metabolism and protein quality control system. Three proteins were found significantly regulated in both patient groups: adenylate kinase 4 (AK4), nucleoside diphosphate kinase A (NME1) and aldehyde dehydrogenase family 4 member A1 (ALDH4A1). Proteins AK4 and NME1 deserve further investigation because of their involvement in energy reprogramming, cell survival and proliferation with relevance for SCAD deficiency and related metabolic disorders.


Subject(s)
Acyl-CoA Dehydrogenase/deficiency , Butyryl-CoA Dehydrogenase/genetics , Lipid Metabolism, Inborn Errors/genetics , Mitochondria/genetics , Mitochondrial Proteins/biosynthesis , Acyl-CoA Dehydrogenase/genetics , Acyl-CoA Dehydrogenase/metabolism , Butyryl-CoA Dehydrogenase/metabolism , Cells, Cultured , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression Regulation , Humans , Lipid Metabolism, Inborn Errors/metabolism , Lipid Metabolism, Inborn Errors/pathology , Male , Mitochondria/pathology , Oxidative Stress/genetics , Proteomics , Tandem Mass Spectrometry
15.
J Bacteriol ; 195(16): 3704-13, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23772070

ABSTRACT

The butyrogenic genes from Clostridium difficile DSM 1296(T) have been cloned and expressed in Escherichia coli. The enzymes acetyl-coenzyme A (CoA) C-acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, phosphate butyryltransferase, and butyrate kinase and the butyryl-CoA dehydrogenase complex composed of the dehydrogenase and two electron-transferring flavoprotein subunits were individually produced in E. coli and kinetically characterized in vitro. While most of these enzymes were measured using well-established test systems, novel methods to determine butyrate kinase and butyryl-CoA dehydrogenase activities with respect to physiological function were developed. Subsequently, the individual genes were combined to form a single plasmid-encoded operon in a plasmid vector, which was successfully used to confer butyrate-forming capability to the host. In vitro and in vivo studies demonstrated that C. difficile possesses a bifurcating butyryl-CoA dehydrogenase which catalyzes the NADH-dependent reduction of ferredoxin coupled to the reduction of crotonyl-CoA also by NADH. Since the reoxidation of ferredoxin by a membrane-bound ferredoxin:NAD(+)-oxidoreductase enables electron transport phosphorylation, additional ATP is formed. The butyryl-CoA dehydrogenase from C. difficile is oxygen stable and apparently uses oxygen as a co-oxidant of NADH in the presence of air. These properties suggest that this enzyme complex might be well suited to provide butyryl-CoA for solventogenesis in recombinant strains. The central role of bifurcating butyryl-CoA dehydrogenases and membrane-bound ferredoxin:NAD oxidoreductases (Rhodobacter nitrogen fixation [RNF]), which affect the energy yield of butyrate fermentation in the clostridial metabolism, is discussed.


Subject(s)
Butyrates/metabolism , Butyryl-CoA Dehydrogenase/metabolism , Clostridioides difficile/metabolism , Electron-Transferring Flavoproteins/metabolism , Escherichia coli/metabolism , Oxygen , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Butyryl-CoA Dehydrogenase/genetics , Cloning, Molecular , Clostridioides difficile/enzymology , Clostridioides difficile/genetics , Electron-Transferring Flavoproteins/genetics , Escherichia coli/genetics , Gene Expression Regulation, Bacterial/physiology , Recombinant Proteins
16.
Appl Biochem Biotechnol ; 169(3): 950-9, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23292245

ABSTRACT

Acetogen Clostridum sp. MT1962 produced 287 mM acetate (p < 0.005) and 293 mM ethanol (p < 0.005) fermenting synthesis gas blend 60% CO and 40% H2 in single-stage continuous fermentation. This strain was metabolically engineered to the biocatalyst Clostridium sp. MTButOH1365. The engineered biocatalyst lost production of ethanol and acetate while initiated the production of 297 mM of n-butanol (p < 0.005). The metabolic engineering comprised Cre-lox66/lox71-based elimination of phosphotransacetylase and acetaldehyde dehydrogenase along with integration to chromosome synthetic thiolase, 3-hydroxy butyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase, butyraldehyde dehydrogenase, and NAD-dependent butanol dehydrogenase. This is the first report on elimination of acetate and ethanol production genes and expression of synthetic gene cluster encoding n-butanol biosynthesis pathway in acetogen biocatalyst for selective fuel n-butanol production with no antibiotic support for the introduced genes.


Subject(s)
1-Butanol/metabolism , Clostridium/enzymology , Clostridium/metabolism , Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/metabolism , Butyryl-CoA Dehydrogenase/genetics , Butyryl-CoA Dehydrogenase/metabolism , Enoyl-CoA Hydratase/genetics , Enoyl-CoA Hydratase/metabolism , Fermentation , Oxidoreductases/genetics , Oxidoreductases/metabolism
17.
Chem Biol Interact ; 202(1-3): 111-5, 2013 Feb 25.
Article in English | MEDLINE | ID: mdl-23200746

ABSTRACT

The short-chain dehydrogenases/reductases (SDRs) constitute one of the largest protein superfamilies known today. The members are distantly related with typically 20-30% residue identity in pair-wise comparisons. Still, all hitherto structurally known SDRs present a common three-dimensional structure consisting of a Rossmann fold with a parallel beta sheet flanked by three helices on each side. Using hidden Markov models (HMMs), we have developed a semi-automated subclassification system for this huge family. Currently, 75% of all SDR forms have been assigned to one of the 464 families totalling 122,940 proteins. There are 47 human SDR families, corresponding to 75 genes. Most human SDR families (35 families) have only one gene, while 12 have between 2 and 8 genes. For more than half of the human SDR families, the three-dimensional fold is known. The number of SDR members increases considerably every year, but the number of SDR families now starts to converge. The classification method has paved the ground for a sustainable and expandable nomenclature system. Information on the SDR superfamily is continuously updated at http://sdr-enzymes.org/.


Subject(s)
Butyryl-CoA Dehydrogenase/classification , Fatty Acid Synthases/classification , NADH, NADPH Oxidoreductases/classification , Butyryl-CoA Dehydrogenase/chemistry , Butyryl-CoA Dehydrogenase/genetics , Butyryl-CoA Dehydrogenase/metabolism , Fatty Acid Synthases/chemistry , Fatty Acid Synthases/genetics , Fatty Acid Synthases/metabolism , Humans , NADH, NADPH Oxidoreductases/chemistry , NADH, NADPH Oxidoreductases/genetics , NADH, NADPH Oxidoreductases/metabolism , Terminology as Topic
18.
J Pediatr Endocrinol Metab ; 25(7-8): 795-7, 2012.
Article in English | MEDLINE | ID: mdl-23155713

ABSTRACT

Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is a rare autosomal recessive inborn error of mitochondrial fatty acid oxidation. It is caused by rare mutations as well as polymorphic susceptibility variants. We describe here the case of a 1-year-old male patient who had growth and mental retardation, seizures, and recurring fever since infancy. Urinary gas chromatography/mass spectrometry (GC/MS) showed elevated levels of ethylmalonic acid. Plasma acylcarnitines on tandem mass spectrometry (MS/MS) and elevations of C4-cartinitine are consistently present. The two polymorphic susceptibility variants of the short-chain acyl-CoA dehydrogenase (SCAD) gene, c.625G>A and c.322G>A, were detected. Because of its highly variable clinical characteristics, there are no related reports in China. This report broadens the phenotype and genotype of SCADD in China and underlines the difficulty of diagnosis.


Subject(s)
Lipid Metabolism, Inborn Errors/diagnosis , Acyl-CoA Dehydrogenase/deficiency , Acyl-CoA Dehydrogenase/genetics , Butyryl-CoA Dehydrogenase/genetics , China , Genotype , Humans , Infant , Intellectual Disability/complications , Intellectual Disability/diagnosis , Intellectual Disability/genetics , Lipid Metabolism, Inborn Errors/complications , Lipid Metabolism, Inborn Errors/genetics , Male , Polymorphism, Single Nucleotide/physiology , Seizures/complications , Seizures/congenital , Seizures/diagnosis , Seizures/genetics
19.
J Ind Microbiol Biotechnol ; 39(8): 1101-7, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22776992

ABSTRACT

A homobutanol fermentation pathway was engineered in a derivative of Escherichia coli B (glucose [glycolysis] => 2 pyruvate + 2 NADH; pyruvate [pyruvate dehydrogenase] => acetyl-CoA + NADH; 2 acetyl-CoA [butanol pathway enzymes] + 4 NADH => butanol; summary stoichiometry: glucose => butanol). Initially, the native fermentation pathways were eliminated from E. coli B by deleting the genes encoding for lactate dehydrogenase (ldhA), acetate kinase (ackA), fumarate reductase (frdABCD), pyruvate formate lyase (pflB), and alcohol dehydrogenase (adhE), and the pyruvate dehydrogenase complex (aceEF-lpd) was anaerobically expressed through promoter replacement. The resulting strain, E. coli EG03 (ΔfrdABCD ΔldhA ΔackA ΔpflB Δ adhE ΔpdhR ::pflBp6-aceEF-lpd ΔmgsA), could generate 4 NADH for every glucose oxidized to two acetyl-CoA through glycolysis and the pyruvate dehydrogenase complex. However, EG03 lost its ability for anaerobic growth due to the lack of NADH oxidation pathways. When the butanol pathway genes that encode for acetyl-CoA acetyltransferase (thiL), 3-hydroxybutyryl-CoA dehydrogenase (hbd), crotonase (crt), butyryl-CoA dehydrogenase (bcd, etfA, etfB), and butyraldehyde dehydrogenase (adheII) were cloned from Clostridium acetobutylicum ATCC 824, and expressed in E. coli EG03, a balanced NADH oxidation pathway was established for homobutanol fermentation (glucose => 4 NADH + 2 acetyl-CoA => butanol). This strain was able to convert glucose to butanol (1,254 mg l(-1)) under anaerobic condition.


Subject(s)
1-Butanol/metabolism , Bioreactors , Butanols/metabolism , Escherichia coli/metabolism , Fermentation , Metabolic Engineering , 3-Hydroxyacyl CoA Dehydrogenases/genetics , 3-Hydroxyacyl CoA Dehydrogenases/metabolism , Acetyl-CoA C-Acetyltransferase/genetics , Acetyl-CoA C-Acetyltransferase/metabolism , Alcohol Dehydrogenase/genetics , Alcohol Dehydrogenase/metabolism , Biofuels , Butyryl-CoA Dehydrogenase/genetics , Butyryl-CoA Dehydrogenase/metabolism , Clostridium acetobutylicum/enzymology , Clostridium acetobutylicum/genetics , Enoyl-CoA Hydratase/genetics , Enoyl-CoA Hydratase/metabolism , Escherichia coli/classification , Escherichia coli/genetics , Glucose/metabolism , Glycolysis , NAD/metabolism , Oxidation-Reduction , Pyruvate Dehydrogenase Complex/genetics , Pyruvate Dehydrogenase Complex/metabolism
20.
Sheng Wu Gong Cheng Xue Bao ; 27(9): 1317-25, 2011 Sep.
Article in Chinese | MEDLINE | ID: mdl-22117515

ABSTRACT

To explore the physiological role and biocatalytic properties of short-chain dehydrogenases from Pseudomonas fluorescens GIM1.49, we cloned the structural gene pfd and characterized its over-expressed product. The length of gene pfd was 684 bp encoding a short-chain dehydrogenase with 227 amino acid residues and calculated molecular mass of 24.2 kDa. The recombinant plasmid pET28b-pfd was constructed and functionally expressed in Escherichia coli BL21(DE3), resulting in the over-production of recombinant short-chain dehydrogenase PFD with a size of 28 kDa. The enzyme could oxidize alcohols including 4-chloro-3-hydroxbutanoate ester and reduce 4-chloro-acetoacetate ester using either NAD(H) or NADP(H) as coenzyme. The enzyme showed the highest activity against 4-chloro-3-hydroxbutanoate ester as substrate, with Km of 186.40 mmol/L and Vmax of 89.56 U/mg. When catalying the oxidative reaction, its optimal temperature was 12 degrees C and optimal pH was 10.5, in contrast to the values of 24 degrees C and pH 8.8 in the reductive reaction. The enzyme had high solvent tolerance and its activity was improved by the addition of Ca2+ (1 mmol/L) or EDTA (5 mmol/L). These results indicated that the enzyme from Pseudomonas fluorescens GIM1.49 was a novel short-chain dehydrogenase and might play a role in oxidative degradation of halogenated secondary alcohols.


Subject(s)
Bacterial Proteins/metabolism , Butyryl-CoA Dehydrogenase/metabolism , Pseudomonas fluorescens/enzymology , Alcohols/metabolism , Bacterial Proteins/genetics , Butyryl-CoA Dehydrogenase/genetics , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Genetic Vectors/genetics , Oxidation-Reduction , Pseudomonas fluorescens/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
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