acyl-CoA Dehydrogenase Enzyme Research Articles

Functional annotation of putative fadE9 of Mycobacterium tuberculosis as isobutyryl-CoA dehydrogenase involved in valine catabolism

Members of the Acyl-CoA dehydrogenase (ACADs) family of enzymes play a crucial role in cholesterol and steroid catabolism and are widely studied in the oldest known human pathogen, Mycobacterium tuberculosis (Mtb). However, there is a paucity of information on ACADs involved in branched chain amino acid catabolism. Here we characterized one of the putative ACAD enzyme, fadE9, as “Isobutyryl CoA Dehydrogenase (IBDH)” using a combined computational and experimental approach, guided by homology modeled structural information, affirming its role in valine catabolism. Multiple sequence alignment and phylogenetic analysis place it in a separate cluster from a recently identified family of α2β2-heterotetramer ACADs in Mtb, based on the position of the conserved Arg247 and catalytic Glu368 residues. The conserved Arg247 was predicted to play an essential role at the center of H-bonding network of reaction center and was confirmed by the reduced activity of R247K mutant. Thus, in addition to the finding of an architecturally distinct α2β2-heterotetramer among ACADs, these studies also highlight the differences between MtIBDH, fadE9 from the other ACADs that are involved in cholesterol and steroid catabolism of Mtb.

CML Patients with Primary Resistance or Suboptimal Response to TKI Therapy Have Variants in Genes Affecting Drug Absorption and Metabolism

BACKGROUNDChronic myeloid leukemia in chronic phase (CML CP) is successfully treated with tyrosine kinase inhibitors (TKIs) but a proportion of patients show primary or secondary resistance to TKI therapy. BCR-ABL1 mutations are the primary cause for secondary resistance to TKI therapy, but the causes for primary resistance are poorly known. Putative variants in genes affecting drug absorption, distribution, metabolism and excretion (ADME) have not been comprehensively analyzed. We set out to screen CML CP patients who either had primary resistance to TKI therapy or suboptimal response with large ADME gene panel to examine if these genes play a role in poor TKI responses.METHODSWe selected samples from 21 CML CP patients treated in our institute who either had primary resistance (N=6) or responded suboptimallyto TKI treatment (n=15) but did not have BCR-ABL1 mutations. DNA samples from the time of diagnosis were sequenced with massively parallel sequencing using a comprehensive custom ADME gene panel covering exons and introns of 382 genes. Over 200 healthy volunteers who had been sequenced with the same gene panel served as population controls. Loss-of-function and novel variants were validated using capillary sequencing.We tested the differences of variant allele frequencies between CML patients and healthy controls by performing the Cochran-Armitage trend test using additive coding (0,1,2) for genotypes homozygous for the major allele, heterozygous, and homozygous for the minor allele, respectively. CML patients with optimal response (n=24) were used as a control group.RESULTSSequencing of 21 poor responding CML CP patients revealed a total of 658 nonsynonymousgene variants. Majority of the variants (94%) were missense variants whose effects on gene function are uncertain. Eigthvariants were previously unreported. 18 variants were predicted to lead to a complete loss-of-function of the genes affected. Principal component analysis did not reveal clear global differences between the CML patients and healthy controls in the frequencies of ADME gene variants. However, the Cochran-Armitage trend test showed a number of variants whose frequencies in CML samples were significantly different from the healthy controls (table 1). Two of the loss-of-function variants were in the top 10 variants, whose frequencies differed between CML patients and healthy controls. ACAD11 rs41272317 variant was observed in 14.2% of patients whereas its frequency in healthy controls was 0.5% (p=0.003). Similarly DPYD rs3918290 was observed in 14.2% of patients and 3.3% of healthy controls (p=0.006). ACAD11 encodes an acyl-CoA dehydrogenase enzyme with a preference for carbon chain lengths between 20 and 26, and has been shown to be an important metabolic target for p53 pro-survival function. DPYD codesdihydropyrimidine dehydrogenase, which is involved in the metabolism ofpyrimidines and the anticancer agent 5-fluorouracil. Sanger sequencing confirmed both variants in patient samples. From the top10 missense variants, SLC15A1 rs144553892 variant was significantly more often observed in poor responding CML patients than in healthy controls (14.2 and 0.9%, respectively, p=0.003). SLC15A1 is a solute carrier family 15 member 1 which has a known function as a co-transporter foroligopeptides. Recently solute carriers have been identified to have significant impact on cancer therapy. Screening of additional CML patients is ongoing to confirm the frequency of these variants in optimally responding CML patients.CONCLUSIONSCML patients with primary resistance or suboptimal response to TKI therapy have mutations in genes affecting drug intake and metabolism, which can be causally associated with poor therapy responses. Validation in larger patient cohorts is warranted. [Display omitted] DisclosuresPorkka:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Ariad: Research Funding.

200: Parity is not associated with increased atherosclerosis in a mouse model

atherosclerosis in a mouse model Hai-Lang Duong, Kristine Lain, Deborah Howatt, Debra Rateri, Alan Daugherty, Thomas Curry University of Kentucky, Obstetrics and Gynecology, Lexington, KY, Norton Healthcare, Maternal-Fetal Medicine, Louisville, KY, University of Kentucky, Saha Cardiovascular Research Center, Lexington, KY OBJECTIVE: The correlation of parity to cardiovascular (CV) risk later in life has not been clearly elucidated. Using a genetically modified strain of mice susceptible to atherosclerosis, we hypothesized that there is increased atherogenesis in parous mice compared to nulliparous mice. STUDY DESIGN: A total of 38 age-matched, female, low density lipoprotein receptor (LDLr)-/mice were initially fed normal laboratory diet. At 2 months of age, 19 mice were impregnated and delivered pups three consecutive times. The other 19 mice were never bred. At 8 months of age, all mice were fed a fat-enriched diet, which promotes hypercholesterolemia. After 11 weeks on this diet, the mice were euthanized. The intimal surface of the aorta was exposed and the total area of intima from the aortic arch through the thorax was measured. Quantification of atherosclerotic lesions on the intimal surface was performed using en face analysis. For this analysis, atherosclerotic lesions were measured in the arch and thoracic regions; lesion area (LA) was then calculated as a percentage of the total intimal surface area. Non-fasting, serum total cholesterol (TC) levels were also measured. Statistical analyses used either the Wilcoxon rank sum test or the Student’s t-test. RESULTS: LA in the combined arch and thorax regions of the parous mice (median 19.5%) did not differ from that of the nulliparous mice (median 18.8%, p 0.49, Table 1). In the parous mice, LA within the arch region was 34.9%, compared to 33.8% (p 0.60) in the nulliparous mice. Within the region of the thorax, LA measured 0.74% in the parous mice, compared to 0.79% (p 0.50) in the control mice. The two groups of mice did not differ in weight (p 0.14) or serum TC (p 0.75). CONCLUSION: Using a well-established mouse model for atherogenesis, we observed no difference in aortic atherosclerosis in parous mice compared to nulliparous mice. Our results suggest that, in this model, there is no effect of pregnancy on the development of atherosclerosis. Higher parity or exposure to an atherogenic diet during pregnancy may be necessary to reveal a link between parity and CV disease. 201 Beta oxidation of fatty acids in term human placentas Hector Mendez-Figueroa, Edward Chien, Nicole L. Nesbitt, Sivakama S. Bharathi, Eric Goetzman Women & Infants Hospital of RI and the Warren Alpert Medical School of Brown University, Department of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, Providence, RI, Childrens Hospital of Pittsburgh, Department of Pediatrics, Pittsburgh, PA OBJECTIVE: Fatty acid (FA) metabolism in human placentas has not been well illustrated. Distribution across gestation as well as the enzyme activity level has not been elucidated. We propose to determine normal expression and function of enzymes involved in FA metabolism in term human placentas as well as the impact labor has on the pathway STUDY DESIGN: Samples were collected within 10 minutes of delivery from patients presenting for: a) scheduled non-labored cesarean section and b) normal vaginal delivery 37 weeks. 1) Expression: Enzyme detection was done via western blot against: 5 Acyl-CoA dehydrogenase (ACAD) enzymes VLCAD, LCAD, MCAD, SCAD, ACAD9; Long and short-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD, SCHAD), Long chain 3-Ketoacyl CoA thiolase (LKAT) and complex II and V of the electron transport chain (ETC). 2) Function: A) Overall pathway function was tested using 3H palmitic and 3H octanoic acid via an explant placental tissue protocol. B) ACAD activities: VLCAD, LCAD, MCAD and SCAD function was measured with the anaerobic electron transfer flavoprotein (ETF) fluorescence reduction assay. C) Activity of the ETC complex I was assessed the commercially available Complex I “dipstick assay” D) ATP concentration was measured using a colorimetric kit RESULTS: Oxidation rates for C16 and C8 were roughly 20X less than that seen in fed-state mouse liver (C16 49.3 / 4.3, C8 435 / 50 pmoles/mg/hr). C16 metabolism in non-laboring placentas was significantly increased compared to labor. ATP concentration was significantly decreased with labor (0.61 vs. 0.41, p 0.027). Key FA oxidation enzymes are not only expressed in placenta but also maintain their enzymatic activities (Figure 1, Table 1) CONCLUSION: Term human placentas metabolize FA at a rate approximately 20X less than liver. C16 metabolism is reduced with the onset of labor, probably secondary to a decrease in available ATP. MCAD and SCAD enzymatic activity is not absent (or nearly absent) in human placentas as previously suggested. The presence of all five ACAD enzymes indicate that the FAO pathway plays an important role in placentas. www.AJOG.org Clinical Obstetrics, Medical-Surgical-Disease, Neonatology, Physiology-Endocrinology Poster Session I

A role for taurine in mitochondrial function.

The mitochondrial pH gradient across the inner-membrane is stabilised by buffering of the matrix. A low-molecular mass buffer compound has to be localised in the matrix to maintain its alkaline pH value. Taurine is found ubiquitously in animal cells with concentrations in the millimolar range and its pKa value is determined to 9.0 (25°C) and 8.6 (37°C), respectively. Localisation of such a low-molecular buffer in the mitochondrial matrix, transforms the matrix into a biochemical reaction chamber for the important matrix-localised enzyme systems. Three acyl-CoA dehydrogenase enzymes, which are pivotal for beta-oxidation of fatty acids, are demonstrated to have optimal activity in a taurine buffer. By application of the model presented, taurine depletion caused by hyperglycemia could provide a link between mitochondrial dysfunction and diabetes.

Long chain fatty acid oxidation defects in children: importance of detection and treatment options

Mitochondrial beta oxidation plays a major role in energy production. Long chain fatty acid oxidation defects include deficiency of the trifunctional protein (rare) or more commonly defects of the long chain 3-hydroxy acyl-CoA dehydrogenase enzyme (LCHAD). These long chain defects have variable presentations, they may present in the neonate or infant with sudden death, hepatopathy (Reyes disease), hypoketotic hypoglycaemia, rhabdomyolysis, myopathy, cardiomyopathy and with late complications such as peripheral neuropathy, pigmentary retinopathy, retinal degeneration and progressive visual loss. The correct diagnosis at presentation is not only life saving but also allows for the appropriate dietary and other intervention, which may have major effects on outcome. Three case reports of patients with long chain fatty acid oxidation defects who have shown significant benefits from treatment are reported. These paediatric presentations illustrate the clinical heterogeneity of long chain fatty acid oxidation defects and opportunities for effective management if correctly diagnosed.

Arginine 387 of Human Isovaleryl-CoA Dehydrogenase Plays a Crucial Role in Substrate/Product Binding

Isovaleryl-CoA dehydrogenase (IVD) is a homotetrameric flavoenzyme, which catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA and transfers electrons to the electron-transferring flavoprotein, and is a member of the acyl-CoA dehydrogenase (ACD) enzyme family. Human IVD crystal structure with a bound substrate analogue shows the guanidino group of Arg387, a conserved residue among other members of the ACD enzyme family, juxtaposed to a phosphate oxygen of the 4′-phosphopantothiene moiety of the substrate analogue. Site-directed mutagenesis was used to investigate the role of Arg387 in substrate binding and enzyme function. Replacing this residue with Lys, Ala, Gln, or Glu resulted in stable proteins. Spectrophotometric substrate binding assays indicated that the Arg387Lys mutant was able to form the charge-transfer complex intermediate with similar efficiency to wild type, while the rest of the mutants were significantly less able to properly form this intermediate. However, the Km of the isovaleryl-CoA for the Arg387Lys mutant was 20.3 compared to 1.5 μM for the wild type. The Km for the rest of the mutants were 75.6, 195, and 550 μM, respectively. The catalytic efficiency per mole of FAD was 20.3, 3.3, 2.0, and 0.34 for the mutants, respectively, compared to 260 μM−1 min−1 for the wild type. These results substantiate the important role of Arg387 in anchoring the substrate, and are consistent with the hypothesis that residues distant from the active site are important for stabilizing the enzyme:substrate/product complex, and could play an important role in the mechanism of the enzyme-catalyzed reaction.

The medium-/long-chain fatty acyl-CoA dehydrogenase (fadF) gene of Salmonella typhimurium is a phase 1 starvation-stress response (SSR) locus.

Salmonella enterica serovar Typhimurium (S. typhimurium) is an enteric pathogen that causes significant morbidity in humans and other mammals. During their life cycle, salmonellae must survive frequent exposures to a variety of environmental stresses, e.g. carbon-source (C) starvation. The starvation-stress response (SSR) of S. typhimurium encompasses the genetic and physiological realignments that occur when an essential nutrient becomes limiting for bacterial growth. The function of the SSR is to produce a cell capable of surviving long-term starvation. This paper reports that three C-starvation-inducible lac fusions from an S. typhimurium C-starvation-inducible lac fusion library are all within a gene identified as fadF, which encodes an acyl-CoA dehydrogenase (ACDH) specific for medium-/long-chain fatty acids. This identification is supported by several findings: (a) significant homology at the amino acid sequence level with the ACDH enzymes from other bacteria and eukaryotes, (b) undetectable beta-oxidation levels in fadF insertion mutants, (c) inability of fad insertion mutants to grow on oleate or decanoate as a sole C-source, and (d) inducibility of fadF::lac fusions by the long-chain fatty acid oleate. In addition, the results indicate that the C-starvation-induction of fadF is under negative control by the FadR global regulator and positive control by the cAMP:cAMP receptor protein complex and ppGpp. It is also shown that the fadF locus is important for C-starvation-survival in S. typhimurium. Furthermore, the results demonstrate that fadF is induced within cultured Madin-Darby canine kidney (MDCK) epithelial cells, suggesting that signals for its induction (C-starvation and/or long-chain fatty acids) may be present in the intracellular environment encountered by S. typhimurium. However, fadF insertion mutations did not have an overt effect on mouse virulence.

Carnitine effects on coenzyme A profiles in rat liver with hypoglycin inhibition of multiple dehydrogenases.

To examine the changes in coenzyme A profile and the possible corrective effects of carnitine supplementation in the genetic disorders of mitochondrial beta-oxidation, we carried out experiments using an inhibitor of multiple acyl-CoA dehydrogenase enzymes, methylenecyclopropaneacetic acid (MCPA), in rat hepatocytes. MCPA irreversibly inhibited ketone synthesis from straight-chain fatty acids (butyrate, octanoate, palmitate) and branched-chain fatty acids (alpha-ketoisocaproate) with a parallel 70-90% reduction of hepatocyte acetyl-CoA levels. Alone, MCPA or substrates halved free CoA levels to 15% of total CoA and doubled short- and medium-chain acyl-CoA levels to 30% of total CoA. With MCPA plus substrates combined, free CoA levels were 10% of total CoA, and short- and medium-chain acyl-CoA levels were 45% of total CoA. Comparable changes in CoA profiles were found in a patient with a severe genetic defect in beta-oxidation. Neither the suppression of ketogenesis nor the alterations in CoA profiles induced by MCPA inhibition could be corrected by carnitine supplementation.

Cloning and characterization of human very-long-chain acyl-CoA dehydrogenase cDNA, chromosomal assignment of the gene and identification in four patients of nine different mutations within the VLCAD gene.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) is one of four straight-chain acyl-CoA dehydrogenase (ACD) enzymes, which are all nuclear encoded mitochondrial flavoproteins catalyzing the initial step in fatty acid beta-oxidation. We have used the very fast, Rapid Amplification of cDNA Ends (RACE) based strategy to obtain the sequence of cDNAs encoding human VLCAD from placenta and fibroblasts. Alignment of the predicted amino acid sequence of human VLCAD with those of the other human ACD enzymes revealed extensive sequence homology. Moreover, human VLCAD and human acyl-CoA oxidase showed extensive sequence homology corroborating the notion that these genes are evolutionarily related. Southern blot analysis of genomic DNA from hybrid cell lines was used to localize the VLCAD gene to human chromosome 17p11.2-p11.13105. Using Northern and Western blot analysis to investigate the tissue specific distribution of VLCAD mRNA and protein in several human tissues we showed that VLCAD is most abundant in heart and skeletal muscle. This agrees well with the fact that cardiac and muscle symptoms are characteristic for patients with VLCAD deficiency. Northern blot analysis and sequencing of cloned PCR amplified VLCAD cDNA from four unrelated patients with VLCAD deficiency showed that VLCAD mRNA was undetectable in one patient and that the other three have mutations in both VLCAD alleles. Western blot analysis of patient fibroblasts showed that the identified mutations result in severely reduced amounts of VLCAD protein. None of the patients harbored identical mutations suggesting that the mutational heterogeneity in VLCAD deficiency is large.

Inactivation of Short-Chain Acyl-Coenzyme A Dehydrogenase from Pig Liver by 2-Pentynoyl-Coenzyme A

2-Pentynoyl-CoA is a mechanism-based inactivator of the flavoprotein short-chain acyl-CoA dehydrogenase from pig liver. Inactivation is associated with the formation of an intermediate absorbing at 800 nm and results in the incorporation of 0.86 ± 0.13 molecules of radiolabeled inhibitor per subunit. A rapid procedure was devised to isolate the labeled peptide. A glutamate residue was identified as the target of 2-pentynoyl-CoA treatment and proved homologous to the proposed catalytic base, GLU376, in the corresponding medium-chain acyl-CoA dehydrogenase sequence. These results are discussed in terms of the lack of conservation of this glutamate residue in the acyl-CoA dehydrogenase enzyme family.

Acyl-CoA dehydrogenase enzymes during early postnatal development in the rat.

Fatty acid oxidation rates tend to increase with age in most developing tissues. In skeletal muscle, heart, and liver of developing rats, we measured activities of three acyl-CoA dehydrogenase enzymes, which constitute the first step in the mitochondrial beta-oxidation sequence. In skeletal muscle, activities of all three enzymes increased with age. In heart muscle, palmityl-CoA dehydrogenase increased, while the other two enzymes changed only minimally. In liver, palmityl-CoA dehydrogenase activity steadily increased with age. Decanoyl- and butyryl-CoA dehydrogenase also increased with age, but much more irregularly. We also examined the electrophoretic characteristics of these enzyme proteins in the three tissues. There were no changes in their electrophoretic patterns during development.

Fatty acyl-CoA dehydrogenase enzymes in human skeletal muscle

An electrophoretic and enzyme-substrate staining technique for acyl-CoA dehydrogenase (ACD) enzymes was developed for use with small (< 100 mg) tissue samples. Based on their electrophoretic mobility and substrate staining specificity, at least two and perhaps three chain-length specific enzymes for dehydrogenation of saturated fatty acids were found in human skeletal muscle. ACD enzymes staining with octanoyl-CoA or palmitoyl-CoA were identified by this technique in human skeletal muscle, heart, and liver, but the ACD enzyme staining with butyryl-CoA was difficult to detect and was definitely visualized only in skeletal muscle.