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Search results for: Malonyl CoA decarboxylase (mutant)

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#18487439   2008/05/16 To Up

Reduced heart size and increased myocardial fuel substrate oxidation in ACC2 mutant mice.

The cardiac-enriched isoform of acetyl-CoA carboxylase (ACC2) is a key regulator of mitochondrial fatty acid (FA) uptake via carnitine palmitoyltransferase 1 (CPT1). To test the hypothesis that oxidative metabolism is upregulated in hearts from animals lacking ACC2 (employing a transgenic Acc2-mutant mouse), we assessed cardiac function in vivo and determined rates of myocardial substrate oxidation ex vivo. When examined by echocardiography, there was no difference in systolic function, but left ventricular mass of the Acc2-mutant (MUT) mouse was significantly reduced ( approximately 25%) compared with wild-types (WT). Reduced activation of the mammalian target of rapamycin (mTOR) and its downstream target p70S6K was found in MUT hearts. Exogenous oxidation rates of oleate were increased approximately 22%, and, unexpectedly, exogenous glucose oxidation rates were also increased in MUT hearts. Using a hyperinsulinemic-euglycemic clamp, we found that glucose uptake in MUT hearts was increased by approximately 83%. Myocardial triglyceride levels were significantly reduced in MUT vs. WT while glycogen content was the same. In parallel, transcript levels of PPARalpha and its target genes, pyruvate dehydrogenase kinase-4 (PDK-4), malonyl-CoA decarboxylase (MCD), and mCPT1, were downregulated in MUT mice. In summary, we report that 1) Acc2-mutant hearts exhibit a marked preference for the oxidation of both glucose and FAs coupled with greater utilization of endogenous fuel substrates (triglycerides), 2) attenuated mTOR signaling may result in reduced heart sizes observed in Acc2-mutant mice, and 3) Acc2-mutant hearts displayed normal functional parameters despite a significant decrease in size.
M Faadiel Essop, Heidi S Camp, Cheol Soo Choi, Saumya Sharma, Ryan M Fryer, Glenn A Reinhart, Patrick H Guthrie, Assia Bentebibel, Zeiwei Gu, Gerald I Shulman, Heinrich Taegtmeyer, Salih J Wakil, Lutfi Abu-Elheiga

1215 related Products with: Reduced heart size and increased myocardial fuel substrate oxidation in ACC2 mutant mice.

100 UG4/120 Packing /sleeve/bo1 mg4/120 Packing /sleeve/bo4/120 Packing /sleeve/boSize: 1 mL 100 mM x 20.5mg

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#17535268   2007/05/29 To Up

Fatal malonyl CoA decarboxylase deficiency due to maternal uniparental isodisomy of the telomeric end of chromosome 16.

Malonic aciduria is a rare autosomal recessive disorder caused by deficiency of malonyl-CoA decarboxylase, encoded by the MLYCD gene. We report on a patient with clinical presentation in the neonatal period. Metabolic investigations led to a diagnosis of malonyl-CoA decarboxylase deficiency, confirmed by decreased activity in cultured fibroblasts. High doses of carnitine and a diet low in lipids led to a reduction in malonic acid excretion, and to an improvement in his clinical conditions, but at the age of 4 months he died suddenly and unexpectedly. No autopsy was performed. Molecular analysis of the MLYCD gene performed on the proband's RNA and genomic DNA identified a previously undescribed mutation (c.772-775delACTG) which was homozygous. This mutation was present in his mother but not in his father; paternity was confirmed by microsatellite analysis. A hypothesis of maternal uniparental disomy (UPD) was investigated using fourteen microsatellite markers on chromosome 16, and the results confirmed maternal UPD. Maternal isodisomy of the 16q24 region led to homozygosity for the MLYCD mutant allele, causing the patient's disease. These findings are relevant for genetic counselling of couples with a previously affected child, since the recurrence risk in future pregnancies is dramatically reduced by the finding of UPD. In addition, since the patient had none of the clinical manifestations previously associated with maternal UPD 16, this case provides no support for the existence of maternally imprinted genes on chromosome 16 with a major effect on phenotype.
S Malvagia, L Papi, A Morrone, M A Donati, F Ciani, E Pasquini, G la Marca, H R Scholte, M Genuardi, E Zammarchi

1689 related Products with: Fatal malonyl CoA decarboxylase deficiency due to maternal uniparental isodisomy of the telomeric end of chromosome 16.

250ul1.00 flask 5 G250ul250ul250ul10 IU

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#16739991   // To Up

Mass spectrometric identification of K210 essential for rat malonyl-CoA decarboxylase catalysis.

Proteomic technology provides useful tools to detect protein modification sites in vivo and in vitro. In this work, we applied proteomics to identify an essential amino acid residue involved in Malonyl-CoA Decarboxylase (MCD) catalysis. A reaction with acetic anhydride and MCD, under mild conditions without acetyl CoA as a substrate, resulted in the acetylation of six lysyl residues, K210, K58, K167, K316, K388, and K444. When acetyl CoA was added to the reaction, K210 was protected from acetylation, indicating a potential role for this residue in catalysis. In addition, K210 was the only lysyl residue, out of six, that was not endogenously acetylated. Because K210, K308, and K388 are conserved across species, they were site-specifically mutated to methionine which is size-wise similar to lysine but not protonated. The K308M and K388M MCD mutants retained 60% of their enzyme activities, whereas the K210M mutant was completely inactive. These results strongly suggest that K210 is an essential residue in rat MCD catalysis and is a likely proton donor to the alpha carbon of malonyl-CoA. Therapeutic inhibition of MCD may be a viable approach to treating various clinical pathologies associated with defective fatty acid metabolism.
Hyung Wook Nam, Gha Young Lee, Yu Sam Kim

2145 related Products with: Mass spectrometric identification of K210 essential for rat malonyl-CoA decarboxylase catalysis.

250ul250ul250ul250ul60 IU100 mg 500 ml 100ml96 Well100 mg0.2 mg96 Samples

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#15019286   // To Up

Isolation of two distinct populations of recombinant antibody molecules specific for rat malonyl-CoA decarboxylase from a semi-synthetic human scFv display library using Ex-phage system.

A semi-synthetic human scFv phage display library by randomizing amino acid residues at CDR3H was constructed using pIGT3 phagemid vector. Recombinant phages were rescued by super-infecting the JS5 E. coli library stock with Ex-phage, the mutant M13KO7 helper phage containing amber mutations at gIII. The library was composed of 2 x 10(8) independent clones, and selected for the specific binders against malonyl-CoA decarboxylase (MCD) by panning. Five soluble scFv clones specific for MCD were finally identified and classified into two groups based on the difference in their binding pattern to MCD. Two clones (M4 and M8) showed good binding reactivity to MCD in ELISA but not in Western blot, whereas, the rest three clones (M23, M28 and M41) reacted to the antigen in Western blot but not in ELISA implying they bound to somewhat different epitopes on MCD. DNA sequencing analysis of M4, M8, M23 and M28 showed that VH of all clones were belonged to VH3 subgroup. On the other hand, M4 and M8 utilized VLkappa subgroup I, and M23 and M28 used VLkappa subgroup IV, suggesting that difference in binding pattern between M4/M8 and M23/M28 against MCD might come from the different VL gene utilization. In conclusion, human monoclonal scFv antibodies specific for MCD were successfully isolated and we demonstrated that distinct populations of recombinant antibodies specific to the target antigen could be isolated by Ex-phage system.
Hyun-jung Baek, Byung-woong Hur, Jin-won Cho, Hee-kyung Lee, Nam-il Kim, Mi-young Oh, Sang-hoon Cha

2699 related Products with: Isolation of two distinct populations of recombinant antibody molecules specific for rat malonyl-CoA decarboxylase from a semi-synthetic human scFv display library using Ex-phage system.

25 µg25 µg100 TESTS4 Membranes/Box0.2 mg0.25 mg4 Membranes/Box4 Arrays/Slide4 Arrays/Slide4 Membranes/Box0.1 mg4 Membranes/Box

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#11502177   // To Up

beta-Ketoacyl-[acyl carrier protein] synthase I of Escherichia coli: aspects of the condensation mechanism revealed by analyses of mutations in the active site pocket.

beta-Ketoacyl-[acyl carrier protein (ACP)] synthase forms new carbon-carbon bonds in three steps: transfer of an acyl primer from ACP to the enzyme, decarboxylation of the elongating substrate and its condensation with the acyl primer substrate. Six residues of Escherichia coli beta-ketoacyl-ACP synthase I (KAS I) implicated in these reactions were subjected to site-directed mutagenesis. Analyses of the abilities of C163A, C163S, H298A, D306A, E309A, K328A, and H333A to carry out the three reactions lead to the following conclusions. The active site Cys-163 is not required for decarboxylation, whereas His-298 and His-333 are indispensable. Neither of the histidines is essential for increasing the nucleophilicity of Cys-163 to enable transfer of the acyl primer substrate. Maintenance of the structural integrity of the active site by Asp-306 and Glu-309 is required for decarboxylation but not for transfer. One function of Lys-328 occurs very early in catalysis, potentially before transfer. These results in conjunction with structural analyses of substrate complexes have led to a model for KAS I catalysis [Olsen, J. G., Kadziola, A., von Wettstein-Knowles, P., Siggaard-Andersen, M., and Larsen, S. (2001) Structure 9, 233-243]. Another facet of catalysis revealed by the mutant analyses is that the acyl primer transfer activity of beta-ketoacyl-ACP synthase I is inhibited by free ACP at physiological concentrations. Differences in the inhibitory response by individual mutant proteins indicate that interaction of free ACP with Cys-163, Asp-306, Glu-309, Lys-328, and His-333 might form a sensitive regulatory mechanism for the transfer of acyl primers.
K A McGuire, M Siggaard-Andersen, M G Bangera, J G Olsen, P von Wettstein-Knowles

1704 related Products with: beta-Ketoacyl-[acyl carrier protein] synthase I of Escherichia coli: aspects of the condensation mechanism revealed by analyses of mutations in the active site pocket.

1100 μg5mg100 U2ug5ug2ug

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#11006298   // To Up

Mechanism of chalcone synthase. pKa of the catalytic cysteine and the role of the conserved histidine in a plant polyketide synthase.

Polyketide synthases (PKS) assemble structurally diverse natural products using a common mechanistic strategy that relies on a cysteine residue to anchor the polyketide during a series of decarboxylative condensation reactions that build the final reaction product. Crystallographic and functional studies of chalcone synthase (CHS), a plant-specific PKS, indicate that a cysteine-histidine pair (Cys(164)-His(303)) forms part of the catalytic machinery. Thiol-specific inactivation and the pH dependence of the malonyl-CoA decarboxylation reaction were used to evaluate the potential interaction between these two residues. Inactivation of CHS by iodoacetamide and iodoacetic acid targets Cys(164) in a pH-dependent manner (pK(a) = 5.50). The acidic pK(a) of Cys(164) suggests that an ionic interaction with His(303) stabilizes the thiolate anion. Consistent with this assertion, substitution of a glutamine for His(303) maintains catalytic activity but shifts the pK(a) of the thiol to 6.61. Although the H303A mutant was catalytically inactive, the pH-dependent incorporation of [(14)C]iodoacetamide into this mutant exhibits a pK(a) = 7.62. Subsequent analysis of the pH dependence of the malonyl-CoA decarboxylation reaction catalyzed by wild-type CHS and the H303Q and C164A mutants also supports the presence of an ion pair at the CHS active site. Structural and sequence conservation of a cysteine-histidine pair in the active sites of other PKS implies that a thiolate-imidazolium ion pair plays a central role in polyketide biosynthesis.
J M Jez, J P Noel

1383 related Products with: Mechanism of chalcone synthase. pKa of the catalytic cysteine and the role of the conserved histidine in a plant polyketide synthase.

1100 100.00 ul

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#10512619   // To Up

Conversion of a beta-ketoacyl synthase to a malonyl decarboxylase by replacement of the active-site cysteine with glutamine.

beta-Ketoacyl synthases involved in the biosynthesis of fatty acids and polyketides exhibit extensive sequence similarity and share a common reaction mechanism, in which the carbanion participating in the condensation reaction is generated by decarboxylation of a malonyl or methylmalonyl moiety; normally, the decarboxylation step does not take place readily unless an acyl moiety is positioned on the active-site cysteine residue in readiness for the ensuing condensation reaction. Replacement of the cysteine nucleophile (Cys-161) with glutamine, in the beta-ketoacyl synthase domain of the multifunctional animal fatty acid synthase, completely inhibits the condensation reaction but increases the uncoupled rate of malonyl decarboxylation by more than 2 orders of magnitude. On the other hand, replacement with Ser, Ala, Asn, Gly, and Thr compromises the condensation reaction without having any marked effect on the decarboxylation reaction. The affinity of the beta-ketoacyl synthase for malonyl moieties, in the absence of acetyl moieties, is significantly increased in the Cys161Gln mutant compared to that in the wild type and is similar to that exhibited by the wild-type beta-ketoacyl synthase in the presence of an acetyl primer. These results, together with modeling studies of the Cys --> Gln mutant from the crystal structure of the Escherichia coli beta-ketoacyl synthase II enzyme, suggest that the side chain carbonyl group of the Gln-161 can mimic the carbonyl of the acyl moiety in the acyl-enzyme intermediate so that the mutant adopts a conformation analogous to that of the acyl-enzyme intermediate. Catalysis of the decarboxylation of malonyl-CoA requires the dimeric form of the Cys161Gln fatty acid synthase and involves prior transfer of the malonyl moiety from the CoA ester to the acyl carrier protein domain and subsequent release of the acetyl product by transfer back to a CoA acceptor. These results suggest that the role of the Cys --> Gln beta-ketoacyl synthases found in the loading domains of some modular polyketide synthases likely is to act as malonyl, or methylmalonyl, decarboxylases that provide a source of primer for the chain extension reactions catalyzed by associated modules containing fully competent beta-ketoacyl synthases.
A Witkowski, A K Joshi, Y Lindqvist, S Smith

1545 related Products with: Conversion of a beta-ketoacyl synthase to a malonyl decarboxylase by replacement of the active-site cysteine with glutamine.

100μg1 mL100 ul200 5 μg100μg100 ul100 μg

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