Acid synthases

Fatty acids are an important energy source, for they yield over twice as much energy as an equal mass of carbohydrate or protein. In humans, the primary dietary source of fatty acids is triacylglycerols. This lecture will describe the metabolism of fatty acids. The two main components of fatty acid metabolism are β oxidation and fatty acid synthesis. Upon completion of this lecture, you will understand that the fatty-acid breakdown reactions of β oxidation result in the formation of reduced cofactors and acetyl-CoA molecules, which can be further catabolized to release free energy.

64p zingzing09 21-10-2012 111 11   Download

Liver metabolism is influenced by hormones and nutrients. Amino acids such as glutamine or leucine induce an anabolic response, which resembles that of insulin in muscle and adipose tissue. In this work, the signalling pathways and the effects of insulin were compared to those of glutamine and leucine in isolated hepatocytes from normal and streptozotocin-diabetic rats.

9p system191 01-06-2013 50 5   Download

An oxidosqualene cyclase cDNA, LcIMS1, was isolated from cultured cells of Luffa cylindrica Roem. by heterologous hybridization with cDNA of Glycyrrhiza glabra b-amyrin synthase. Expression of LcIMS1 in yeast lacking endogenous oxidosqualene cyclase activity resulted in the accumulation of isomultiflorenol, a triterpene. This is consistent with LcIMS1 encoding isomultiflorenol synthase, an oxidosqualene cyclase involved in bryonolic acid biosynthesis in cultured Luffa cells.

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By replacing specific amino acids at positions 112, 147 and 152 of the human aldosterone synthase (CYP11B2) with the corresponding residues from human, mouse or rat 11b-hydroxylase (CYP11B1), we have been able to investigate whether these residues belong to structural determinants of individual enzymatic activities. When incubated with 11-deoxycorticosterone (DOC), the 11b-hydroxylation activity of the mutants was most effectively increased by combining D147E and I112P (sixfold increase).

10p research12 01-06-2013 39 5   Download

The eukaryotic glyoxylate cycle has been previously hypothesized to occur in the peroxisomal compartment, which in the yeast Saccharomyces cerevisiae additionally represents the sole site for fatty acid b-oxidation. The subcellular location of the key glyoxylate-cycle enzyme malate synthase 1 (Mls1p), an SKL-terminated protein, was examined in yeast cells grown on di€erent carbon sources.

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We are interested in constructing a model for the substrate-bindingsiteof fattyacidelongase-13-ketoacylCoAsynthase (FAE1 KCS),the enzyme responsible forproductionof very long chain fatty acids of plant seed oils.Arabidopsis thaliana andBrassica napusFAE1 KCS enzymes are highly homo-logous but the seed oil content of these plants suggests that their substrate specificities differ with respect to acyl chain length.

10p research12 29-04-2013 35 3   Download

The pathway of the oxidation of propionate to pyruvate in Escherichia coliinvolves five enzymes, only two of which, methylcitrate synthase and 2-methylisocitrate lyase, have been thoroughly characterized. Here we report that the isomerization of (2S,3S)-methylcitrate to (2R,3S)-2-methyl-isocitrate requires anovel enzyme,methylcitratedehydratase (PrpD), and the well-known enzyme, aconitase (AcnB), of the tricarboxylic acid cycle.

11p tumor12 22-04-2013 51 4   Download

Site-directedmutagenesis was used to investigate the control of 2-oxoacid cosubstrate selectivity by deacetoxycephalo-sporin C synthase. The wild-type enzyme has a requirement for 2-oxoglutarate and cannot efficiently use hydrophobic 2-oxoacids (e.g. 2-oxohexanoic acid, 2-oxo-4-methyl-penta-noic acid) as the cosubstrate. The followingmutant enzymes were produced: R258A, R258L, R258F, R258H and R258K. All of the mutants have broadened cosubstrate selectivity and were able to utilize hydrophobic 2-oxoacids.

7p tumor12 20-04-2013 44 3   Download

The plant enzyme arbutin synthase isolated from cell sus-pension cultures ofRauvolfia serpentinaand heterologously expressed inEscherichia coliis a member of the NRD1b family of glycosyltransferases. This enzyme was used to prove, by site-directed mutagenesis, suggested catalytic domains and reaction mechanisms proposed for enzyme-catalyzed glycosylation. Replacement of amino acids far from the NRD domain do not significantly affect arbutin synthase activity.

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Acetohydroxyacid synthase (AHAS, EC 4.1.3.18) catalyses the first step in branched-chain amino acid biosynthesis and is the target for sulfonylurea and imidazolinone herbicides, which act as potent and specific inhibitors. Mutants of the enzyme have been identified that are resistant to particular herbicides.However, the selectivityof thesemutants towards various sulfonylureas and imidazolinones has not been determined systematically.

10p fptmusic 16-04-2013 27 3   Download

(All-E) prenyl diphosphate synthases catalyze the con-secutive condensation of isopentenyl diphosphates with allylic prenyl diphosphates, producing products with vari-ous chain-lengths that are unique for each enzyme. Some short-chain (all-E) prenyl diphosphate synthases, i.e. farnesyl diphosphate synthases and geranylgeranyl diphosphate synthases contain characteristic amino acid sequences around the allylic substrate binding sites, which have been shown to play a role in determining the chain-length of the product....

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Eleven of the codons specifying the amino acids of the flexible catalytic loop [KRRPRPNVAEVM(197–208)] of Bacillus subtilis phosphoribosyl diphosphate synthase have been changed individually to specify alanine. The resulting variant enzyme forms, as well as the wildtype enzyme, were produced in anEscherichia colistrain lacking endogenous phosphoribosyl diphosphate synthase activity and purified to near homogeneity. The B. subtilisphosphoribosyl diphosphate synthase mutant variants K197A and R199A were studied in detail....

9p fptmusic 11-04-2013 31 3   Download

Depolarization and repolarization phases (D and R phases, respectively) of mitochondrial potential fluctuations induced by photoactivation of the fluorescent probe tetramethylrhodamine methyl ester (TMRM) were ana-lyzed separately and investigated using specific inhibitors and substrates. The frequency of R phases was significantly inhibited by oligomycin and aurovertin (mitochondrial ATP synthase inhibitors), rotenone (mitochond-rial complex I inhibitor) and iodoacetic acid (inhibitor of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase)....

11p awards 06-04-2013 52 2   Download

Five truncation mutants of chloroplast ATP synthasecsubunit from spin-ach (Spinacia oleracea) lacking 8, 12, 16, 20 or 60 N-terminal amino acids were generated by PCR by a mutagenesis method. The recombinantc genes were overexpressed inEscherichia coliand assembled withabsub-units into a native complex. The wild-type (WT)abcassembly i.e.abcWT exhibited high Mg 2+ -dependent and Ca 2+ -dependent ATP hydrolytic activity.

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GTP is an allosteric activator of CTP synthase and acts to increase thekcat for the glutamine-dependent CTP synthesis reaction. GTP is suggested, in part, to optimally orient the oxy-anion hole for hydrolysis of glutamine that takes place in the glutamine amidotransferase class I (GATase) domain of CTP synthase.

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Escherichia colifatty acid cyclopropane synthase (CFAS) was overproduced and purified as a His6 -tagged protein. This recombinant enzyme is as active as the native enzyme with aKmof 90lMforS-AdoMet and a specific activity of 5·10 )2 lmolÆmin )1 Æmg )1 . The enzyme is devoid of organic or metal cofactors and is unable to catalyze the wash-out of themethyl protons ofS-AdoMet to the solvent, data that do not support the ylidemechanism. Inactivationof the enzyme by 5,5¢-dithiobis-(2-nitrobenzoic acid) (DTNB), a pseudo first-order process with a rate constant of 1.

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The amino acid sequence of 5-phospho-a-D-ribosyl 1-diphosphate synthase from the thermophile Bacillus caldolyticusis 81% identical to the amino acid sequence of 5-phospho-a-D-ribosyl 1-diphosphate synthase from the mesophileBacillus subtilis.Nevertheless the enzyme fromthe two organisms possesses very different thermal properties. TheB. caldolyticusenzyme has optimal activity at 60–65
C and a half-life of 26 min at 65
C, compared to values of 46
Cand60sat65
C, respectively, for theB. subtilis enzyme. ...

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The amino acid residue tryptophan 27 of 6,7-dimethyl-8-ribityllumazine synthase of the yeastSchizosaccharomyces pombewas replacedby tyrosine. The structures of theW27Y mutant protein in complex with riboflavin, the substrate analogue 5-nitroso-6-ribitylamino-2,4(1H,3H)-pyrimidin-edione, and the product analogue 6-carboxyethyl-7-oxo-8-ribityllumazine,weredeterminedbyX-raycrystallography at resolutions of 2.7–2.8 A ˚ .

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We describe a strategy for systematic amplification of chitin synthase genes (chs) in the filamentous ascomycetes plant-pathogenBotrytis cinereausing PCR with multiple degen-erate primers designed on specific and conserved sequence motifs. Eight distinctchsgeneswere isolated, namedBcchs I, II, IIIa, IIIb, IV, V, VIandVII. They probably constitute the entire chsmultigenic family of this fungus, as revealed by careful analysis of six euascomycetes genomes.

12p dell39 03-04-2013 39 4   Download

The expression of the colonic mitochondrial 3-hydroxy 3-methyl glutaryl CoA (mHMGCoA) synthase,a key con-trol site of ketogenesis from butyrate,is lower in germ-free (GF) than in conventional (CV) rats. In contrast,the activity of glutaminase is higher. The objective of this study was to investigate whether the intestinal flora can affect gene expression through short chain fatty acid (SCFA) and butyrate production. GF rats were inoculated with a con-ventional flora (Ino-CV) orwitha bacterial strainproducing butyrate (Clostridium paraputrificum,Ino-Cp) or not (Bifidobacterium breve,Ino-Bb).

9p dell39 03-04-2013 35 4   Download