Self-acylation properties of type II fatty acid biosynthesis acyl carrier protein

Acyl carrier protein (ACIP) plays a central role in many metabolic processes inside the cell, and almost 4% of the total enzymes inside the cell require it as a cofactor. Here, we report self-acylation properties in ACPs from Plasmodium falciparum and Brassica napus that are essential components of type II fatty acid biosynthesis (FAS II), disproving the existing notion that this phenomenon is restricted only to ACPs involved in polyketide biosynthesis. We also provide strong evidence to suggest that catalytic self-acylation is intrinsic to the individual ACP. Mutational analysis of these ACPs revealed the key residue(s) involved in this phenomenon. We also demonstrate that these FAS 11 ACPs exhibit a high degree of selectivity for self-acylation employing only dicarboxylic acids as substrates. A plausible mechanism for the self-acylation reaction is also proposed.

Backbone chemical shift assignments of the acyl-acyl carrier protein intermediates of the fatty acid biosynthesis pathway of Plasmodium falciparum

We report the backbone chemical shift assignments of the acyl-acyl carrier protein (ACP) intermediates of the fatty acid biosynthesis pathway of Plasmodium falciparum. The acyl-ACP intermediates butyryl (C4), -octanoyl (C8), -decanoyl (C10), -dodecanoyl (C12) and -tetradecanoyl (C14)-ACPs display marked changes in backbone HN, Cα and Cβ chemical shifts as a result of acyl chain insertion into the hydrophobic core. Chemical shift changes cast light on the mechanism of expansion of the acyl carrier protein core.

How pantothenol intervenes in Coenzyme-A biosynthesis of Mycobacterium tuberculosis

Coenzyme A is an indispensable cofactor for all organisms and holds a central position in a number of pathways. Prokaryotic enzymes involved in the synthesis of CoA are quite different from their mammalian counterparts; hence, they are good targets for the development of antimicrobials to treat many diseases. There are antimicrobials that act by inhibiting CoA biosynthesis. It has been suggested that pantothenol exhibits antibacterial activity by competitively inhibiting pantothenate kinase, a key regulatory enzyme for CoA synthesis. Contrary to these suggestions, in this paper, we demonstrate that pantothenol acts as a substrate for Mycobacterium tuberculosis and Escherichia coli pantothenate kinases. The product, 4'-phosphopantothenol, thus formed inhibits competitively the utilization of 4'-phosphopantothenate by CoaBC. Thus, it is the failure of CoaBC to utilize 4'-phosphopantothenol as a substrate that accounts for the bactericidal activity of pantothenol. (C) 2007 Elsevier Inc. All rights reserved.

Heme oxygenase-1 in cardiovascular diseases

Myocardial infarction (MI) and heart failure are major causes of morbidity and mortality worldwide. Treatment of MI involves early restoration of blood flow to limit infarct size and preserve cardiac function. MI leads to left ventricular remodeling, which may eventually progress to heart failure, despite the established pharmacological treatment of the disease. To improve outcome of MI, new strategies for protecting the myocardium against ischemic injury and enhancing the recovery and repair of the infarcted heart are needed. Heme oxygenase-1 (HO-1) is a stress-responsive and cytoprotective enzyme catalyzing the degradation of heme into the biologically active reaction products biliverdin/bilirubin, carbon monoxide (CO) and free iron. HO-1 plays a key role in maintaining cellular homeostasis by its antiapoptotic, anti-inflammatory, antioxidative and proangiogenic properties. The present study aimed, first, at evaluating the role of HO-1 as a cardioprotective and prohealing enzyme in experimental rat models and at investigating the potential mechanisms mediating the beneficial effects of HO-1 in the heart. The second aim was to evaluate the role of HO-1 in 231 critically ill intensive care unit (ICU) patients by investigating the association of HO-1 polymorphisms and HO-1 plasma concentrations with illness severity, organ dysfunction and mortality throughout the study population and in the subgroup of cardiac patients. We observed in an experimental rat MI model, that HO-1 expression was induced in the infarcted rat hearts, especially in the infarct and infarct border areas. In addition, pre-emptive HO-1 induction and CO donor pretreatment promoted recovery and repair of the infarcted hearts by differential mechanisms. CO promoted vasculogenesis and formation of new cardiomyocytes by activating c-kit+ stem/progenitor cells via hypoxia-inducible factor 1 alpha, stromal cell-derived factor 1 alpha (SDF-1a) and vascular endothelial growth factor B, whereas HO-1 promoted angiogenesis possibly via SDF-1a. Furthermore, HO-1 protected the heart in the early phase of infarct healing by increasing survival and proliferation of cardiomyocytes. The antiapoptotic effect of HO-1 persisted in the late phases of infarct healing. HO-1 also modulated the production of extracellular matrix components and reduced perivascular fibrosis. Some of these beneficial effects of HO-1 were mediated by CO, e.g. the antiapoptotic effect. However, CO may also have adverse effects on the heart, since it increased the expression of extracellular matrix components. In isolated perfused rat hearts, HO-1 induction improved the recovery of postischemic cardiac function and abrogated reperfusion-induced ventricular fibrillation, possibly in part via connexin 43. We found that HO-1 plasma levels were increased in all critically ill patients, including cardiac patients, and were associated with the degree of organ dysfunction and disease severity. HO-1 plasma concentrations were also higher in ICU and hospital nonsurvivors than in survivors, and the maximum HO-1 concentration was an independent predictor of hospital mortality. Patients with the HO-1 -413T/GT(L)/+99C haplotype had lower HO-1 plasma concentrations and lower incidence of multiple organ dysfunction. However, HO-1 polymorphisms were not associated with ICU or hospital mortality. The present study shows that HO-1 is induced in response to stress in both experimental animal models and severely ill patients. HO-1 played an important role in the recovery and repair of infarcted rat hearts. HO-1 induction and CO donor pretreatment enhanced cardiac regeneration after MI, and HO-1 may protect against pathological left ventricular remodeling. Furthermore, HO-1 induction potentially may protect against I/R injury and cardiac dysfunction in isolated rat hearts. In critically ill ICU patients, HO-1 plasma levels correlate with the degree of organ dysfunction, disease severity, and mortality, suggesting that HO-1 may be useful as a marker of disease severity and in the assessment of outcome of critically ill patients.

Biosynthesis of isoleucine and valine in Mycobacterium tuberculosis H37 Rv

The enzymes involved in the biosynthesis of isoleucine and valine have been shown to be present in cell-free extracts of Mycobacterium tuberculosis H37Rv. In addition to the known enzymes of the pathway, cell-free extracts of this organism contain a new enzyme. When cell-free extracts were incubated with acetolactate and Image -ascorbic acid, without reduced nicotinamide adenine dinucleotide phosphate, the isomer of acetolactate, viz., α-keto-β-hydroxyisovalerate, was found to accumulate and was identified by different methods. The reaction is enzymic, and Image -ascorbic acid cannot be replaced by other reducing agents such as hydroquinone, 2,6-dichlorophenol indophenol, or glutathione; by derivatives of Image -ascorbic acid such as dehydroascorbic acid or dimethyl ascorbic acid; or by cobamide coenzyme. Since the extracts also isomerize α-acetohydroxybutyrate to α-keto-β-hydroxy-β-methylvalerate, the enzyme catalyzing the reaction has been termed “acetohydroxy acid isomerase.” This is the first time that the presence of acetohydroxy acid isomerase has been reported in any biological system and that a specific metabolic role has been assigned for Image -ascorbic acid. The extract also possesses reductase activity to convert α-keto-β-hydroxyisovalerate to α,β-dihydroxyisovalerate in the presence of reduced nicotinamide adenine dinucleotide phosphate.

Studies on the biosynthesis of β-n-oxalyl-l-α,β-diaminopropionic acid, the Lathyrus sativus neurotoxin

The biosynthesis of β-N-oxalyl-l-α,β-diaminopropionic acid (ODAP), HOOC· CO·NH·CH2·CH(NH2·COOH is of interest, since this neurotoxin has been isolated from the seeds of Lathyrus sativus, the consumption of which causes the disease neurolathyrism in humans. The concentration of this non-protein amino acid in the seeds increases on germination. When the seeds are germinated in the presence of [14C2]- oxalic acid, the isolated ODAP is labelled exclusively in the oxalyl moiety. An oxalyl- CoA synthetase requiring the obligatory presence of ATP, CoA and Mg2+ can be demonstrated in crude extracts of the seedlings. When l-α,β-diaminopropionic acid is incubated with the enzyme in the presence of the components for oxalyl activation, net formation of ODAP can be shown. The enzymic reaction is specific to the β-amino group of l-α,β-diaminopropionic acidm and the higher homologues like α,γ-diaminobutyric acid, ornithine and lysine are inactive in this system. ODAP is not formed with α,β-diaminopropionic acid when the enzyme extract is prepared from Pisum sativum although oxalyl-CoA formation can be demonstrated.

Biosynthesis of sulfated mucopolysaccharides in relation to vitamins A and K

1.(1) Incorporation of Na235SO4 into acid mucopolysaccharides of intestine and colon tissue has been studied in normal, vitamin A-deficient and excess vitamin A-fed rats. 2. (2) Vitamin A deficiency resulted in a significant decrease in [35S]sulfate incorporation into mucopolysaccharides isolated from intestines of male rats. There was no significant change in the total mucopolysaccharides per gram of fresh tissue. 3. (3) When rats are fed excessive amounts of retinyl acetate, increased [35S]sulfate incorporation into mucopolysaccharides of rat intestine and colon is observed. 4. (4) Supplementation of vitamin K1 to rats fed excessive amounts of vitamin A restores the incorporation of [35S]sulfate into the acid mucopolysaccharides to the normal level. 5. (5) The implications of these findings with special reference to the role of vitamins A and K in the synthesis of sulfated mucopolysaccharides are discussed.

Biosynthesis of ubiquinone and ubichromenol in vitamin A-deficient rats

1. The concentrations of ubiquinone and ubichromenol increased in the livers, but not in the intestines and kidneys, of rats maintained on a diet deficient in vitamin 2. After short time intervals (e.g. 2 h) following administration of the tracer, incorporation of [2-14C]mevalonate into ubiquinone and ubichromenol in livers of vitamin A-deficient rats was lower than for normal animals; this was in contrast to later times (e.g. 72 h) when it was higher. 3. The “newly synthesized” ubiquinone in livers of vitamin A-deficient rats was distributed in all the cell fractions without specific localization. 4. Absorbed exogenous [14C]ubiquinone and [14C]ubichromenol were retained in the livers of vitamin A-deficient rats to a larger extent and for a longer time than in the normal animals. 5. The results suggest that the accumulation of ubiquinone and ubichromenol in the livers of vitamin A-deficient rats is due to lowered catabolism and not to increased rate of synthesis.

Biosynthesis of isoleucine and valine in mycobacterium tuberculosis H37Rv*1: II. Purification and properties of acetohydroxy acid isomerase

Acetohydroxy acid isomerase (AHA isomerase) was purified about 110-fold and separated from reductase and acetohydroxy acid isomeroreductase. The AHA isomerase was found to be homogeneous by agar and polyacrylamide gel electrophoreses at different pHs. The properties of AHA isomerase have been studied. The purified enzyme showed requirement for Image -ascorbic acid and sulfate ions for its activity. Synthetic ascorbic acid sulfate could replace Image -ascorbic acid and sulfate. α-Methyllactate and α-ketoisovalerate were found to inhibit AHA isomerase activity competitively whereas Image -valine and Image -isoleucine had no significant inhibitory effect. p-Hydroxymercuribenzoate inhibited AHA isomerase activity and the inhibition was reversed by β-mercaptoethanol.

Biosynthesis of coenzyme Q in microorganisms

Incorporation of mevalonate-2-C14, acetate-1-C14, and formate-C14 into the lipids of microorganisms was studied. In the case of four bacteria tested—Agrobacterium tumefaciens, Azotobacter vinelandii, Escherichia coli, and a Pseudomonas species—the various homologues of coenzyme Q present were not labeled with any of the tracers used, although significant amounts of radioactivity were present in the lipids. Both acetate and mevalonate were incorporated into coenzyme Q and sterol of the moulds, Aspergillus niger, Neurospora crassa, Penicillium chrysogenum, and Gibberella fujickuroi, and a yeast, Torulopsis utilis. Mevalonate was incorporated into the side chain but not the ring, whereas acetate was incorporated into both. It appears that the mevalonate pathway for the synthesis of coenzyme Q is operative only in those organisms which also contain other isoprene compounds such as sterol and carotene.

Biosynthesis of valine and isoleucine in plants II. Dihydroxyacid dehydratase from Phaseolus radiatus

1. 1. An enzyme catalysing the conversion of α,β-dihydroxyisovalerate and α,β-dihydroxy-β-methylvalerate to α-ketoisovalerate and α-keto-β-methylvalerate has been partially purified from green gram (Phaseolus radiatus), and its characteristics studied. 2. 2. A natural inhibitor, heat stable and inorganic in nature, was observed in the crude extracts. 3. 3. The observed Km values for α-β-dihydroxyisovalerate and α,β-dihydroxy-β-methylvalerate were 2.4 · 10-3 M and 9 · 10-4 M, respectively. 4. 4. The enzyme required the presence of a divalent metal ion (Mg2+, Mn2+ or Fe2+) for maximal activity. Heavy metals like Ag+ and Hg2+ were inhibitory. 5. 5. The optimal activity was around pH 8.0 and the optimum temperature at 52°. The activation energy is found to be 12 600 cal/mole. 6. 6. The enzyme was inhibited by p-hydroxymercuribenzoate, N-ethylmaleimide and sulphydryl compounds like cysteine, glutathione, 2-mercaptoethanol and 2,3-dimercaptopropanol. The inhibition by p-hydroxymercuribenzoate could not be reversed by any of the sulfhydryl compounds tested.