Biosynthesis of Long-Chain Alcohols by Developing and Regenerating Rat Sciatic Nerve

Cell-free preparations of rat sciatic nerve were found to catalyze the reduction of fatty acid to alcohol in the presence of NADPH as reducing cofactor. The reductase was membrane-bound and associated primarily with the microsomal fraction. When fatty acid was the substrate, ATP, coenzyme A (CoA), and Mg2+ were required, indicating the formation of acyl CoA prior to reduction. When acyl CoA was used as substrate, the presence of albumin was required to inhibit acyl CoA hydro-lase activity. Fatty acid reductase activity was highest with palmitic and stearic acids, and somewhat lower with lauric and myristic acids. It was inhibited by sulfhydryl reagents, indicating the participation of thiol groups in the reduction. Only traces of long-chain aldehyde could be detected or trapped as semicarbazone. Fatty acid reductase activity in rat sciatic nerve was highest between the second and tenth days after birth and decreased substantially thereafter. Microsomal preparations of sciatic nerve from 10-day-old rats exhibited about four times higher fatty acid reductase activity than brain or spinal cord microsomes from the same animals. Wallerian degeneration and regeneration of adult rat sciatic nerve resulted in enhanced fatty acid reductase activity, which reached a maximum at about 12 days after crush injury.

Inhibition of the biosynthesis of sterols by phenyl and phenolic compounds in rat liver

The presence of mitochondria increased the incorporation of [2-14C]mevalonate into sterols in a cell-free system from rat liver. Various phenyl and phenolic compounds inhibited the incorporation of mevalonate when added in vitro. p-Hydroxycinnamate, a metabolite of tyrosine, was the most powerful inhibitor among the compounds tested. Catechol, resorcinol and quinol were inhibitory at high concentrations. Organic acids lacking an aromatic ring were not inhibitory. Two hypocholesterolaemic drugs, Clofibrate (α-p-chlorophenoxyisobutyrate) and Clofenapate [α,4-(p-chlorophenyl)phenoxyisobutyrate], which are known to affect some step before the formation of mevalonate in the biosynthesis of cholesterol in vivo, showed inhibition at a step beyond the formation of mevalonate in vitro. The presence of the aromatic ring and the carboxyl group in a molecule appears to be necessary for the inhibition.

Amine biosynthesis in Lathyrus sativus seedlings

The biosynthesis of certain amines in Lathyrus sativus seedlings was studied in isolated shoots and cotyledons. In shoots, arginine was about 14 times more efficient than ornithine for the synthesis of agmatine, putrescine, spermidine and spermine. Isotope dilution experiments, and the changes in specific activities of the 4 amines with time when 14C-arginine served as the precursor, indicated that putrescine and the polyamines were formed mainly from arginine, via agmatine. Similar experiments showed that cadaverine was formed at least in part from homoarginine, though lysine was ca 4 times more effective as a precursor. The pattern of changes in specific activity of homoagmatine and cadaverine with time when 14C-homoarginine served as the precursor support the conclusion that homoarginine and arginine follow analogous metabolic routes in the biosynthesis of putrescine and cadaverine respectively.

Inhibition of the biosynthesis of isoprenoid compounds by phenolic acids in the rat brain

The incorporation of [2-14C]mevalonate into nonsaponifiable lipids by rat brain homogenates is inhibited by phenolic acids derived from tyrosine. The phenyl acids derived from phenylalanine are inhibitory only at very high concentrations compared with phenolic acids. The brain is more sensitive to inhibition by the phenolic acids than the liver. These studies indicate a possible role for phenolic acids in the impairment of cerebral sterol metabolism in phenylketonuria.

Role of heme in the synthesis of cytochrome c oxidase in Neurospora crassa

The role of heme in the synthesis of cytochrome c oxidase has been investigated in the mold Neurospora crassa. Iron-deficient cultures of the mold have low levels of cytochrome oxidase and delta-aminolevulinate dehydratase, the latter being the rate-limiting enzyme of the heme-biosynthetic pathway in this organism. Addition of iron to the iron-deficient cultures results in an immediate increase in the levels of delta-aminolevulinate dehydratase followed by an increase in the rate of heme synthesis and cytochrome oxidase levels. The rate of precursor labeling of the mitochondrial subunits of cytochrome oxidase is decreased preferentially under conditions of iron deficiency and addition of iron corrects this picture. Exogenous hemin addition which prevents iron-mediated induction of delta-aminolevulinate dehydratase also inhibits the increase in the activity of cytochrome oxidase and the enhanced precursor labeling of the mitochondrial subunits of cytochrome oxidase. Protein synthesis on mitoribosomes measured in vivo and in vitro is decreased under conditions of heme deficiency. Hemin addition in vitro to mitochondrial lysates prepared from heme-deficient mycelia restores a near normal rate of protein synthesis. It is concluded that heme is required for the optimal rate of translation on mitoribosomes.

The regulation of the biosynthesis of ubiquinone in the rat

The urinary excretion of p-hydroxybenzoate was not altered by ubiquinone feeding, but, although decreased considerably, was not eliminated in protein deficiency. The incorporation of p-hydroxy[U-14C]benzaldehyde into ubiquinone in vivo increased in cold-exposed and p-chlorophenoxyisobutyrate (clofibrate)-fed rats, and these changes were parallel with the changes in the incorporation of [2-14C]mevalonate under these conditions. Starvation, cholesterol feeding and cholic acid feeding resulted in the decreased incorporation of p-hydroxy[U-14C]benzaldehyde into ubiquinone, confirming the decreased ubiquinone synthesis. Feeding exogenous ubiquinone increased the hepatic ubiquinone concentration, but did not cause any decrease in the incorporation of p-hydroxy[U-14C]benzaldehyde into ubiquinone, indicating the absence of a feedback control.

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.

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.