Expression, purification, and characterization of biol: A carbon-carbon bond cleaving cytochrome P450 involved in biotin biosynthesis in Bacillus subtilis

Pimelic acid formation for biotin biosynthesis in Bacillus subtilis has been proposed to involve a cytochrome P450 encoded by the gene biol. We have subcloned bioI and overexpressed the encoded protein, BioI. A purification protocol was developed utilizing ion exchange, gel filtration, and hydroxyapatite chromatography, Investigation of the purified BioI by UV-visible spectroscopy revealed spectral properties characteristic of a cytochrome P450 enzyme. BioI copurifies with acylated Escherichia coil acyl carrier protein (ACP), suggesting that in vivo a fatty acid substrate may be presented to BioI as an acyl-ACP. A combination of electrospray mass spectrometry of the intact acyl-ACP and GCMS indicated a range of fatty acids were bound to the ACP. A catalytically active system has been established employing E. coli flavodoxin reductase and a novel, heterologous flavodoxin as the redox partners for BioI. In this system, BioI cleaves a carbon-carbon bond of an acyl-ACP to generate a pimeloyl-ACP equivalent, from which pimelic acid is isolated after base-catalyzed saponification. A range of free fatty acids have also been explored as potential alternative substrates for BioI, with C16 binding most tightly to the enzyme. These fatty acids are also metabolized to dicarboxylic acids, but with less regiospecificity than is observed with acyl-ACPs. A possible mechanism for this transformation is discussed. These results strongly support the proposed role for BioI in biotin biosynthesis. In addition, the production of pimeloyl-ACP explains the ability of BioI to function as a pimeloyl CoA source in E. coli, which, unlike B. subtilis, is unable to utilize free pimelic acid for biotin production. (C) 2000 Academic Press.

Antioxidant behaviour of thia fatty acids

Eight thia fatty acids and other sulfides have been studied as inhibitors of autoxidation of arachidonic acid. The inhibitors extend the lag phase of the oxidation, to varying degrees. A carboxyl group in the vicinity of the sulfur reduces the antioxidant activity, while unsaturated sulfides are more effective than their saturated analogues. The results are consistent with the sulfides acting to reduce fatty acid hydroperoxides, which otherwise accumulate during the early stages of reaction and propagate the free-radical oxidation process.

The structures of Langmuir-Blodgett films of fatty acids and their salts

Recent advances in several experimental techniques have enabled detailed structural information to be obtained for floating (Langmuir) monolayers and Langmuir-Blodgett films. These techniques are described briefly and their application to the study of films of fatty acids and their salts is discussed. Floating monolayers on aqueous subphases have been shown to possess a complex polymorphism with phases whose structures may be compared to those of smectic mesophases. However, only those phases that exist at high surface pressures are normally used in Langmuir-Blodgett (LB) deposition. In single LB monolayers of fatty acids and fatty acid salts the acyl chains are in the all-cans conformation with their long axes normal to the substrate. The in-plane molecular packing is hexagonal with long-range bond orientational order and short-range positional order: known as the hexatic-B structure. This structure is found irrespective of the phase of the parent floating monolayer. The structures of multilayer LB films are similar to the structures of their bulk crystals, consisting of stacked bilayer lamellae. Each lamella is formed from two monolayers of fatty acid molecules or ions arranged head to head and held together by hydrogen bonding between pairs of acids or ionic bonding through the divalent cations. With acids the acyl chains are tilted with respect to the substrate normal and have a monoclinic structure, whereas the salts with divalent cations may have the chains normal to the substrate or tilted. The in-plane structures are usually centred rectangular with the chains in the trans conformation and packed in a herringbone pattern, Multilayer films of the acids show only a single-step order-disorder transition at the malting point, This temperature tends to rise as the number of layers increases. Complex changes occur when multilayer films of the salts are heated. Disorder of the chains begins at low temperatures but the arrangement of the head groups does not alter until the melting temperature is reached, Slow heating to a temperature just below the melting temperature gives, with some salts, a radical change in phase. The lamellar structure disappears and a new phase consisting of cylindrical rods lying parallel to the substrate surface and stacked in a hexagonal pattern is formed, In each rod the cations are aligned along the central axis surrounded by the disordered acyl chains. (C) 2001 Elsevier Science B,V. All rights reserved.

Hepatocyte [H-3]-palmitate uptake: effect of albumin surface charge modification

The role of plasma proteins on the cellular uptake of lipophilic substrates has perplexed investigators for many years. We tested the hypothesis that an ionic interaction between the protein-ligand complex and hepatocyte surface may be responsible for supplying more ligand to the cell for uptake. The surface-charged groups on albumin were modified to yield proteins having a range of isoelectric points (ALB, ALBs, ALBm, ALBe had values of 4.8-5.0, 4.5-4.7, 3.0-3.5, 8.4-8.6, respectively). [H-3]-Palmitate uptake studies were performed with adult rat hepatocyte suspensions using similar unbound ligand fractions in the presence of the different binding proteins. Mass spectrometry, isoelectric focusing (pI), and heptane : water partitioning were used to determine protein molecular weight, pI, and protein-palmitate equilibrium binding constant, respectively. Hepatocyte [H-3]-palmitate clearance in the presence of ALBs and ALBm were significantly lower (p < 0.05) than ALB, whereas [H-3]-palmitate clearance in the presence of ALBe was significantly higher (p < 0.05) than ALB. The data were consistent with the notion that ionic interactions between extracellular protein-ligand complexes and the hepatocyte surface facilitate the uptake of long-chain fatty acids.

Effects of rosiglitazone and linoleic acid on human preadipocyte differentiation

Background Peroxisome proliferator activated receptor gamma (PPARgamma) is a ligand-activated transcription factor known to be central to both adipose tissue development and insulin action. Growth of adipose tissue requires differentiation of preadipocytes with acquisition of specific cellular functions including insulin sensitivity, leptin secretion and the capacity to store triglyceride. Dietary fatty acids and members of the thiazolidinedione class of compounds have been reported to influence adipogenesis at the transcriptional level. Here, we compare the effects of a dietary fatty acid, linoleic acid, and a thiazolidinedione, rosiglitazone, on biochemical and functional aspects of human preadipocyte differentiation in vitro . Materials and methods Human omental and subcutaneous preadipocytes were subcultured 2-3 times and subsequently differentiated for 21 days in the presence of either linoleic acid or rosiglitazone. Differentiation was assessed using a number of biochemical and functional parameters. Results Omental and subcutaneous preadipocytes differentiated in the presence of linoleic acid showed marked cytoplasmic triacylglycerol accumulation however, no biochemical markers of differentiation (LPL expression, G3PDH gene expression and enzyme activity and leptin expression or secretion) were detected. In contrast, treatment of these cells with rosiglitazone induced full biochemical differentiation as judged by all markers assessed, despite comparatively little lipid accumulation. The rosiglitazone effects were subcutaneous depot-specific. Cells treated with linoleic acid showed decreased glucose uptake cf rosiglitazone-treated cells. A luciferase reporter assay demonstrated that rosiglitazone potently activates h-peroxisome proliferator activated receptor gamma while linoleic acid had no effect. Conclusions These studies demonstrate that (a) human preadipocytes have the potential to accumulate triacylglycerol irrespective of their stage of biochemical differentiation; (b) while omental preadipocytes are refractory to biochemical differentiation in vitro , they are able to accumulate triacylglycerol; and (c) rosiglitazone and linoleic acid may exert their effects via different biochemical pathways.

The peroxisome proliferator-activated receptor beta/delta agonist, GW501516, regulates the expression of genes involved in lipid catabolism and energy uncoupling in skeletal muscle cells

Lipid homeostasis is controlled by the peroxisome proliferator-activated receptors (PPARalpha, -beta/delta, and -gamma) that function as fatty acid-dependent DNA-binding proteins that regulate lipid metabolism. In vitro and in vivo genetic and pharmacological studies have demonstrated PPARalpha regulates lipid catabolism. In contrast, PPARgamma regulates the conflicting process of lipid storage. However, relatively little is known about PPARbeta/delta in the context of target tissues, target genes, lipid homeostasis, and functional overlap with PPARalpha and -gamma. PPARbeta/delta, a very low-density lipoprotein sensor, is abundantly expressed in skeletal muscle, a major mass peripheral tissue that accounts for approximately 40% of total body weight. Skeletal muscle is a metabolically active tissue, and a primary site of glucose metabolism, fatty acid oxidation, and cholesterol efflux. Consequently, it has a significant role in insulin sensitivity, the blood-lipid profile, and lipid homeostasis. Surprisingly, the role of PPARbeta/delta in skeletal muscle has not been investigated. We utilize selective PPARalpha, -beta/delta, -gamma, and liver X receptor agonists in skeletal muscle cells to understand the functional role of PPARbeta/delta, and the complementary and/or contrasting roles of PPARs in this major mass peripheral tissue. Activation of PPARbeta/delta by GW501516 in skeletal muscle cells induces the expression of genes involved in preferential lipid utilization, beta-oxidation, cholesterol efflux, and energy uncoupling. Furthermore, we show that treatment of muscle cells with GW501516 increases apolipoprotein-A1 specific efflux of intracellular cholesterol, thus identifying this tissue as an important target of PPARbeta/delta agonists. Interestingly, fenofibrate induces genes involved in fructose uptake, and glycogen formation. In contrast, rosiglitazone-mediated activation of PPARgamma induces gene expression associated with glucose uptake, fatty acid synthesis, and lipid storage. Furthermore, we show that the PPAR-dependent reporter in the muscle carnitine palmitoyltransferase-1 promoter is directly regulated by PPARbeta/delta, and not PPARalpha in skeletal muscle cells in a PPARgamma coactivator-1-dependent manner. This study demonstrates that PPARs have distinct roles in skeletal muscle cells with respect to the regulation of lipid, carbohydrate, and energy homeostasis. Moreover, we surmise that PPARgamma/delta agonists would increase fatty acid catabolism, cholesterol efflux, and energy expenditure in muscle, and speculate selective activators of PPARbeta/delta may have therapeutic utility in the treatment of hyperlipidemia, atherosclerosis, and obesity.