J. Kalervo Hiltunen

The biochemistry of peroxisomal β-oxidation in the yeast Saccharomyces cerevisiae

Peroxisomal fatty acid degradation in the yeast Saccharomyces cerevisiae requires an array of β-oxidation enzyme activities as well as a set of auxiliary activities to provide the β-oxidation machinery with the proper substrates. The corresponding classical and auxiliary enzymes of β-oxidation have been completely characterized, many at the structural level with the identification of catalytic residues. Import of fatty acids from the growth medium involves passive diffusion in combination with an active, protein-mediated component that includes acyl-CoA ligases, illustrating the intimate linkage between fatty acid import and activation. The main factors involved in protein import into peroxisomes are also known, but only one peroxisomal metabolite transporter has been characterized in detail, Ant1p, which exchanges intraperoxisomal AMP with cytosolic ATP. The other known transporter is Pxa1p–Pxa2p, which bears similarity to the human adrenoleukodystrophy protein ALDP. The major players in the regulation of fatty acid-induced gene expression are Pip2p and Oaf1p, which unite to form a transcription factor that binds to oleate response elements in the promoter regions of genes encoding peroxisomal proteins. Adr1p, a transcription factor, binding upstream activating sequence 1, also regulates key genes involved in β-oxidation. The development of new, postgenomic-era tools allows for the characterization of the entire transcriptome involved in β-oxidation and will facilitate the identification of novel proteins as well as the characterization of protein families involved in this process.

Saturated Very-Long-Chain Fatty Acids Promote Cotton Fiber and Arabidopsis Cell Elongation by Activating Ethylene Biosynthesis

Fatty acids are essential for membrane biosynthesis in all organisms and serve as signaling molecules in many animals. Here, we found that saturated very-long-chain fatty acids (VLCFAs; C20:0 to C30:0) exogenously applied in ovule culture medium significantly promoted cotton (Gossypium hirsutum) fiber cell elongation, whereas acetochlor (2-chloro-N-[ethoxymethyl]-N-[2-ethyl-6-methyl-phenyl]-acetamide; ACE), which inhibits VLCFA biosynthesis, abolished fiber growth. This inhibition was overcome by lignoceric acid (C24:0). Elongating fibers contained significantly higher amounts of VLCFAs than those of wild-type or fuzzless-lintless mutant ovules. Ethylene nullified inhibition by ACE, whereas C24:0 was inactive in the presence of the ethylene biosynthesis inhibitor (l-[2-aminoethoxyvinyl]-glycine), indicating that VLCFAs may act upstream of ethylene. C24:0 induced a rapid and significant increase in ACO (for 1-aminocyclopropane-1-carboxylic acid oxidase) transcript levels that resulted in substantial ethylene production. C24:0 also promoted Ser palmitoyltransferase expression at a later stage, resulting in increased sphingolipid biosynthesis. Application of C24:0 not only stimulated Arabidopsis thaliana root cell growth but also complemented the cut1 phenotype. Transgenic expression of Gh KCS13/CER6, encoding the cotton 3-ketoacyl-CoA synthase, in the cut1 background produced similar results. Promotion of Arabidopsis stem elongation was accompanied by increased ACO transcript levels. Thus, VLCFAs may be involved in maximizing the extensibility of cotton fibers and multiple Arabidopsis cell types, possibly by activating ethylene biosynthesis.

β-Oxidation – strategies for the metabolism of a wide variety of acyl-CoA esters

Living organisms are exposed to a number of different fatty acids and their various derivatives arising either via endogenous synthesis or from exogenous sources. These hydrophobic compounds can play specific metabolic, structural or endocrinic functions in the organisms before their elimination, which can be metabolism to CO(2) or to more polar lipid metabolites allowing their excretion. Quantitatively, one of the major pathways metabolizing fatty acids is beta-oxidation, which consists of a set of four reactions operating at the carbons 2 or 3 of acyl-CoA esters and shortening of the acyl-chain. To allow the beta-oxidation of acyl groups with various steric variants to proceed, different strategies have been developed. These strategies include evolution of beta-oxidation enzymes as paralogues showing specificity with respect to either chain-length or modified acyl-chain, metabolic compartmentalization in eukaryotic cells, controlling of substrate transport across membranes, development of auxiliary enzyme systems, acquisition of enzymes with adaptive active sites and recruiting and optimizing enzymes from non-homologous sources allowing them to catalyze a parallel set of reactions with different substrate specificities.

Peroxisomes Are Oxidative Organelles

Peroxisomes are multifunctional organelles with an important role in the generation and decomposition of reactive oxygen species (ROS). In this review, the ROS-producing enzymes, as well as the antioxidative defense system in mammalian peroxisomes, are described. In addition, various conditions leading to disturbances in peroxisomal ROS metabolism, such as abnormal peroxisomal biogenesis, hypocatalasemia, and proliferation of peroxisomes are discussed. We also review the role of mammalian peroxisomes in some physiological and pathological processes involving ROS that lead to mitochondrial abnormalities, defects in cell proliferation, and alterations in the central nervous system, alcoholic cardiomyopathy, and aging. Antioxid.

Pxmp2 Is a Channel-Forming Protein in Mammalian Peroxisomal Membrane

Background Peroxisomal metabolic machinery requires a continuous flow of organic and inorganic solutes across peroxisomal membrane. Concerning small solutes, the molecular nature of their traffic has remained an enigma. Methods/Principal Findings In this study, we show that disruption in mice of the Pxmp2 gene encoding Pxmp2, which belongs to a family of integral membrane proteins with unknown function, leads to partial restriction of peroxisomal membrane permeability to solutes in vitro and in vivo. Multiple-channel recording of liver peroxisomal preparations reveals that the channel-forming components with a conductance of 1.3 nS in 1.0 M KCl were lost in Pxmp2 −/− mice. The channel-forming properties of Pxmp2 were confirmed with recombinant protein expressed in insect cells and with native Pxmp2 purified from mouse liver. The Pxmp2 channel, with an estimated diameter of 1.4 nm, shows weak cation selectivity and no voltage dependence. The long-lasting open states of the channel indicate its functional role as a protein forming a general diffusion pore in the membrane. Conclusions/Significance Pxmp2 is the first peroxisomal channel identified, and its existence leads to prediction that the mammalian peroxisomal membrane is permeable to small solutes while transfer of “bulky” metabolites, e.g., cofactors (NAD/H, NADP/H, and CoA) and ATP, requires specific transporters.

The crystal structure of dienoyl-CoA isomerase at 1.5 A resolution reveals the importance of aspartate and glutamate sidechains for catalysis.

BACKGROUND The degradation of unsaturated fatty acids is vital to all living organisms. Certain unsaturated fatty acids must be catabolized via a pathway auxiliary to the main beta-oxidation pathway. Dienoyl-coenzyme A (dienoyl-CoA) isomerase catalyzes one step of this auxiliary pathway, the isomerization of 3-trans,5-cis-dienoyl-CoA to 2-trans,4-trans-dienoyl-CoA, and is imported into both mitochondria and peroxisomes. Dienoyl-CoA isomerase belongs to a family of CoA-binding proteins that share the enoyl-CoA hydratase/isomerase sequence motif. RESULTS The crystal structure of rat dienoyl-CoA isomerase has been determined at 1.5 A resolution. The fold closely resembles that of enoyl-CoA hydratase and 4-chlorobenzoyl-CoA dehalogenase. Dienoyl-CoA isomerase forms hexamers made up of two trimers. The structure contains a well ordered peroxisomal targeting signal type-1 which is mostly buried in the inter-trimer space. The active-site pocket is deeply buried and entirely hydrophobic, with the exception of the acidic residues Asp176, Glu196 and Asp204. Site-directed mutagenesis of Asp204 revealed that this residue is essential for catalysis. In a molecular modeling simulation, a molecule of 3-trans,5-cis-octadienoyl-CoA was docked into the active site. CONCLUSIONS The structural data, supported by the mutagenesis data, suggest a reaction mechanism where Glu196 acts as a proton acceptor and Asp204 acts as a proton donor. Asp176 is paired with Glu196 and is important for optimizing the catalytic proton transfer properties of Glu196. In the predicted mode of substrate binding, an oxyanion hole stabilizes the transition state by binding the thioester oxygen. The presence of a buried peroxisomal targeting signal suggests that dienoyl-CoA isomerase is prevented from reaching its hexameric structure in the cytosol.

Mitochondrial fatty acid synthesis and respiration

Recent studies have revealed that mitochondria are able to synthesize fatty acids in a malonyl-CoA/acyl carrier protein (ACP)-dependent manner. This pathway resembles bacterial fatty acid synthesis (FAS) type II, which uses discrete, nuclearly encoded proteins. Experimental evidence, obtained mainly through using yeast as a model system, indicates that this pathway is essential for mitochondrial respiratory function. Curiously, the deficiency in mitochondrial FAS cannot be complemented by inclusion of fatty acids in the culture medium or by products of the cytosolic FAS complex. Defects in mitochondrial FAS in yeast result in the inability to grow on nonfermentable carbon sources, the loss of mitochondrial cytochromes a/a3 and b, mitochondrial RNA processing defects, and loss of cellular lipoic acid. Eukaryotic FAS II generates octanoyl-ACP, a substrate for mitochondrial lipoic acid synthase. Endogenous lipoic acid synthesis challenges the hypothesis that lipoic acid can be provided as an exogenously supplied vitamin. Purified eukaryotic FAS II enzymes are catalytically active in vitro using substrates with an acyl chain length of up to 16 carbon atoms. However, with the exception of 3-hydroxymyristoyl-ACP, a component of respiratory complex I in higher eukaryotes, the fate of long-chain fatty acids synthesized by the mitochondrial FAS pathway remains an enigma. The linkage of FAS II genes to published animal models for human disease supports the hypothesis that mitochondrial FAS dysfunction leads to the development of disorders in mammals.

Lipoic Acid Synthesis and Attachment in Yeast Mitochondria

Lipoic acid is a sulfur-containing cofactor required for the function of several multienzyme complexes involved in the oxidative decarboxylation of α-keto acids and glycine. Mechanistic details of lipoic acid metabolism are unclear in eukaryotes, despite two well defined pathways for synthesis and covalent attachment of lipoic acid in prokaryotes. We report here the involvement of four genes in the synthesis and attachment of lipoic acid in Saccharomyces cerevisiae. LIP2 and LIP5 are required for lipoylation of all three mitochondrial target proteins: Lat1 and Kgd2, the respective E2 subunits of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, and Gcv3, the H protein of the glycine cleavage enzyme. LIP3, which encodes a lipoate-protein ligase homolog, is necessary for lipoylation of Lat1 and Kgd2, and the enzymatic activity of Lip3 is essential for this function. Finally, GCV3, encoding the H protein target of lipoylation, is itself absolutely required for lipoylation of Lat1 and Kgd2. We show that lipoylated Gcv3, and not glycine cleavage activity per se, is responsible for this function. Demonstration that a target of lipoylation is required for lipoylation is a novel result. Through analysis of the role of these genes in protein lipoylation, we conclude that only one pathway for de novo synthesis and attachment of lipoic acid exists in yeast. We propose a model for protein lipoylation in which Lip2, Lip3, Lip5, and Gcv3 function in a complex, which may be regulated by the availability of acetyl-CoA, and which in turn may regulate mitochondrial gene expression.

Htd2p/Yhr067p is a yeast 3-hydroxyacyl-ACP dehydratase essential for mitochondrial function and morphology

Among the recently recognized aspects of mitochondrial functions, in yeast as well as humans, is their ability to synthesize fatty acids in a malonyl‐CoA dependent manner. We describe here the identification of the 3‐hydroxyacyl‐ACP dehydratase involved in mitochondrial fatty acid synthesis. A colony‐colour‐sectoring screen was applied in Saccharomyces cerevisiae in a search for mutants that, when grown on a non‐fermentable carbon source, were unable to lose a plasmid that carried a chimeric construct coding for mitochondrially localized bacterial analogue. Our mutants, which are respiratory deficient, lack cytochromes and display abnormal mitochondrial morphology,  were  found  to  have  a lesion in the yeast YHR067w/RMD12 gene. The Yhr067p is predicted to be a member of the thioesterase/thioester dehydratase‐isomerase superfamily enzymes. Hydratase 2 activity in mitochondrial extracts from cells overexpressing YHR067w was increased. These overexpressing cells also display a striking mitochondrial enlargement phenotype. We conclude that YHR067w encodes a novel mitochondrial 3‐hydroxyacyl‐thioester dehydratase 2 and suggest renaming it HTD2. The mitochondrial phenotypes of the null and overexpression mutants suggest a crucial role of YHR067w in maintenance of mitochondrial respiratory competence and morphology in yeast.

Subcellular distribution of phosphagens in isolated perfused rat heart

~~p~rt~e~t of ~~d~~~l B~o~h~?~~istr~l, ~~~~~~~s~ty of Zulu, K~j~~~~i~tie 52 A, SF-95225 Oufu 22, Finland It has been suggested that the oxygen consunlption of the mitochondrial respiratory chain is regulated by the extranlitochondrial ATP/ADP X P, ratio and that a near-equilibrium exists between the oxidation-reduction reactions of the mitochondrial respiratory chain and the phosphorylation of cytosolic ADP [ 11. Data on the situation in intact tissues have been largely obtained by measurements of total average concentra. tions of the reactants [2], but corroboration of the near-equilibrium hypothesis [3] of respiratory control needs data on the concentrations of the reactants in specified intracellular compartments. The isolated perfused rat heart has been employed in studies on the regulation of cellular respiration, and total average concentrations of ATP, ADP, Pi, creatine and creatine phosphate used in calculations of the extramitochondrial phosphorylation potential [4,5]. Here, advantage has been taken of the method of tissue fractionation in non-aqueous media [6] to measure the cytosolic and m~tochondrial phospl~orylation potentials. The results show that the error in estimating the cytosolic phosphorylation potential from total tissue concentration of the reactants is quite small when calculated from the creatine kinase equilibrium. The results are compatible with a near-equilibrium between the mitochondrial respiratory chain and the cytosolic adenylate system. Changes in the distribution of the adenylates across the mitochondrial membrane upon transitions in the cellular ATP consumption indicate that the ~nitochondrial membrane potential and transmembrane pH difference also change.