Proteomic Identification and Characterization of a Novel Peroxisomal Adenine Nucleotide Transporter Supplying ATP for Fatty Acid β-Oxidation in Soybean and Arabidopsis

We have identified the novel protein Glycine max PEROXISOMAL ADENINE NUCLEOTIDE CARRIER (Gm PNC1) by proteomic analyses of peroxisomal membrane proteins using a blue native/SDS-PAGE technique combined with peptide mass fingerprinting. Gm PNC1, and the Arabidopsis thaliana orthologs At PNC1 and At PNC2, were targeted to peroxisomes. Functional integration of Gm PNC1 and At PNC2 into the cytoplasmic membranes of intact Escherichia coli cells revealed ATP and ADP import activities. The amount of Gm PNC1 in cotyledons increased until 5 d after germination under constant darkness and then decreased very rapidly in response to illumination. We investigated the physiological functions of PNC1 in peroxisomal metabolism by analyzing a transgenic Arabidopsis plant in which At PNC1 and At PNC2 expression was suppressed using RNA interference. The pnc1/2i mutant required sucrose for germination and suppressed the degradation of storage lipids during postgerminative growth. These results suggest that PNC1 contributes to the transport of adenine nucleotides that are consumed by reactions that generate acyl-CoA for peroxisomal fatty acid β-oxidation during postgerminative growth.     

Two Modes of Regulation of the Fatty Acid Elongase ELOVL6 by the 3-Ketoacyl-CoA Reductase KAR in the Fatty Acid Elongation Cycle

Fatty acids (FAs) are diverse molecules, and such diversity is important for lipids to exert their functions under several environmental conditions. FA elongation occurs at the endoplasmic reticulum and produces a variety of FA species; the FA elongation cycle consists of four distinct enzyme reactions. For this cycle to be driven efficiently, there must exist coordinated regulation of protein components of the FA elongation machinery. However, such regulation is poorly understood. In the present study, we performed biochemical analyses using the FA elongase ELOVL6 and the 3-ketoacyl-CoA reductase KAR, which catalyze the first and second steps of the FA elongation cycle, respectively. In vitro FA elongation assays using membrane fractions demonstrated that ELOVL6 activity was enhanced ∼10-fold in the presence of NADPH, although ELOVL6 itself did not require NADPH for its catalysis. On the other hand, KAR does use NADPH as a reductant in its enzyme reaction. Activity of purified ELOVL6 was enhanced by ∼3-fold in the presence of KAR. This effect was KAR enzyme activity-independent, since it was observed in the absence of NADPH and in the KAR mutant. However, ELOVL6 enzyme activity was further enhanced in a KAR enzyme activity-dependent manner. Therefore, KAR regulates ELOVL6 via two modes. In the first mode, KAR may induce conformational changes in ELOVL6 to become structure that can undergo catalysis. In the second mode, conversion of 3-ketoacyl-CoA to 3-hydroxyacyl-CoA by KAR may facilitate release of the product from the presumed ELOVL6–KAR complex.     

The Arabidopsis pxa1 Mutant Is Defective in an ATP-Binding Cassette Transporter-Like Protein Required for Peroxisomal Fatty Acid β-Oxidation

Peroxisomes are important organelles in plant metabolism, containing all the enzymes required for fatty acid beta-oxidation. More than 20 proteins are required for peroxisomal biogenesis and maintenance. The Arabidopsis pxa1 mutant, originally isolated because it is resistant to the auxin indole-3-butyric acid (IBA), developmentally arrests when germinated without supplemental sucrose, suggesting defects in fatty acid beta-oxidation. Because IBA is converted to the more abundant auxin, indole-3-acetic acid (IAA), in a mechanism that parallels beta-oxidation, the mutant is likely to be IBA resistant because it cannot convert IBA to IAA. Adult pxa1 plants grow slowly compared with wild type, with smaller rosettes, fewer leaves, and shorter inflorescence stems, indicating that PXA1 is important throughout development. We identified the molecular defect in pxa1 using a map-based positional approach. PXA1 encodes a predicted peroxisomal ATP-binding cassette transporter that is 42% identical to the human adrenoleukodystrophy (ALD) protein, which is defective in patients with the demyelinating disorder X-linked ALD. Homology to ALD protein and other human and yeast peroxisomal transporters suggests that PXA1 imports coenzyme A esters of fatty acids and IBA into the peroxisome for beta-oxidation. The pxa1 mutant makes fewer lateral roots than wild type, both in response to IBA and without exogenous hormones, suggesting that the IAA derived from IBA during seedling development promotes lateral root formation.     

β-β′ Subunits of Ribonucleic Acid Polymerase in Episome-Free Minicells of Escherichia coli

Episome-free minicells of Escherichia coli, previously shown to lack ribonucleic acid polymerase activity, do contain the beta-beta' subunits of the polymerase.     

Purification and Characterization of Yeast β-Glucosidases

Constitutive beta-glucosidases from Saccharomyces fragilis (Y-18) and S. dobzhanskii (Y-19) precipitated at the same concentration of ammonium sulfate. The partially purified enzymes had similar activation energies, molecular weights, affinities for certain natural and synthetic beta-glucosides, and optimal pH values for substrate hydrolysis, and they were stable over approximately the same pH range. The enzymes, however, could be clearly distinguished by other criteria. Affinities of the synthetic, sulfur-containing beta-glucosides for Y-18 enzyme were many times greater than for Y-19 enzyme. The latter enzyme was more resistant to heat. The two enzymes eluted from diethylaminoethyl cellulose at different concentrations of sodium chloride. In precipitin tests, homologous enzyme-antisera systems were highly specific. The beta-glucosidase synthesized by a hybrid, S. fragilis x S. dobzhanskii (Y-42), was unique. Characterization of this enzyme produced values which were intermediate to those for the enzymes from the parental yeast strains. Heat-inactivation slopes and Lineweaver-Burk plots for the Y-42 enzyme were anomalous. It is suggested that hydrolytic activity in Y-42 preparations is due to a spectrum of hybrid enzyme molecules composed of varying amounts of two distinct polypeptides. It is further suggested that these polypeptides may be identical to those synthesized by the parental Y-18 and Y-19 yeast strains.     

Cross Talk between β1 and αV Integrins: β1 Affects β3 mRNA Stability

There is increasing evidence that a fine-tuned integrin cross talk can generate a high degree of specificity in cell adhesion, suggesting that spatially and temporally coordinated expression and activation of integrins are more important for regulated cell adhesive functions than the intrinsic specificity of individual receptors. However, little is known concerning the molecular mechanisms of integrin cross talk. With the use of beta(1)-null GD25 cells ectopically expressing the beta(1)A integrin subunit, we provide evidence for the existence of a cross talk between beta(1) and alpha(V) integrins that affects the ratio of alpha(V)beta(3) and alpha(V)beta(5) integrin cell surface levels. In particular, we demonstrate that a down-regulation of alpha(V)beta(3) and an up-regulation of alpha(V)beta(5) occur as a consequence of beta(1)A expression. Moreover, with the use of GD25 cells expressing the integrin isoforms beta(1)B and beta(1)D, as well as two beta(1) cytoplasmic domain deletion mutants lacking either the entire cytoplasmic domain (beta(1)TR) or only its "variable" region (beta(1)COM), we show that the effects of beta(1) over alpha(V) integrins take place irrespective of the type of beta(1) isoform, but require the presence of the "common" region of the beta(1) cytoplasmic domain. In an attempt to establish the regulatory mechanism(s) whereby beta(1) integrins exert their trans-acting functions, we have found that the down-regulation of alpha(V)beta(3) is due to a decreased beta(3) subunit mRNA stability, whereas the up-regulation of alpha(V)beta(5) is mainly due to translational or posttranslational events. These findings provide the first evidence for an integrin cross talk based on the regulation of mRNA stability.     

Microbial Transformation of β-Ionone and β-Methylionone

Aspergillus niger JTS 191 was selected from many microorganisms tested as capable of converting ionones to other compounds having aromas. The individual transformation products from β-ionone were isolated and identified by comparison with synthetically derived compounds. The major products were (R)-4-hydroxy-β-ionone and (S)-2-hydroxy-β-ionone. 2-Oxo-, 4-oxo-, 3,4-dehydro-, 2,3-dehydro-4-oxo-, 3,4-dehydro-2-oxo-, (S)-2-acetoxy-, (R)-4-acetoxy-, and 5,6-epoxy-β-ionone and 4-(2,3,6-trimethylphenyl)-but-3-en-2-one were also identified. Analogous transformation products of β-methylionone also were identified. Based on gas-liquid chromatographic analysis during the fermentation, we propose two main oxidative pathways of β-ionone. The results of this study suggest that these transformations of β-ionones may be useful as tobacco-flavoring compounds.     

Characterization of β-Ketoacyl-Acyl Carrier Protein Synthase III from Streptomyces glaucescens and Its Role in Initiation of Fatty Acid Biosynthesis

The Streptomyces glaucescens fabH gene, encoding β-ketoacyl-acyl carrier protein (β-ketoacyl-ACP) synthase (KAS) III (FabH), was overexpressed in Escherichia coli, and the resulting gene product was purified to homogeneity by metal chelate chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified protein revealed an M_r of 37,000, while gel filtration analysis determined a native M_r of 72,000 ± 3,000 (mean ± standard deviation), indicating that the enzyme is homodimeric. The purified recombinant protein demonstrated both KAS activity and acyl coenzyme A (acyl-CoA):ACP transacylase (ACAT) activity in a 1:0.12 ratio. The KAS and ACAT activities were both sensitive to thiolactomycin inhibition. The KAS activity of the protein demonstrated a K_m value of 3.66 μM for the malonyl-ACP substrate and an unusual broad specificity for acyl-CoA substrates, with K_m values of 2.4 μM for acetyl-CoA, 0.71 μM for butyryl-CoA, and 0.41 μM for isobutyryl-CoA. These data suggest that the S. glaucescens FabH is responsible for initiating both straight- and branched-chain fatty acid biosynthesis in Streptomyces and that the ratio of the various fatty acids produced by this organism will be dictated by the ratios of the various acyl-CoA substrates that can react with FabH. Results from a series of in vivo directed biosynthetic experiments in which the ratio of these acyl-CoA substrates was varied are consistent with this hypothesis. An additional set of in vivo experiments using thiolactomycin provides support for the role of FabH and further suggests that a FabH-independent pathway for straight-chain fatty acid biosynthesis operates in S. glaucescens.     

Functional Characterization of Polyunsaturated Fatty Acid Delta 6-Desaturase and Elongase Genes from the Black Seabream (Acanthopagrus schlegelii)

Fatty acid delta 6-desaturase (D6DES) and elongases are key enzymes in the synthesis of polyunsaturated fatty acids (PUFAs) including arachidonic acid (ARA) and eicosapentaenoic acid (EPA) from microorganisms to higher animals. To identify the genes encoding D6DES and elongases for PUFAs, we isolated each cDNA with a high similarity to the D6DES and ELOVL5-like elongases of mammals and fishes via degenerate PCR and RACE-PCR from Acanthopagrus schlegelii. A recombinant vector expressing AsD6DES was subsequently constructed and transformed into Saccharomyces cerevisiae to test the enzymatic activity toward n-6 and n-3 fatty acids in the PUFA biosynthesis. The heterologously expressed AsD6DES produced γ-linolenic acid (GLA, C_18:3 n-6) and stearidonic acid (STA, C_18:4 n-3) at conversion rates of 26.3–35.6 % from exogenous linoleic acid (LA, C_18:2 n-6) and α-linolenic acid (ALA, C_18:3 n-3) substrates, respectively. When AsELOVL5 was expressed in yeast, it conferred an ability to elongate GLA to di-homo-γ-linolenic acid (DGLA, C_20:3 n-6). In addition, AsELOVL5 showed an ability to convert ARA (C_20:4 n-6) and EPA (C_20:5 n-3) to dodecylthioacetic acid (DTA, C_22:4 n-6) and docosapentaenoic acid (DPA, C_22:5 n-3), respectively. In these results, the AsD6DES encodes a delta 6-fatty acid desaturase and the AsELOVL5 encoding a long-chain fatty acid elongase shows activity to enlongate C₁₈Δ6/C₂₀Δ5, but not C₂₂.     

Occurrence of β-Aminoglutaric Acid in Marine Bacteria

β-Aminoglutaric acid, a nonprotein amino acid isomer of glutamic acid, was found in the free amino acid pool of a marine bacterium, Alteromonas luteoviolacea. It was also found in a mixed culture of fermenting bacteria enriched from an anoxic marine sediment.     

Turnover of Fatty Acids during Natural Senescence of Arabidopsis, Brachypodium, and Switchgrass and in Arabidopsis β-Oxidation Mutants

During leaf senescence, macromolecule breakdown occurs and nutrients are translocated to support growth of new vegetative tissues, seeds, or other storage organs. In this study, we determined the fatty acid levels and profiles in Arabidopsis (Arabidopsis thaliana), Brachypodium distachyon, and switchgrass (Panicum virgatum) leaves during natural senescence. In young leaves, fatty acids represent 4% to 5% of dry weight and approximately 10% of the chemical energy content of the leaf tissues. In all three species, fatty acid levels in leaves began to decline at the onset of leaf senescence and progressively decreased as senescence advanced, resulting in a greater than 80% decline in fatty acids on a dry weight basis. During senescence, Arabidopsis leaves lost 1.6% of fatty acids per day at a rate of 2.1 μg per leaf (0.6 μg mg⁻¹ dry weight). Triacylglycerol levels remained less than 1% of total lipids at all stages. In contrast to glycerolipids, aliphatic surface waxes of Arabidopsis leaves were much more stable, showing only minor reduction during senescence. We also examined three Arabidopsis mutants, acx1acx2, lacs6lacs7, and kat2, which are blocked in enzyme activities of β-oxidation and are defective in lipid mobilization during seed germination. In each case, no major differences in the fatty acid contents of leaves were observed between these mutants and the wild type, indicating that several mutations in β-oxidation that cause reduced breakdown of reserve oil in seeds do not substantially reduce the degradation of fatty acids during leaf senescence.     

Staphylococcal β-Hemolysin I. Purification of β-Hemolysin

The purification of staphylococcal β-hemolysin was accomplished by the successive use of three protein fractionation methods. The first method employed was a double precipitation with the use of ammonium sulfate at 65% saturation. The second phase of purification used Sephadex G-100 column fractionation. The third phase utilized either carboxymethyl cellulose or diethylaminoethyl cellulose fractionation. The last two fractionation methods both resulted in the separation of a relatively high concentration of cationic hot-cold lysin and a low concentration of anionic hot-cold lysin. Because of the low concentration of the anionic component, its purity could not be assessed. However, the purity of the cationic component was demonstrated by immunodiffusion, microimmunoelectrophoresis, and by disc polyacrylamide gel electrophoresis. In addition, antisera against purified cationic β-hemolysin yielded one line of precipitate when tested against the original crude β-hemolysin. The purified cationic β-hemolysin was stable in the lyophilized state. Crude β-hemolysin was dermonecrotic, whereas purified cationic β-hemolysin was not dermonecrotic even after Mg⁺⁺ activation.     

NADPH–Cytochrome P450 Reductase Mediates the Fatty Acid Desaturation of ω3 and ω6 Desaturases from Mortierella alpina

Fatty acid desaturases play an important role in maintaining the appropriate structure and function of biological membranes. The biochemical characterization of integral membrane desaturases, particularly ω3 and ω6 desaturases, has been limited by technical difficulties relating to the acquisition of large quantities of purified proteins, and by the fact that functional activities of these proteins were only tested in an NADH-initiated reaction system. The main aim of this study was to reconstitute an NADPH-dependent reaction system in vitro and investigate the kinetic properties of Mortierella alpina ω3 and ω6 desaturases in this system. After expression and purification of the soluble catalytic domain of NADPH–cytochrome P450 reductase, the NADPH-dependent fatty acid desaturation was reconstituted for the first time in a system containing NADPH, NADPH–cytochrome P450 reductase, cytochrome b5, M. alpina ω3 and ω6 desaturase and detergent. In this system, the maximum activity of ω3 and ω6 desaturase was 213.4 ± 9.0 nmol min⁻¹ mg⁻¹ and 10.0 ± 0.5 nmol min⁻¹ mg⁻¹, respectively. The highest k_cat/K_m value of ω3 and ω6 desaturase was 0.41 µM⁻¹ min⁻¹ and 0.09 µM⁻¹ min⁻¹ when using linoleoyl CoA (18:2 ω6) and oleoyl CoA (18:1 ω9) as substrates, respectively. M. alpina ω3 and ω6 desaturases were capable of using NADPH as reductant when mediated by NADPH–cytochrome P450 reductase; although, their efficiency is distinguishable from NADH-dependent desaturation. These results provide insights into the mechanisms underlying ω3 and ω6 fatty acid desaturation and may facilitate the production of important fatty acids in M. alpina.     

Fatty Acid Production from Amino Acids and α-Keto Acids by Brevibacterium linens BL2

Low concentrations of branched-chain fatty acids, such as isobutyric and isovaleric acids, develop during the ripening of hard cheeses and contribute to the beneficial flavor profile. Catabolism of amino acids, such as branched-chain amino acids, by bacteria via aminotransferase reactions and α-keto acids is one mechanism to generate these flavorful compounds; however, metabolism of α-keto acids to flavor-associated compounds is controversial. The objective of this study was to determine the ability of Brevibacterium linens BL2 to produce fatty acids from amino acids and α-keto acids and determine the occurrence of the likely genes in the draft genome sequence. BL2 catabolized amino acids to fatty acids only under carbohydrate starvation conditions. The primary fatty acid end products from leucine were isovaleric acid, acetic acid, and propionic acid. In contrast, logarithmic-phase cells of BL2 produced fatty acids from α-keto acids only. BL2 also converted α-keto acids to branched-chain fatty acids after carbohydrate starvation was achieved. At least 100 genes are potentially involved in five different metabolic pathways. The genome of B. linens ATCC 9174 contained these genes for production and degradation of fatty acids. These data indicate that brevibacteria have the ability to produce fatty acids from amino and α-keto acids and that carbon metabolism is important in regulating this event.