Isozymes of Ipomoea batatas catechol oxidase differ in catalase-like activity.

The amino acid sequences of two isozymes of catechol oxidase from sweet potatoes (Ipomoea batatas) were determined by Edman degradation of BrCN cleavage fragments of the native protein and by sequencing of amplified cDNA fragments. Sequence alignment and phylogenetic analysis of plant catechol oxidases revealed about 80% equidistance between the two I. batatas catechol oxidases and approximately 40--60% to catechol oxidases of other plants. When H(2)O(2) was applied as substrate the 39 kDa isozyme, but not the 40 kDa isozyme, showed catalase-like activity. The structure of the 40 kDa isozyme was modeled on the basis of the published crystal structure of the 39 kDa isozyme [T. Klabunde et al., Nat. Struct. Biol. 5 (1998) 1084]. The active site model closely resembled that of the 39 kDa isozyme determined by crystallography, except for a mutation of Thr243 (40 kDa isozyme) to Ile241 (39 kDa isozyme) close to the dimetal center. This residue difference affects the orientation of the Glu238/236 residue, which is thought to be responsible for the catalase-like activity of the 39 kDa isozyme for which a catalytic mechanism is proposed.     

Rapid methods for the characterization of unilamellar phospholipid vesicles. Application to studies on fatty acid donor and acceptor properties of membranes and fatty acid binding proteins

Vesicles having diameters from 20 to 200 nm were prepared from egg-yolk phosphatidylcholine (PC) and were separated as well as analyzed by methods that can be carried out with standard laboratory equipment. Gel-chromatography on Sephacryl S 1000 was adapted for expeditious size analysis of vesicles as well as for isolation of vesicle populations having a narrow range of diameters. The internal volume of vesicles was derived from enzymic tests for PC and for glucose encapsulated. Size analysis and enzymic determinations provided a convenient check for the lamellarity of membranes produced.Fatty acids and fatty acid binding proteins (FABPs) must interact in vivo in the presence of cellular membranes. As a model, interactions between unilamellar vesicles, anthroyloxypalmitic acid (A16:0) and FABPs were studied with the aid of gel-chromatographic methods elaborated and of fluorescence spectroscopy. FABP from bovine heart donated A16:0 to membranes, whereas FABP from bovine liver removed this fatty acid from vesicle membranes. The results revealed characteristic differences between cardiac and hepatic FABPs with regard to binding a fatty acid.     

Biosynthesis of cyclopentenylglycine from α-ketopimelate in Idesia polycarpa callus cultures

The nonproteinogenic amino acid, cyclopentenylglycine, is found in certain Flacourtiaceae. This compound may be synthesized by two C₁-chain elongations of -ketoglutarate via -ketopimelate (C₅+2C₁) or by condensation of C₄ and C₃ units (C₄+C₃), a pathway not involving -ketopimelate. The following experimental design elucidated the biosynthetic pathway: Idesia polycarpa callus cultures were freshly established from leaf petioles; synthetic -[1,2-¹⁴C]ketopimelate was added to the medium and cultures were incubated for 3 weeks. After isolation and separation of free amino acids from the tissues, the radioactivity incorporated into cyclopentenylglycine was determined. The results establish -ketopimelate as a precursor for cyclopentenylglycine, thus providing evidence for the C₅+2C₁ biosynthetic path.     

Dynamics of Galactolipids and Plastids in Nonphotosynthetic Cells of Glycine max Suspension Cultures : A Morphological and Biochemical Study

The age-dependent interrelationship of galactolipids and plastids in heterotrophic cell suspension cultures of Glycine max (soybean) was studied with regard to aging of nonphotosynthetic cells. Cells were propagated in the dark and under illumination with white light, and were harvested at days 7 (end of logarithmic phase), 14, and 21 (extended stationary phase). Electron microscopy revealed in dark-grown cells a proliferating decay of the amyloplast-type plastids, which could be correlated to a decrease of galactolipids. This trend was dramatically reversed in irradiated cultures, where the plastids of day 21 cells appeared rejuvenated. A concomitant increase of galactolipid content in the cells was observed, yet chlorophyll synthesis and photosynthetic activity were not induced. The dynamics of galactolipid contents did not correlate with total lipid contents in dark-grown as well as in irradiated cultures. [³H]Galactose served as a radioactive probe for the subcellular localization of galactolipids by electron microscopic autoradiography. Apart from plastids, galactolipids may also be constituents of the plasma membrane. The results render the heterotrophic cell suspension culture a suitable model to study the impact of senescence on plastids of nonphotosynthetic cells.     

Localisation and characterization of the fatty acid synthesizing system in cells of Glycine max (soubean) suspension cultures

In course of a study of fatty acid synthetase in higher plants, non-green cell suspension cultures of Glycine max (soybean) served as model tissues. For the first time, a fatty acid synthesizing system was characterized in cell cultures of higher plants and was found to be solely located in proplastids of the cells. Optimum activity of the fatty acid synthesizing system in proplastids was observed between pH 8.0 and 8.2; with [1-14C]acetate as substrate, cofactors required were CoA, ATP, Mn2+, Mg2+, HCO3-, NADH and NADPH. The system was more sensitive towards NADH than NADHP. [1-14C]Acetate,[2-14C]-malonate and [3-14C]pyruvate served as precursors for fatty acids, indicating the presence of pyruvate dehydrogenase activity in proplastids. In disrupted proplastids, [2-14C]malonylCoA was a better precursor than [1-14C]acetylCoA. After incubation of proplastids with [2-14C]malonate, a small shift, from palmitic acid to higher homologs, of label incorporated was observed, as compared to incorporation of label from [1-14C]acetate and [3-14C]pyruvate. Under the conditions of the experiment, only small amounts of polyunsaturated fatty acids, the main fatty acid components of this organelle, were synthesized. In respect to fatty acid synthesis, the non-green cell suspension culture resembles photosynthetic leaf tissue.     

The impact of genetic stress by ATGL deficiency on the lipidome of lipid droplets from murine hepatocytes

We showed earlier that nutritional stress like starvation or high-fat diet resulted in phenotypic changes in the lipidomes of hepatocyte lipid droplets (LDs), representative for the pathophysiological status of the mouse model. Here we extend our former study by adding genetic stress due to knockout (KO) of adipocyte triglyceride lipase (ATGL), the rate limiting enzyme in LD lipolysis. An intervention trial for 6 weeks with male wild-type (WT) and ATGL-KO mice was carried out; both genotypes were fed lab chow or were exposed to short-time starvation. Isolated LDs were analyzed by LC-MS/MS. Triacylglycerol, diacylglycerol, and phosphatidylcholine lipidomes, in that order, provided the best phenotypic signatures characteristic for respective stresses applied to the animals. This was evidenced at lipid species level by principal component analysis, calculation of average values for chain-lengths and numbers of double bonds, and by visualization in heat maps. Structural backgrounds for analyses and metabolic relationships were elaborated at lipid molecular species level. Relating our lipidomic data to nonalcoholic fatty liver diseases of nutritional and genetic etiologies with or without accompanying insulin resistance, phenotypic distinction in hepatocyte LDs dependent on insulin status emerged. Taken together, lipidomes of hepatocyte LDs are sensitive responders to nutritional and genetic stress.     

Towards novel biocatalysts via protein design: the case of lipases

Abstract: During the past 3 years, the tertiary structures of several lipases have been solved by X-ray analysis. The structures revealed unique features such as hydrophobic 'patches' on the surface, presumably involved in lipid supersubstrate binding, and a lid structure which covers the active site in the absence of substrate. Only very recently the first X-ray structure of a bacterial lipase has been solved, and further structural features different from lipases of eukaryotic origin became apparent. Many lipase genes have been cloned and sequenced recently, and expression systems for the preparation of recombinant enzymes in good yields are available. As an example, the lipase from Rhizopus oryzae has been successfully expressed by us in Escherichia coli , and the resulting inclusion bodies were renatured in high yields. Consequently, the mechanism of action of lipases is now being studied via site-directed mutagenesis, and the rational design of lipases for the selective transformation of substrates is presently addressed in several laboratories.     

Intracellular lipid binding proteins and nuclear receptors involved in branched-chain fatty acid signaling

Branched-chain fatty acids are potent regulators of gene expression. Among them are the vitamin A-derived retinoic acids, which are involved in cell growth and differentiation, and the chlorophyll-derived phytol metabolites such as phytanic and pristanic acids, which affect catabolic lipid metabolism. Gene expression regulated by these signaling molecules is mediated by two protein families. These are, on the one hand, the intracellular lipid binding proteins, i.e. cellular retinoic acid binding protein and liver-type fatty acid binding protein, which are responsible for ligand-transport to the nucleus. On the other hand are the ligand-activated nuclear receptors, i.e. the retinoic acid receptors for retinoic acids and the peroxisome proliferator-activated receptors for the phytol metabolites. In this review, we discuss the cross-talk between the two protein families and how this cross-talk contributes to targeted signaling with branched-chain fatty acids.     

Crystal structure and thermodynamic analysis of human brain fatty acid-binding protein

Expression of brain fatty acid-binding protein (B-FABP) is spatially and temporally correlated with neuronal differentiation during brain development. Isothermal titration calorimetry demonstrates that recombinant human B-FABP clearly exhibits high affinity for the polyunsaturated n-3 fatty acids alpha-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, and for monounsaturated n-9 oleic acid (K(d) from 28 to 53 nm) over polyunsaturated n-6 fatty acids, linoleic acid, and arachidonic acid (K(d) from 115 to 206 nm). B-FABP has low binding affinity for saturated long chain fatty acids. The three-dimensional structure of recombinant human B-FABP in complex with oleic acid shows that the oleic acid hydrocarbon tail assumes a "U-shaped" conformation, whereas in the complex with docosahexaenoic acid the hydrocarbon tail adopts a helical conformation. A comparison of the three-dimensional structures and binding properties of human B-FABP with other homologous FABPs, indicates that the binding specificity is in part the result of nonconserved amino acid Phe(104), which interacts with double bonds present in the lipid hydrocarbon tail. In this context, analysis of the primary and tertiary structures of human B-FABP provides a rationale for its high affinity and specificity for polyunsaturated fatty acids. The expression of B-FABP in glial cells and its high affinity for docosahexaenoic acid, which is known to be an important component of neuronal membranes, points toward a role for B-FABP in supplying brain abundant fatty acids to the developing neuron.