Akihiko Kawaguchi

Mechanism of action of the antibiotic thiolactomycin inhibition of fatty acid synthesis of Escherichia coli.

Thiolactomycin, an antibiotic with the structure of (4S)-(2E,5E)-2,4,6-trimethyl-3-hydroxy-2,5,7-octatriene-4-thiolide, inhibits the incorporation of [14C]acetate into cellular fatty acids of Escherichia coli. This antibiotic inhibits the fatty acid synthetase system of E. coli. However, the fatty acid synthetases from Saccharomyces cerevisiae, Candida albicans and rat liver are insensitive to thiolactomycin. This effect may account for the antibacterial activity of thiolactomycin and for its low toxicity in animals.

Positional distribution of fatty acids in lipids of the marine diatom phaeodactylum tricornutum

Abstract The composition of fatty acids and lipids in the marine diatom, Phaeodactylum tricornutum was determined. The Lipids consisted of monogalactosyldiacylglycerol, digalactosyldiacylglycerol, sulphoquinovosyldiacylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphtidylinositol, triacylglycerol and minor unidentified ones. At the early stationary phase of growth, the total fatty acids were mainly 20:5, 16:1, 16:0 and 16:3. 20:5 was distributed in polar lipids, particularly in monogalactosyldiacylglycerol, phosphatidylcholine and phosphatidylglycerol. This fatty acid was exclusively located at the sn -1 position of the glycerol moiety in all polar lipids except for phosphatidylcholine. In phosphatidylcholine 20:5 was distributed at both the sn -1 and sn -2 positions. 16:3 was concentrated at the sn 2 position of monogalactosyldiacylglycerol and trans -16:1 ( n -13) was dominant at the sn -2 position of phosphatidylglycerol. C 18 fatty acids, the minor fatty acids in P. tricornutum , were confined to the sn -2 position of phosphatidylcholine.

Contribution of lowered unsaturation levels of chloroplast lipids to high temperature tolerance of photosynthesis in Chlamydomonas reinhardtii

Abstract A mutant of Chlamydomonas reinhardtii designated as hf-9 is impaired in fatty acid desaturation of chloroplasts, and showed lowered unsaturation levels of chloroplast lipids, as compared with the parent (Sato et al., Eur. J. Biochem., 230 (1995) 987–993). The effects of temperature on photosynthesis were compared between hf-9 and the parent for investigation whether or not unsaturation levels of chloroplast lipids are correlated with the thermal properties of photosynthesis. Growth rates determined by turbidity were higher in the parent than in hf-9at both 10 and 24°C, while similar for the parent and hf-9 at 39°C. The cells grown at 24°C revealed that both activities of CO2-dependent oxygen evolution and photosystem II were higher in the parent than in hf-9 in the range between 7 and 40°C. In contrast, hf-9 surpassed the parent in both activities at 45°C. Optimal temperatures for both activities were at around 35°C and 40°C in the parent and hf-9, respectively. Incubation of the cells at 41 and 45°C demonstrated that the activity of photosystem II in hf-9 was more tolerant to the high temperatures than that in the parent. These results suggest that lowered unsaturation levels of chloroplast lipids constributed to high temperature tolerance of photosystem II, and eventually to that of photosynthesis.

Glycerolipid synthesis in Chlorella kessleri 11h. I. Existence of a eukaryotic pathway.

The fatty acid distributions at the sn-1 and sn-2 positions in major chloroplast lipids of Chlorella kessleri 11h, monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG), were determined to show the coexistence of both C16 and C18 acids at the sn-2 position, i.e. of prokaryotic and eukaryotic types in these galactolipids. For investigation of the biosynthetic pathway for glycerolipids in C. kessleri 11h, cells were fed with [14C]acetate for 30 min, and then the distribution of the radioactivity among glycerolipids and their constituent fatty acids during the subsequent chase period was determined. MGDG and DGDG were labeled predominantly as the sn-1-C18-sn-2-C16 (C18/C16) species as early as by the start of the chase, which suggested the synthesis of these lipids within chloroplasts via a prokaryotic pathway. On the other hand, the sn-1-C18-sn-2-C18 (C18/C18) species of these galactolipids gradually gained radioactivity at later times, concomitant with a decrease in the radioactivity of the C18/C18 species of phosphatidylcholine (PC). The change at later times can be explained by the conversion of the C18/C18 species of PC into galactolipids through a eukaryotic pathway. The results showed that C. kessleri 11h, distinct from most of other green algal species that were postulated mainly to use a prokaryotic pathway for the synthesis of chloroplast lipids, is similar to a group of higher plants designated as 16:3 plants in terms of the cooperation of prokaryotic and eukaryotic pathways to synthesize chloroplast lipids. We propose that the physiological function of the eukaryotic pathway in C. kessleri 11h is to supply chloroplast membranes with 18:3/18:3-MGDG for their functioning, and that the acquisition of a eukaryotic pathway by green algae was favorable for evolution into land plants.

Cerulenin resistance in a cerulenin-producing fungus. Isolation of cerulenin insensitive fatty acid synthetase.

Abstract Cerulenin, an antifungal antibiotic isolated from a culture filtrate of Cephalosporium caerulens , is a potent inhibitor of fatty acid synthetase systems. This antibiotic specifically blocks the activity of β-ketoacyl thioester synthetase (condensing enzyme). The mechanism of the resistance of C. caerulens to cerulenin was investigated. The rate of growth in medium containing up to 100 gmg/ml cerulenin was as rapid as that in cerulenin-free medium. At a cerulenin concentration of 300 μg/ml, the rate of growth was still more than half that of the control. The addition of cerulenin (200 μg/ml) to a culture of growing cells has almost no effect on the incorporation of [ 14 C ]acetate into cellular lipids. Fatty acid synthetase was purified from C. caerulens to homogeneity. Properties of this fatty acid synthetase were almost the same as those of yeast fatty acid synthetase except for the sensitivity to cerulenin. C. caerulens synthetase is much less sensitive to cerulenin than fatty acid synthetases from other sources. These findings suggested that the insensitivity of C. caerulens fatty acid synthetase plays an important role in the cerulenin resistance of this fungus.

Glycerolipid synthesis in Chlorella kessleri 11 h. II. Effect of the CO2 concentration during growth.

In the accompanying paper, we demonstrated that Chlorella kessleri uses prokaryotic and eukaryotic pathways to synthesize sn-1-C18-sn-2-C16 (C18/C16, prokaryotic lipids) and sn-1-C18-sn-2-C18 (C18/C18, eukaryotic lipids) species, respectively, in chloroplast lipids such as monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG). In this study, to examine the effect of CO2 on lipid metabolism, we compared the fatty acid distributions at the sn-1 and sn-2 positions of each major lipid, i.e. MGDG, DGDG, phosphatidylcholine (PC), and phosphatidylethanolamine (PE), and the patterns of incorporation of [14C]acetate into fatty acids and lipids in vivo between cells of C. kessleri grown under ordinary air (low-CO2 cells) and ones grown under CO2-enriched air (high-CO2 cells). Low-CO2 cells, as compared with high-CO2 cells, showed elevated contents of 18:3(9,12,15), especially at both the sn-1 and sn-2 positions of MGDG and DGDG, and also at the sn-2 position of PC and PE. When the cells were labeled with [14C]acetate, slower rates of 18:3 synthesis in the respective major lipids with lower incorporation of 14C into total membrane lipids were observed in low-CO2 cells than in high-CO2 cells. These results thus indicate that the higher unsaturation levels in low-CO2 cells are at least partially due to repressed fatty acid synthesis, which promotes the desaturation of pre-existing fatty acids, rather than to up-regulation of desaturation activity. It was also noted that, in both MGDG and DGDG, the contents of eukaryotic lipids were higher at the expense of prokaryotic lipids in low-CO2 cells than in high-CO2 cells, suggesting relatively greater metabolic flow in the eukaryotic pathway compared to the prokaryotic pathway for galactolipid synthesis in low-CO2 cells. We propose that, together with the repression of fatty acid synthesis, the increased synthesis of C18/C18 species of galactolipids, which are suitable substrates for chloroplast desaturation, through the eukaryotic pathway, contributes to the higher contents of 18:3 in low-CO2 cells than in high-CO2 cells.

METABOLISM OF SOLANACEOUS ALKALOIDS IN TRANSGENIC PLANT TERATOMAS INTEGRATED WITH GENETICALLY ENGINEERED GENES

Abstract Transgenic hairy roots and shooty teratomas of Atropa belladonna L., Nicotiana tabacum L. and Solanum tuberosum L. were obtained with Agrobacterium Ti and Ri plasmids. The hairy roots of A. belladonna, N. tabacum and S. tuberosum accumulated tropane alkaloids, nicotine alkaloids and steroidal alkaloids, respectively. The shooty teratomas of these plant species failed to produce these alkaloids. However, the shooty teratomas had the abilities to store and metabolize the alkaloids. These results indicate that the biosynthesis and metabolism of these solanaceous alkaloids are regulated in correlation to tissue differentiation of the plants.

Greening induced production of (+)-lupanine in tissue culture of Thermopsis lupinoides

Abstract We have established three different types of tissue culture of Thermopsis lupinoides (Leguminosae): white callus, adventitious roots and green callus. Only the green callus accumulated lupin alkaloid, and its concentrations in the callus were correlated to the amounts of chlorophyll in the cells. (+)-Lupanine (6R,7S,9S,11S) was the sole alkaloid produced in green callus, although the differentiated plant contains not only (+)-lupanine but also α-pyridone-type alkaloids of the opposite absolute configuration (7R,9R,11R). These results suggest that the production of (+)-lupanine is related to the greening of the tissue but that of the enantiomeric alkaloids, e.g. (−)-lupanine, (−)-anagyrine, is not.

Stereochemical studies of hydrogen incorporation from nucleotides with fatty acid synthetase from Brevibacterium ammoniagenes.

The biosynthesis of fatty acids from malonyl-CoA and acetyl-CoA was investigated with an enzyme preparation which was purified 100-fold from Brevibacterium ammoniagenes. Fatty acids synthesized in the presence of D2O and stereospecifically deuterated NADPH and NADH were isolated and analyzed by mass chromatography to examine the localization of deuterium in the molecule. The following results were obtained: 1) HB hydrogen of NADPH was used for beta-ketoacyl reductase. 2) HB hydrogen of NADH was used for enoyl reductase. 3) Hydrogen atoms from water were found on the even-numbered methylene carbon atoms (2-hydrogen atoms per carbon atom) and some were also found on the odd-numbered methylene carbon. 4) Hydrogen atoms from NADPH were found on odd-numbered methylene carbon atoms (1-hydrogen per carbon). 5) Hydrogen atoms from NADH were also found on the odd-numbered methylene carbon atoms, but the number of incorporated hydrogen atoms was less than expected. The exchange of HB hydrogen of NADH with water catalyzed by enoyl reductase was suspected. 6) The exchange of methylene hydrogen atoms of malonyl-CoA with proton of water was suggested by 13C NMR analysis.

Structure of bacterial fatty acid synthetase from brevibacterium ammoniagenes

Hydrodynamic measurements and a cross-linking study with dimethyl suberimidate have shown that the native fatty acid synthetase from Brevibacterium ammoniagenes is a hexameric protein having a molecular weight of 1.56 . 10(6). The subunits of the enzyme are identical in size (Mr 2.6 . 10(5). The negatively stained fatty acid synthetase had an electron microscopic image of ellipsoidal structure with major and minor axes approximately equal to 270 A and 180 A, respectively. The electron microscopic image is similar to that of the yeast enzyme, which is quite distinct from the B. ammoniagenes enzyme with respect to the subunit composition. The inactivated enzyme prepared by dialysis against a lower ionic strength solution was partially reactivated by raising the ionic strength. Ellipsoidal images similar to those of the native enzyme were found in the electron micrograph of the reactivated enzyme. Sucrose density gradient centrifugation of the reactivated enzyme sample showed that the active component had almost the same sedimentation coefficient as the native hexamer. These results indicate that the enzyme is active only in its hexameric state.