Mechanism of fatty acid desaturation in the green alga Chlorella vulgaris.

The hypothesis that the Delta9 desaturase of Chlorella vulgaris might operate by a synchronous mechanism has been tested using a kinetic isotope effect (KIE) approach. Thus the intermolecular primary deuterium KIE on the individual C-H bond cleavage steps involved in Delta9 desaturation have been determined by incubating growing cultures of C. vulgaris (strain 211/8K) with mixtures of the appropriate regiospecifically deuterated fatty acid analogues. Our analysis shows that the introduction of a double bond between C-9 and C-10 occurs in two discrete steps as the cleavage of the C9-H bond is very sensitive to isotopic substitution (kH/kD = 6.6 +/- 0.3) whereas a negligible isotope effect (kH/kD = 1.05 +/- 0.05) was observed for the C10-H bond-breaking step. Similar results were obtained for linoleic acid biosynthesis (Delta12 desaturation). These data clearly rule out a synchronous mechanism for these reactions.     

The stereochemistry of desaturations of long-chain fatty acids in Chlorella vulgaris

1. A study was made of the stereospecificity of hydrogen removal in the sequential desaturations performed by intact cells of Chlorella vulgaris in the biosynthesis of oleic acid, linoleic acid and alpha-linolenic acid. 2. By use of erythro- and threo-9,10-(2)H(2)-, -12,13-(2)H(2)- and -15,16-(2)H(2)-labelled precursors, it was demonstrated that the pair of hydrogen atoms removed from each of these positions had the cis relative configuration. 3. That the hydrogen atoms removed in oleic acid and linoleic acid formation were of the d absolute configuration was proved by use of d- and l-9-(3)H-and -12-(3)H-labelled precursors. 4. The presence of a substantial kinetic isotope effect of deuterium at both positions of the putative double bond was indicated, suggesting that the mechanism of desaturation involves simultaneous concerted removal of the pair of hydrogen atoms.     

A D-amino acid oxidase from Chlorella vulgaris.

A procedure has been developed for the partial purification from Chlorella vulgaris of an enzyme which catalyzes the formation of HCN from D-histidine when supplemented with peroxidase of a metal with redox properties. Some properties of the enzyme are described. Evidence is presented that the catalytic activity for HCN formation is associated with a capacity for catalyzing the oxidation of a wide variety of D-amino acids. With D-leucine, the best substrate for O2 consumption, 1 mol of ammonia is formed for half a mol of O2 consumed in the presence of catalase. An inactive apoenzyme can be obtained by acid ammonium sulfate precipitation, and reactivated by added FAD. On the basis of these criteria, the Chlorella enzyme can be classified as a D-amino acid oxidase (EC 1.4.3.3). Kidney D-amino acid oxidase and snake venom L-amino acid oxidase, which likewise form HCN from histidine on supplementation with peroxidase, have been compared with the Chlorella D-amino acid oxidase. The capacity of these enzymes for causing HCN formation from histidine is about proportional to their ability to catalyze the oxidation of histidine.     

Antibacterial targets in fatty acid biosynthesis

The fatty acid biosynthesis pathway is an attractive but still largely unexploited target for the development of new antibacterial agents. The extended use of the antituberculosis drug isoniazid and the antiseptic triclosan, which are inhibitors of fatty acid biosynthesis, validates this pathway as a target for antibacterial development. Differences in subcellular organization of the bacterial and eukaryotic multienzyme fatty acid synthase systems offer the prospect of inhibitors with host versus target specificity. Platensimycin, platencin, and phomallenic acids, newly discovered natural product inhibitors of the condensation steps in fatty acid biosynthesis, represent new classes of compounds with antibiotic potential. An almost complete catalog of crystal structures for the enzymes of the type II fatty acid biosynthesis pathway can now be exploited in the rational design of new inhibitors, as well as the recently published crystal structures of type I FAS complexes.     

Stereospecificity of hydrogen transfer by pyridine nucleotide-dependent enoyl reductases in fatty acid synthesis: Studies with enzymes obtained from developing castor bean seeds and Chlorella vulgaris

The mechanism of hydrogen incorporation into fatty acids wasinvestigated with fatty acid synthetase systems from developingcastor bean seeds and Chlorella vulgaris. Fatty acids synthesizedin the presence of D₂O or stereospecifically deuterium-labeledNADPH or NADH were isolated and analyzed by mass spectrometryto examine the localization of deuterium atoms in the molecule.The stereospecificities of ß-ketoacyl-acyl carrierprotein (ACP) reductase and enoyl-ACP reductase for reducedpyridine nucleotide were determined with acetoacetyl-ACP andcrotonyl-ACP as substrates. The products were also analyzedby gas chromatography-mass spectrometry. The following resultswere obtained:

1. ß-Ketoacyl-ACP reductases from both castor bean seedsand C. vulgaris used the B-side hydrogen of NADPH.
2. Enoyl-ACPreductase from C. vulgaris required NADH for the activity.
3. Enoyl-ACPreductase from castor bean seeds used the A-side hydrogenofNADPH, whereas that from C. vulgaris used the B-side hydrogenof NADH.
4. When stearate was synthesized with the crude fattyacid synthetasefraction from castor bean seeds, hydrogen atomsfrom water werefound on the even-numbered methylene carbonatoms (two hydrogenatoms per carbon atom) and some were foundon the odd-numberedmethylene carbon atoms. Hydrogen atoms fromthe B-side of NADPHwere found on the odd-numbered methylenecarbon atoms (one hydrogenatom per carbon atom). Hydrogen atomsfrom the A-side of NADPHwere also found on the odd-numberedmethylene carbon atoms,but the number of incorporated hydrogenatoms was less thanexpected.

(Received October 17, 1979; )     

Relationship between fatty acid binding proteins,acetyl-CoA formation and fatty acid synthesis in developing human placenta

The relationship between fatty acid binding proteins, ATP citrate lyase activity and fatty acid synthesis in developing human placenta has been studied. Fatty acid binding proteins reverse the inhibitory efect of palmitoyl-CoA and oleate on ATP citrate lyase and fatty acid synthesis. In the absence of these inhibitors fatty acid binding proteins activate ATP citrate lyase and stimulate [ 1-¹⁴ C] acetate incorporation into placental fatty acids indicating binding of endogenous inhibitors by these proteins. Thus these proteins regulate the supply of acetyl-CoA as well as the synthesis of fatty acids from that substrates. As gestation proceeds and more lipids are required by the developing placenta fatty acid binding protein content, activity of ATP citrate lyase and rate of fatty acid synthesis increase indicating a cause and efect relationship between the demand of lipids and supply of precursor fatty acids during human placental development.     

Methionine Transport in Chlorella vulgaris

Absorption of ³⁵S-l-methionine by Chlorella vulgaris was measured at concentrations that ranged from 0.1 to 10.0 μmoles/ml. A brief, rapid phase of uptake was followed by a more prolonged, slower phase that was linear only at the lowest concentrations. The radioactivity accumulated by the end of 1 hour's incubation at an exogenous level of 0.1 μmole/ml was retained by the cells despite the inclusion of 10 μmoles/ml of nonradioactive methionine in the rinse medium. As the exogenous concentration was raised, the ratio of intracellular soluble radioactivity to exogenous radioactivity decreased. Analysis of the accumulated, soluble radioactivity showed that 90% was in the form of methionine and that about 10% had been converted to a compound with properties of S-adenosylmethionine. Azide and ethionine were the most effective of the inhibitors tested.     

Branched-chain fatty acid biosynthesis in Escherichia coli

β-Ketoacyl-acyl carrier protein (ACP) synthase III (KASIII) catalyzes the first elongation step in straight-chain fatty acid (SCFA) biosynthesis in Escherichia coli. Overproduction of the corresponding KASIII gene, or the Brassica napus KASIII gene has previously been observed to lead to an increase in the amount of shorter-chain fatty acids produced by E. coli. In this study it is shown that overexpression of the KASIII gene, which initiates branched-chain fatty acid (BCFA) in Streptomyces glaucescens, does not lead to a change in the fatty acid profiles of E. coli. E. coli produces trace levels of BCFAs when grown in the presence of isobutyric acid, but the amounts of these are not significantly altered by expression of the S. glaucescens KASIII gene. In contrast, the amounts of BCFAs produced from isobutyryl CoA in vitro by E. coli cell-free extracts can be increased at least four-fold by the presence of the S. glaucescens KASIII. These observations suggest that in vivo production of isopalmitate by E. coli expressing the S. glaucescens KASIII is limited by availability of the appropriate BCFA biosynthetic primers. Journal of Industrial Microbiology & Biotechnology (2001) 27, 246–251. Received 10 January 2001/ Accepted in revised form 13 July 2001     

Fatty acid synthesis by spinach chloroplasts III. Relationship between fatty acid synthesis and photophosphorylation

The modes of actions of photosynthetic inhibitors on photosynthesisand fatty acid synthesis were examined. DCMU, an electron transport inhibitor, inhibited fatty acidsynthesis and photophosphorylation to the same extent, suggestingdependence of fatty acid synthesis on photosynthesis. The samewas also the case with FCCP, a photophosphorylation uncoupler.In contrast, NH₄Cl and phlorizin at concentrations completelysuppressing ATP formation, only partially inhibited the fattyacid synthesis. These facts suggest that a certain level ofhigh-energy intermediate (state) is responsible for the lightenhancement of fatty acid synthesis. This idea is further supportedby the fact that the partial inhibition of fatty acid synthesisby NH₄Cl was relieved by addition of DCCD at low concentrationssuppressing the ATP formation but not completely destroyingthe high energy intermediate. The lag period in the initial period of fatty acid synthesiswas shortened by preillumination of chloroplasts, even in theabsence of ADP. This indicates that the light dependent fattyacid synthesis is closely associated with the high-energy intermediate(state), but not directly with ATP formation by photophosphorylation. ¹ Present address: Radioisotope Centre, University of Tokyo,Yayoi, Bunkyo, Tokyo 113, Japan. (Received August 26, 1974; )     

Functional cross-talk between fatty acid synthesis and nonribosomal peptide synthesis in quinoxaline antibiotic-producing streptomycetes

Quinoxaline antibiotics are chromopeptide lactones embracing the two families of triostins and quinomycins, each having characteristic sulfur-containing cross-bridges. Interest in these compounds stems from their antineoplastic activities and their specific binding to DNA via bifunctional intercalation of the twin chromophores represented by quinoxaline-2-carboxylic acid (QA). Enzymatic analysis of triostin A-producing Streptomyces triostinicus and quinomycin A-producing Streptomyces echinatus revealed four nonribosomal peptide synthetase modules for the assembly of the quinoxalinoyl tetrapeptide backbone of the quinoxaline antibiotics. The modules were contained in three protein fractions, referred to as triostin synthetases (TrsII, III, and IV). TrsII is a 245-kDa bimodular nonribosomal peptide synthetase activating as thioesters for both serine and alanine, the first two amino acids of the quinoxalinoyl tetrapeptide chain. TrsIII, represented by a protein of 250 kDa, activates cysteine as a thioester. TrsIV, an unstable protein of apparent Mr about 280,000, was identified by its ability to activate and N-methylate valine, the last amino acid. QA, the chromophore, was shown to be recruited by a free-standing adenylation domain, TrsI, in conjunction with a QA-binding protein, AcpPSE. Cloning of the gene for the QA-binding protein revealed that it is the fatty acyl carrier protein, AcpPSE, of the fatty acid synthase of S. echinatus and S. triostinicus. Analysis of the acylation reaction of AcpPSE by TrsI along with other A-domains and the aroyl carrier protein AcmACP from actinomycin biosynthesis revealed a specific requirement for AcpPSE in the activation and also in the condensation of QA with serine in the initiation step of QA tetrapeptide assembly on TrsII. These data show for the first time a functional interaction between nonribosomal peptide synthesis and fatty acid synthesis.