Seeds of the tung tree (Vernicia fordii) produce large quantities of triacylglycerols (TAGs) containing ∼80% eleostearic acid, an unusual conjugated fatty acid. We present a comparative analysis of the genetic, functional, and cellular properties of tung type 1 and type 2 diacylglycerol acyltransferases (DGAT1 and DGAT2), two unrelated enzymes that catalyze the committed step in TAG biosynthesis. We show that both enzymes are encoded by single genes and that DGAT1 is expressed at similar levels in various organs, whereas DGAT2 is strongly induced in developing seeds at the onset of oil biosynthesis. Expression of DGAT1 and DGAT2 in yeast produced different types and proportions of TAGs containing eleostearic acid, with DGAT2 possessing an enhanced propensity for the synthesis of trieleostearin, the main component of tung oil. Both DGAT1 and DGAT2 are located in distinct, dynamic regions of the endoplasmic reticulum (ER), and surprisingly, these regions do not overlap. Furthermore, although both DGAT1 and DGAT2 contain a similar C-terminal pentapeptide ER retrieval motif, this motif alone is not sufficient for their localization to specific regions of the ER. These data suggest that DGAT1 and DGAT2 have nonredundant functions in plants and that the production of storage oils, including those containing unusual fatty acids, occurs in distinct ER subdomains.

Ovine pulmonary surfactant is bactericidal for Pasteurella haemolytica when surfactant and bacteria mixtures are incubated with normal ovine serum. To isolate this component, surfactant (1 mg/ml) was centrifuged at 100,000 x gav, and the supernatant was fractionated by HPLC. Fractions were eluted with acetonitrile (10-100%)/0.1% trifluoracetic acid and tested for bactericidal activity. Amino acid and sequence analysis of three bactericidal fractions showed that fraction 2 contained H-GDDDDDD-OH, fraction 3 contained H-DDDDDDD-OH, and fraction 6 contained H-GADDDDD-OH. Peptides in 0.14 M NaCl/10 microM ZnCl2 (zinc saline solution) induced killing of P. haemolytica and other bacteria comparable to defensins and beta-defensins [minimal bactericidal concentration (MBC)50 range, 0.01-0.06 mM] but not in 0.14 M NaCl/10 mM sodium phosphate buffer, pH 7.2/0.5 mM CaCl2/0.15 mM MgCl2 (MBC50 range, 2.8-11.5 mM). Bactericidal activity resided in the core aspartate hexapeptide homopolymeric region, and MBC50 values of aspartate dipeptide-to-heptapeptide homopolymers were inversely proportional to the number of aspartate residues in the peptide. P. haemolytica incubated with H-DDDDDD-OH in zinc saline solution was killed within 30 min. Ultrastructurally, cells contained flocculated intracellular constituents. In contrast to cationic defensins and beta-defensins, surfactant-associated anionic peptides are smaller in size, opposite in charge, and are bactericidal in zinc saline solution. They are members of another class of peptide antibiotics containing aspartate, which when present in pulmonary secretions may help clear bacteria as a part of the innate pulmonary defense system.

Biosynthesis of the toxic and carcinogenic aflatoxins by the fungus Aspergillus flavus is a complicated process involving more that 27 enzymes and regulatory factors encoded by a clustered group of genes. Previous studies found that three enzymes, encoded by verA , ver-1 , and aflY , are required for conversion of versicolorin A (VA), to demethylsterigmatocystin. We now show that a fourth enzyme, encoded by the previously uncharacterized gene, aflX ( ordB ), is also required for this conversion. A homolog of this gene, stcQ , is present in the A. nidulans sterigmatocystin (ST) biosynthesis cluster. Disruption of aflX in Aspergillus flavus gave transformants that accumulated ∼4-fold more VA and fourfold less aflatoxin than the untransformed strain. Southern and Northern blot analyses confirmed that aflX was the only gene disrupted in these transformants. Feeding ST or O -methylsterigmatocystin, but not VA or earlier precursor metabolites, restored normal levels of AF production. The protein encoded by aflX is predicted to have domains typical of an NADH-dependent oxidoreductase. It has 27% amino acid identity to a protein encoded by the aflatoxin cluster gene, aflO ( avfA ). Some of domains in the protein are similar to those of epoxide hydrolases.