ABSTRACT
In marine bacteria and some thraustochytrids (marine stramenopiles) long-chain polyunsaturated fatty acids (LC-PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are produced de novo by PUFA synthases. These large, multi-domain enzymes carry out the multitude of individual reactions required for conversion of malonyl-CoA to the final LC-PUFA products. Here we report on the release of fatty acids from the PUFA synthase found in Schizochytrium, a thraustochytrid that has been developed as a commercial source for DHA-enriched biomass and oil. Data from in vitro activity assays indicate that the PUFAs are released from the enzyme as free fatty acids (FFAs). Addition of ATP and Mg(2+) to in vitro assays facilitates appearance of radiolabel from (14)C-malonyl-CoA in a triacylglycerol fraction, suggesting the involvement of acyl-CoA synthetases (ACS). Furthermore, addition of triascin C, an inhibitor of ACSs, to the assays blocks this conversion. When the Schizochytrium PUFA synthase is expressed in Escherichia coli, the products of the enzyme accumulate as FFAs, suggesting that the thioesterase activity required for fatty acid release is an integral part of the PUFA synthase.
Subject(s)
Acetate-CoA Ligase/metabolism , Docosahexaenoic Acids/metabolism , Fatty Acid Synthase, Type II/metabolism , Fatty Acids, Nonesterified/biosynthesis , Fatty Acids, Unsaturated/metabolism , Malonyl Coenzyme A/metabolism , Oomycetes/metabolism , Thiolester Hydrolases/metabolism , Adenosine Triphosphate/metabolism , Enzyme Inhibitors/metabolism , Magnesium/metabolism , Oomycetes/enzymology , Oomycetes/genetics , Plant Oils/metabolism , Triglycerides/metabolismABSTRACT
Yucca glauca in the Colorado shortgrass prairie undergoes a pronounced midday depression in net photosynthesis and stomatal conductance under summer field conditions. This phenomenon can be duplicated in the laboratory using potted plants by simulating a typical summer daily pattern of leaf temperature and leaf-to-air water vapor concentration difference (Δw). The decrease in photosynthetic rate appears to be due primarily to high leaf temperatures, while the decrease in stomatal conductance can be attributed mainly to high Δw values. Stomatal conductance also decreases when leaf temperatures exceed a critical threshold value, even when Δw is artificially maintained at a constant level. The threshold temperature is commonly attained for leaves in situ, but only after substantial stomatal closure has already occurred as a result of high Δw values.The photosynthetic temperature optimum and threshold temperature which promotes stomatal closure increases substantially as the growing season progresses. As a result, the midday depression in photosynthesis occurs at higher temperatures in mid-summer than in late spring. Preliminary evidence suggests that the photosynthetic temperature optimum closely follows the naturally-occurring morning leaf temperatures, while the threshold temperature for stomatal closure matches afternoon leaf temperatures.
