Abstract
The role of the Saccharomyces cerevisae peroxisomal acyl-coenzyme A (acyl-CoA) thioesterase (Pte1p) in fatty acid beta-oxidation was studied by analyzing the in vitro kinetic activity of the purified protein as well as by measuring the carbon flux through the beta-oxidation cycle in vivo using the synthesis of peroxisomal polyhydroxyalkanoate (PHA) from the polymerization of the 3-hydroxyacyl-CoAs as a marker. The amount of PHA synthesized from the degradation of 10-cis-heptadecenoic, tridecanoic, undecanoic, or nonanoic acids was equivalent or slightly reduced in the pte1Delta strain compared with wild type. In contrast, a strong reduction in PHA synthesized from heptanoic acid and 8-methyl-nonanoic acid was observed for the pte1Delta strain compared with wild type. The poor catabolism of 8-methyl-nonanoic acid via beta-oxidation in pte1Delta negatively impacted the degradation of 10-cis-heptadecenoic acid and reduced the ability of the cells to efficiently grow in medium containing such fatty acids. An increase in the proportion of the short chain 3-hydroxyacid monomers was observed in PHA synthesized in pte1Delta cells grown on a variety of fatty acids, indicating a reduction in the metabolism of short chain acyl-CoAs in these cells. A purified histidine-tagged Pte1p showed high activity toward short and medium chain length acyl-CoAs, including butyryl-CoA, decanoyl-CoA and 8-methyl-nonanoyl-CoA. The kinetic parameters measured for the purified Pte1p fit well with the implication of this enzyme in the efficient metabolism of short straight and branched chain fatty acyl-CoAs by the beta-oxidation cycle.
Highlights
Isomerase Dci1p, and 2,4-dienoyl-CoA reductase Sps19p in the degradation of unsaturated fatty acids have been defined [1]
Analysis of Carbon Flux through -Oxidation in pte1⌬ Mutant—The quantity and monomer composition of PHA synthesized from intermediates of the -oxidation cycle was first measured in the wild type and pte1⌬ background for S. cerevisiae cells grown in medium containing either 0.1% 10-cis-heptadecenoic acid or 0.1% tridecanoic acid
PHA synthesized from the degradation of 10-cis-heptadecenoic acid in the pte1⌬ strain reached 66% of the wild type level for cells grown in medium containing only 10-cis-heptadecenoic acid, PHA synthesized from the catabolism of the same fatty acid in pte1⌬ decreased to 19% when 8-methyl-nonanoic acid was added to the medium
Summary
Plasmids, and Culture Conditions—The wild type S. cerevisiae strain BY4742 (mat␣ his3⌬1 leu2⌬0 lys2⌬0 ura3⌬0) and the isogenic mutant pte1⌬ (pte1⌬0::kan) were obtained from EUROSCARF. The plasmid Yiplac128-PHA containing the gene for the PHAC1 synthase from Pseudomonas aeruginosa, modified at the carboxyl end by the addition of a peroxisomal targeting sequence and placed under
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