Differential Activities and Intramolecular Location of Fatty Acid Synthase and 6-Methylsalicylic Acid Synthase Component Enzymes

Abstract

Summary By targeted in vitro mutagenesis, the phosphopantetheine-binding serine residue of yeast fatty acid synthase (FAS) was localized in the β-ketoacyl reductase domain (pos. 180) of FAS subunit α. Attachment of phosphopantetheine to serine-180 requires interaction of this N-terminal site in the protein with a distal, C-terminal domain of the same subunit. Point mutations in this distal region constitute a distinct complementation group (No. VII) and prevent pantetheine-binding even to an intact serine-180. In contrast to other fas2 mutations mapping around serine-180, the in vitro constructed serine-180 → glycine-180 mutation exhibits the intragenic complementation pattern of group VI (ketoacyl synthase-defective) rather than of group VTII (ketoacyl reductase-defective) fas -mutants. Thus, this mutation does not conform with the generally observed clustering of isofunctional fas -mutations within the same domain. Using stereospecifically tritiated NADPH it was shown that the β-ketoacyl reductases of both, FAS and 6-methylsalicylic acid synthase (MSAS), exert the same specificity for the 4-H Si atom of NADPH. However, the two reductases differ with respect to their ketoacyl substrate specificities: while the FAS enzyme reduces both, acetoacetyl- and triacetic acid ester model substrates, the purified MSAS is specific for the triacetyl derivative. The affinities of MSAS (K M = 1.4 x 10 -4 M) and FAS (K M = 8.0 x 10 -4 M) for triacetic acid ethyl ester were determined. Reduction of FAS-bound acetoacetate prevents it from condensation with another malonate to triacetic acid. Reduction of FAS- or MSAS-bound triacetic acid competes with its lactonization and subsequent release from the enzyme. The formation of triacetolactone is therefore always a side-reaction of 6-MSA biosynthesis. With purified MSAS, reduction of triacetic acid ethyl ester but not the subsequent step, i.e. dehydration of the 3-hydroxy acid to 5-oxo-2,3-hexenic acid ethyl ester, could be experimentally verified. It is therefore concluded that the dehydration step occurs at the tetracetic rather than at the triacetic acid level. Cyclization and aromatization of the corresponding intermediate to 6-MSA would then occur at the same time. Enzyme-bound 6-MSA is finally released from the enzyme hydrolytically rather than as a coenzyme A thioester. Hence, the two polyketide synthases, MSAS and FAS, containing a very similar set of catalytic domains produce their specific end products obviously as a consequence of distinctly different substrate specificities of their β-ketoacyl reductase, dehydratase and terminal acyl transferase component enzymes.