Complementation in vitro between Purified Mutant Fatty Acid Synthetase Complexes of Yeast

Abstract

1. By dissociation and subsequent reassociation of appropriate pairs of mutant fatty acid synthetases, hybrid multienzyme complexes were obtained whose overall fatty acid synthetase activities were restored to a considerable extent. The complementation thus achieved in vitro could be both intragenic and intergenic and was, in all cases studied, in agreement with the known complementation characteristics of fatty acid synthetase cr;l.c)2. Similarly, the method of reversible dissociation could be used to reactivate a wild-type fatty acid synthetase which had undergone considerable loss of activity due to prolonged storage. It is believed that only those subunits which have retained their native conformation are used in the reassociation of the complex.3. Specific component enzyme activities were determined with a variety of different mutant fatty acid synthetases and with several hybrid enzymes obtained from them by complementation in vivo or in vitro. The results indicate that the activities of most fatty acid synthetase functional domains are influenced by homologous and/or heterologous subunit interactions within the ct61jh oligomeric complex. Thus, mutational inactivation of any one of the active sites in subunit fl simultancously also alters the other four catalytic sites of this subunit. The lack of complementation occasionally observed between certain mutants of two different ,fas 1 complementation groups may be explained by this effect. Furthermore, alp interactions are indicated by the fact that enoylreductase-deficient mutant enzymes (B defect) always exhibit dramatically increased [Lketoacyl synthase ( x domain) activities.4. The incorporation of F M N in vitro into wild-type Fatty acid synthetase apo-enzyme from which the Ravin had been removed leads to a fully active enzyme complex only when the cofactor is present during the subunit assembly process. However, addition of FMN to a fully associated apo-enzyme restores only 50 of its original overall specific activity. If F M N was replaced by FAD in either of these experiments, reactivation occurred only to an extent of 50-70% of that observed with FMN.

A novel function of yeast fatty acid synthase

“…β‐ketoacyl synthase, β‐ketoacyl reductase or pantetheine‐binding. In heteroallelic crosses, fas2‐mutants with different functional lesions complement each other while those being defective in the same function do not [18,22]. As an exception, complementation between isofunctionally defective fas2‐mutants is observed with a few specific allele combinations.…”

Identification of a starter unit acyl-carrier protein transacylase domain in an iterative type I polyketide synthase

“…The intermediate resolution x-ray structures of fungal and mammalian FAS reveal large distances among chain extension and chain-modifying domains, suggesting great conformational flexibility for the ACP to mediate synthesis of the growing fatty acid. Similarly, biochemical experiments show that the ACPs of fungal and mammalian FASs interact with more than one catalytic chamber (31,32). In the case of NorS, the SAT domain in PksA selects the appropriate acyl length from the associated FAS subunits to divert NorS from fatty acid to polyketide synthesis.…”

Microbial Type I Fatty Acid Synthases (FAS): Major Players in a Network of Cellular FAS Systems

“…Genetic complementation studies with yeast mutants which were specifically defective in one of the various FAS domains revealed that overall FAS activity was always restored, both in vitro and in vivo, whenever two mutations which affected two different catalytic activities were combined (74,140,178). For complementation, it was irrelevant whether the affected domains were located on the same or on two different subunits (140,178). Thus, every catalytic site of yeast FAS is obviously capable of interacting with any other site, at least within an ␣ 2 ␤ 2 FAS subcomplex.…”

“…Determining the molar amounts of substrate bound per mole of enzyme, Even though the individual ␣␤ monomer of yeast FAS is possibly capable of palmitic acid synthesis, cooperation between both identical and nonidentical subunits within the ␣ 6 ␤ 6 oligomer has in fact been demonstrated. Genetic complementation studies with yeast mutants which were specifically defective in one of the various FAS domains revealed that overall FAS activity was always restored, both in vitro and in vivo, whenever two mutations which affected two different catalytic activities were combined (74,140,178). For complementation, it was irrelevant whether the affected domains were located on the same or on two different subunits (140,178).…”