Heterologously expressed acyl carrier protein domain of rat fatty acid synthase functions in Escherichia coli fatty acid synthase and Streptomyces coelicolor polyketide synthase systems.

INTRODUCTION: Fatty acid synthases (FASs) catalyze the de novo biosynthesis of long-chain saturated fatty acids by a process common to eubacteria and eukaryotes, using either a set of monofunctional proteins (Type II FAS) or a polypeptide containing several catalytic functions (Type I FAS). To compare the features of a Type I domain with its Type II counterpart we expressed and characterized an acyl carrier protein (ACP) domain of the Type I rat FAS. RESULTS: An ACP domain of rat FAS was defined that allows expression of a small percentage of active holo-ACP both in Escherichia coli, increasing fivefold upon co-expression with an E. coli holo-ACP synthase, and in Streptomyces coelicolor. The rat ACP domain functions with some components of the E. coli FAS, and can replace the actinorhodin polyketide synthase (PKS) ACP in S. coelicolorA3(2). Purification of the rat ACP domain from E. coli resulted in loss of its functionality. Purified apo-ACP could be converted to its holo-form upon incubation with purified E. coli holo-ACP synthase in vitro, however, suggesting that the loss of functionality was not due to a conformational change. CONCLUSIONS: Functionality of the recombinant rat ACP was shown in distantly related and diverse enzyme systems, suggesting that Type I and Type II ACPs have a similar conformation. A procedure was described that might permit the production of rat FAS holo-ACP for structural and further biochemical characterization.

Reaction mechanisms of homodimeric plant polyketide synthase (stilbenes and chalcone synthase). A single active site for the condensing reaction is sufficient for synthesis of stilbenes, chalcones, and 6'-deoxychalcones.

Stilbene (STS) and chalcone (CHS) synthases are homodimeric, related plant-specific polyketide synthases. Both perform a sequential condensation of three acetate units to a starter residue to form a tetraketide intermediate that is folded to the ring systems specific to the different products. Protein cross-linking and site-directed mutagenesis identified a subunit contact site in position 158, close to the active site (Cys169). This suggested that the active site pockets may be neighboring, possibly alternating in the condensing reactions rather than acting independently. This was investigated by coexpression of active site mutants with differently mutated, inactive proteins. With both STS and CHS, the heterodimers synthesized the end products, indicating that each subunit performed all three condensations. In co-action with a monomeric reductase, CHS also synthesizes 6'-deoxychalcone, but with the chalcone as second product when using plant preparations. The two enzymes expressed as single species in Escherichia coli synthesized both products, and both were also obtained with a CHS heterodimer containing a single active site. The results showed that 6'-deoxychalcone synthesis required no other plant factor and that the formation of two products may be an intrinsic property of the interaction between dimeric CHS and monomeric reductase.

Evidence that stilbene synthases have developed from chalcone synthases several times in the course of evolution.

Chalcone (CHS) and stilbene (STS) synthases are related plant-specific polyketide synthases that are key enzymes in the biosynthesis of flavonoids and of stilbene phytoalexins, respectively. A phylogenetic tree constructed from 34 CHS and four STS sequences revealed that the STS formed no separate cluster but grouped with CHS from the same or related plants. This suggested that STS evolved from CHS several times independently. We attempted to stimulate this by site-directed mutagenesis of an interfamily CHS/STS hybrid, which contained 107 amino acids of a CHS from Sinapis alba (N-terminal) and 287 amino acids of a STS from Arachis hypogaea. The hybrid had no enzyme activity. Three amino acid exchanges in the CHS part (Gln-100 to Glu, Val-103 to Met, Val-105 to Arg) were sufficient to obtain low STS activity, and one additional exchange (Gly-23 to Thr) resulted in 20-25% of the parent STS activity. A kinetic analysis indicated (1) that the hybrids had the same Km for the substrate 4-coumaroyl-CoA but a lower Vmax than the parent STS, and (2) that they had a different substrate preference than the parent STS and CHS. Most of the other mutations and their combinations led to enzymatically inactive protein aggregates, suggesting that the subunit folding and/or the dimerization was disturbed. We propose that STS evolved from CHS by a limited number of amino acid exchanges, and that the advantage gained by this enzyme function favored the selection of plants with improved STS activity.

The role of cysteines in polyketide synthases. Site-directed mutagenesis of resveratrol and chalcone synthases, two key enzymes in different plant-specific pathways.

Resveratrol and chalcone synthases are related plant-specific polyketide synthases that are key enzymes in the biosynthesis of stilbenes and flavonoids, respectively. The stepwise condensing reactions correspond to those in other polyketide and fatty-acid synthases. This predicts that the two proteins also contain cysteines that are essential for enzyme activity because they bind the substrates. We exchanged, in both enzymes, all of the 6 conserved cysteines into alanine by site-directed mutagenesis and tested the mutants after expression of the proteins in the Escherichia coli heterologous system. Only cysteine 169 was essential in both enzymes, and inhibitor studies suggest that it is the main target of cerulenin, an antibiotic reacting with the cysteine in the active center of condensing enzymes. Most of the other exchanges led to reduced activities. In two cases, the enzymes responded differently, suggesting that the cysteines at positions 135 and 195 may be involved in the different product specificity of the two enzymes. The sequences surrounding the essential cysteine 169 revealed no similarity to the active sites of condensing enzymes in other polyketide synthases and in fatty acid biosynthesis. The available data indicate that resveratrol and chalcone synthases represent a group of enzymes that evolved independently of other condensing enzymes.