Kinetic Analysis of the Actinorhodin Aromatic Polyketide Synthase

Abstract

Type II polyketide synthases (PKSs) are bacterial multienzyme systems that catalyze the biosynthesis of a broad range of natural products. A core set of subunits, consisting of a ketosynthase, a chain length factor, an acyl carrier protein (ACP) and possibly a malonyl CoA: ACP transacylase (MAT) forms a "minimal" PKS. They generate a poly-␤-ketone backbone of a specified length from malonyl-CoA derived building blocks. Here we (a) report on the kinetic properties of the actinorhodin minimal PKS, and (b) present further data in support of the requirement of the MAT. Kinetic analysis showed that the apoACP is a competitive inhibitor of minimal PKS activity, demonstrating the importance of proteinprotein interactions between the polypeptide moiety of the ACP and the remainder of the minimal PKS. In further support of the requirement of MAT for PKS activity, two new findings are presented. First, we observe hyperbolic dependence of PKS activity on MAT concentration, saturating at very low amounts (half-maximal rate at 19.7 ؎ 5.1 nM). Since MAT can support PKS activity at less than 1/100 the typical concentration of the ACP and ketosynthase/chain length factor components, it is difficult to rule out the presence of trace quantities of MAT in a PKS reaction mixture. Second, an S97A mutant was constructed at the nucleophilic active site of the MAT. Not only can this mutant protein support PKS activity, it is also covalently labeled by [ 14 C]malonyl-CoA, demonstrating that the serine nucleophile (which has been the target of PMSF inhibition in earlier studies) is dispensible for MAT activity in a Type II PKS system.

Biochemical Characterization of Acyl Carrier Protein (AcpM) and Malonyl-CoA:AcpM Transacylase (mtFabD), Two Major Components ofMycobacterium tuberculosis Fatty Acid Synthase II

“…Surprisingly, this enzyme does not contain the characteristic GXSXG motif of mtFabD, suggesting that other residues replacing the active serine in this motif may be involved in catalysis. This hypothesis is strengthened by recent studies on the MCAT from S. coelicolor (42), where it was observed that a S97A mutant was still covalently labeled by malonyl-CoA, indicating that the serine nucleophile is dispensable for MCAT activity. This suggests that the MCAT from S. coelicolor possesses an alternative nucleophilic group that is capable of substituting for the active site serine in the S97A mutant.…”

“…It has been shown that FabD constitutes a link between both pathways (36,41). The same conclusion was also reached about the possible role in actinorhodin synthesis of MCAT isolated from S. coelicolor (18,42). Therefore, it would be interesting to investigate whether mtFabD may act on both the FAS-II system involved in mycolic acid biosynthesis and in certain PKS systems.…”

Type III polyketide synthases in natural product biosynthesis

“…2A) (13). These two compounds have been reported to be the spontaneously cyclized products of the nascent octaketide chain synthesized by some type II minimal PKSs such as the actinorhodin minimal PKS (14). When 4-coumaroyl-CoA and malonyl-CoA are both used as substrates, OKS not only synthesizes SEK4 and SEK4b but also catalyzes the condensation of 4-coumaroyl-CoA with malonyl-CoA, yielding an unnatural C 21 heptaketide chalcone and a C 19 hexaketide stilbene (Fig.…”

Cloning, Nucleotide Sequence, and Heterologous Expression of the Biosynthetic Gene Cluster for R1128, a Non-steroidal Estrogen Receptor Antagonist

“…protein-protein interactions in chain elongation (34,35). Sequence analysis suggests that ZhuB (the KS), ZhuA (the CLF), ZhuN (the ACP), and the MAT from the Streptomyces fatty acid synthase are the constituents of the R1128 minimal PKS (Fig.…”