Modular polyketide synthases and cis double bond formation: establishment of activated cis-3-cyclohexylpropenoic acid as the diketide intermediate in phoslactomycin biosynthesis.

Phoslactomycins (PLMs), exemplified by PLM B (Figure 1), are a unique class of antitumor, antiviral, and antifungal polyketide natural products.1,2 The antitumor activity of PLMs is attributed to a potent and selective inhibition of protein Ser/Thr phosphatase 2A (PP2A).3 The PLM biosynthetic gene cluster from Streptomyces sp. HK803 has been cloned and sequenced.4 The PLM polyketide synthase (PKS) is a modular PKS comprised of a loading domain and seven extension modules which are responsible for the synthesis of a unique linear unsaturated polyketide structure containing three cis (Z) and one trans (E) double bonds. Modular PKSs which generate unsaturated products typically do so using trans double bonds.5 These double bonds are established by ketoreductase-dehydratase (KR-DH) domains which sequentially carry out ketoreduction and dehydration steps on the 3ketoacyl-ACP products of the KS domains. The dehydration step makes the stereochemical course of the KR-catalyzed step cryptic. Recently, in vitro work using a DH-inactivated module 2 of the pikromycin PKS, which establishes the single trans double bond of pikromycin and methymycin, has shown this KR generates the D-3-hydroxy product.6 A bioinformatic analysis of other cryptic KR-DH domains which generate trans double bonds infers a D-hydroxyl configuration (this analysis is based on an established correlation of diagnostic residues in KR primary sequences and their known stereochemical products).5,7 Polyketide products containing cis double bonds are rare and appear to arise through a variety of mechanisms.8 In many cases, such as modules 7 of PLM and module 4 of the epothilone PKS, the required DH activity is absent from the module.4,9 Modules 1 and 2 of the PLM PKS are intriguing because they have combined KR-DH didomains which appear to establish two conjugated cis double bonds (C12-C13 and C14-C15 of PLM B, respectively).4 Bioinformatic analysis of the primary sequence of these KR domains does not clearly predict a D-hydroxyl configuration (which evidence indicates precedes trans double bond formation) or L-hydroxy configuration (which has been speculated might precede cis double bond formation).7 Thus, in each case, the combined activity of these KR-DH didomains might establish a trans double bond with a subsequent isomerization step to a cis double bond (epimerization domains, in both PKS10 and NRPS11 modules, as well as trans to cis double bond isomerization in retinoid cycle12 have been reported). Alternatively, these KR-DH domains might establish the cis double bond directly. In this work, we have distinguished between these two possibilities by determining the stereochemistry of the polyketide intermediate which is transferred from module 1 to module 2. PLM1 contains a loading domain and the first extension module of the PKS and is predicted to generate either cisor trans-3-cyclohexylpropenoic acid (Figure 2) from an activated cyclohexanecarboxylic acid (CHC) starter unit. We generated a ∆chcA mutant (NP3), blocked in biosynthesis of the starter unit, and demonstrated that it only produces PLM B when grown in the presence of CHC (Table 1). The transand cis-diketide products of PLM1 were synthesized in both the acid (2a and 3a, Figure 2) and N-acetylcysteamine (SNAC) thioester (4a and 5a, Figure 2) forms and added to separate fermentations of this ∆chcA mutant. Surprisingly, compounds 2a-5a all restored PLM B production. PLM B production levels were the highest for the trans products (2a and 4a) and were 40% higher than that observed with either CHC supplementation or the cis-SNAC (5a) (Table 1). The lowest level of PLM B production was observed with the cis-acid (3a). Interestingly, the PLM B isolated from feeding trans-acid 2a had the C14-C15 double bond in the cis configuration, as confirmed by 1H NMR and NOESY experiments. This initial result suggested that the trans-diketide intermediate might be the preferred substrate for PLM2, with a subsequent trans to cis isomerization step. Alternatively, the trans compounds might be converted efficiently to the activated CHC starter unit by fatty acid degradation and subsequently elongated by the entire PLM PKS (in this way, the trans double bond would be lost through degradation and reintroduced as a cis double bond by PLM1) (Figure 2). To distinguish between these two hypotheses, we synthesized and fed the [2-13C]labeled analogues 2b-5b (Figure 2) to the ∆chcA mutant. Mass spectroscopy revealed that isotopic enrichment over natural abundance for the PLM B product was only observed with the cis-SNAC 5b (20% isotope enrichment, Table 2). These data showed that both cis and trans compounds undergo degradation to form the activated CHC starter unit, and that this is the primary route for PLM B production in these experiments. Furthermore, the experiments established that only cis-SNAC (5a, 5b) could prime PLM2 directly. The cis-acid (3a, 3b), which gives the lowest levels of PLM B restoration levels, can be transported into the mutant and degraded to the activated CHC (at about 50% the efficiency of the corresponding trans-diketides) but cannot be activated intact such that it can prime PLM2. A consistent and predictable set of results was obtained by generation and analysis of a plm1 deletion mutant [NP9, see Supporting Information] (Figure 2). PLM B production was abrogated in this mutant and was only significantly restored by growth in the presence of the cis-SNAC compounds 5a and its 13C-labeled counterpart 5b (Table 1). In the case of 5b, the PLM B now contained the same level of isotopic enrichment (>99%) as ‡ Current address: Virginia Commonwealth University. Figure 1. Phoslactomycin B (PLM B). Published on Web 01/27/2007