No abstract

The majority of modular polyketide synthase (PKS) systems which generate unsaturated products do so with trans double bonds. Phoslactomycin B (PLM B) presents a class of antitumor and antiviral natural polyketide products that have unique structural features, including a linear unsaturated backbone with one trans and three cis double bonds. There is substantial evidence that trans double bonds are established by ketoreductase-dehydratase (KR-DH) didomains within a PKS module. In cases where modules containing these didomains appear to generate product containing a cis double bond there is no experimental evidence to determine if they do so directly, or if they also form a trans double bond with a subsequent isomerization step. A critical step in addressing this issue is establishing the stereochemistry of the polyketide intermediate which passes to the subsequent module. Herein, we demonstrate through a series of experiments that an activated cis-3-cyclohexylpropenoic acid is the diketide intermediate which passes from module 1 to module 2 of the PLM PKS. The trans isomer of the diketide intermediate could not be processed directly into PLM B by module 2, but could be converted to PLM B by degradation to cyclohexanecarboxylic acid and elongation by the entire PLM PKS. These observations indicate not only that module 1 with a DH-KR didomain is responsible for establishing C 14 -C 15 cis double bond of PLM B, but that the subsequent modules of the PKS clearly discriminate between the cis and trans-diketide intermediate and do not contain domains capable of catalyzing double bond isomerization.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 3-ketoacyl-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, have shown this KR generates the D-3-hydroxy product. 6 A bioinformatic analysis of other cryptic KR-

Hygromycin A, an antibiotic produced by Streptomyces hygroscopicus NRRL 2388, offers a distinct carbon skeleton structure for development of antibacterial agents targeting the bacterial ribosomal peptidyl transferase. A 31.5 kb genomic DNA region covering the hygromycin A biosynthetic gene cluster has been identified, cloned, and sequenced. The hygromycin gene cluster has 29 ORFs which can be assigned to hygromycin A resistance as well as regulation and biosynthesis of the three key moieties of hygromycin A (5-dehydro-alpha-L-fucofuranose, (E)-3-(3,4-dihydroxyphenyl)-2-methylacrylic acid, and 2L-2-amino-2-deoxy-4,5-O-methylene-neo-inositol. The predicted Hyg26 protein has sequence homology to short-chain alcohol dehydrogenases and is assigned to the final step in production of the 5-dehydro-alpha-L-fucofuranose, catalyzing the reduction of alpha-L-fucofuranose. A hyg26 mutant strain was generated and shown to produce no hygromycin A but 5''-dihydrohygromycin A, 5''-dihydromethoxyhygromycin A, and a 5''-dihydrohygromycin A product lacking the aminocyclitol moiety. To the best of our knowledge, these shunt metabolites of biosynthetic pathway intermediates have not previously been identified. They provide insight into the ordering of the multiple unusual steps which compromise the convergent hygromycin A biosynthetic pathway.

The antifungal phoslactomycins (PLM A-F), produced by Streptomyes sp HK803, are structurally unusual in that three of their four double bonds are in the cis form (Δ 12,13 , Δ 14,15 , Δ 2,3 ). The PLM polyketide synthase (PKS) has the predicted dehydratase catalytic domain in modules 1,2 and 5 required for establishing two of these cis double bonds (Δ 12,13 Δ 14,15 ), as well as the only trans Δ 6,7 double bond. By contrast, the formation of the cis Δ 2,3 in the unsaturated lactone moiety of PLMs has presented an enigma because the predicted dehydratase domain in module 7 is absent. Herein, we have demonstrated that the plmT 2 gene product, with no homology to PKS dehydratase domains, is required for efficient formation of the cis Δ 2,3 alkene. A series of new PLM products in which the C3 hydroxyl group is retained, are made in plmT 2 deletion mutants. In all of these cases, however, the hydroxyl group is esterified with malonic acid. These malonylated PLM products are converted to the corresponding cis Δ 2,3 PLM products and acetic acid by a facile base-catalyzed decarboxylative elimination reaction. Complete or partial restoration of natural PLM production in a plmT 2 deletion mutant can be accomplished by plasmid based expression of plmT 2 or fos ORF4 (a homologous gene from the fostriecin biosynthetic gene cluster), respectively. The data indicate that dehydrataseindependent pathways also function in establishment of unsaturated 6-membered lactone moieties in other PKS pathways, and provide the first biosynthetic insights into the possible routes by which unusual malonylated polyketide products are generated.Modular polyketide synthases (PKSs) generate a vast array of structurally diverse natural products with important biological activities. 1 They are responsible for formation of macrolides, the vast majority of which contain one or more double bonds. 2 The majoritiy of other natural produces made by modular PKSs similarly contain some double bonds. In the majority of cases the alkenes in the polyketide product are in the trans form. The formation of these double bonds has been shown to be directly attributed to the presence of a ketoreductasedehydratase (KR-DH) didomain within the appropriate module. 3, 4 These didomains catalyze a 3-keto reduction and subsequent Δ 2,3 elimination reaction with the PKS-tethered 3-ketoacyl polyketide intermediates.The phoslactomycins (PLMs A-F Figure 1) and fostriecin belong to a class of phosphorylated polyketides that contain multiple double bonds in the cis form. 5, 6 For the PLMs there are three double bonds in the cis form (Δ 12,13 , Δ 14,15 , Δ 2,3 ) and one in the trans form (Δ 6,7 ). 7, 8The PLM biosynthetic gene cluster has been cloned and sequenced and shown to encode a modular PKS. 9 Modules 1 and 2 contain the expected dehydratase DH-KR didomain required for formation of the conjugated diene, while module 5 contains a DH-KR domain likely responsible for formation of the trans Δ 6,7 alkene. The KR-DH domains which generate a trans double bond do so via a...

The antibacterial activity of hygromycin A (HA) arises from protein synthesis inhibition and is dependent upon a methylenedioxy bridged-aminocyclitol moiety. Selective gene deletions and chemical complementation in Streptomyces hygroscopicus NRRL 2388 showed that the hyg18 and hyg25 gene products, proposed to generate a myo-inositol intermediate, are dispensable for HA biosynthesis but contribute to antibiotic yields. Hyg8 and Hyg17, proposed to introduce the amine functionality, are essential for HA biosynthesis. Hyg6 is a methyltransferase acting on the aminocyclitol, and a Deltahyg6 mutant produces desmethylenehygromycin A. Deletion of hyg7, a metallo-dependant hydrolase homolog gene, resulted in methoxyhygromycin A production, demonstrating that the corresponding gene product is responsible for the proposed oxidative cyclization step of methylenedioxy bridge formation. The methyl/methylene group is not required for in vitro protein synthesis inhibition but is essential for activity against Escherichia coli.