Chain initiation on the soraphen-producing modular polyketide synthase from Sorangium cellulosum.

BACKGROUND: Polyketides are structurally diverse natural products with a wide range of useful activities. Bacterial modular polyketide synthases (PKSs) catalyse the production of non-aromatic polyketides using a different set of enzymes for each successive cycle of chain extension. The choice of starter unit is governed by the substrate specificity of a distinct loading module. The unusual loading module of the soraphen modular PKS, from the myxobacterium Sorangium cellulosum, specifies a benzoic acid starter unit. Attempts to design functional hybrid PKSs using this loading module provide a stringent test of our understanding of PKS structure and function, since the order of the domains in the loading and first extension module is non-canonical in the soraphen PKS, and the producing strain is not an actinomycete. RESULTS: We have constructed bimodular PKSs based on DEBS1-TE, a derivative of the erythromycin PKS that contains only extension modules 1 and 2 and a thioesterase (TE) domain, by substituting one or more domains from the soraphen PKS. A hybrid PKS containing the soraphen acyltransferase domain AT1b instead of extension acyltransferase domain AT1 produced triketide lactones lacking a methyl group at C-4, as expected if AT1b catalyses the addition of malonyl-CoA during the first extension cycle on the soraphen PKS. Substitution of the DEBS1-TE loading module AT domain by the soraphen AT1a domain led to the production of 5-phenyl-substituted triketide lactone, as well as the normal products of DEBS1-TE. This 5-phenyl triketide lactone was also the product of a hybrid PKS containing the entire soraphen PKS loading module as well as part of its first extension module. Phenyl-substituted lactone was only produced when measures were simultaneously taken to increase the intracellular supply of benzoyl-CoA in the host strain of Saccharopolyspora erythraea. CONCLUSIONS: These results demonstrate that the ability to recruit a benzoate starter unit can be conferred on a modular PKS by the transfer either of a single AT domain, or of multiple domains to produce a chimaeric first extension module, from the soraphen PKS. However, benzoyl-CoA needs to be provided within the cell as a specific precursor. The data also support the respective roles previously assigned to the adjacent AT domains of the soraphen loading/first extension module. Construction of such hybrid actinomycete-myxobacterial enzymes should significantly extend the synthetic repertoire of modular PKSs.

Evaluating precursor-directed biosynthesis towards novel erythromycins through in vitro studies on a bimodular polyketide synthase.

BACKGROUND: Modular polyketide synthases (PKSs) catalyse the biosynthesis of complex polyketides using a different set of enzymes for each successive cycle of chain extension. Directed biosynthesis starting from synthetic diketides is a potentially valuable route to novel polyketides. We have used a purified bimodular derivative of the erythromycin-producing polyketide synthase (DEBS 1-TE) to study chain extension starting from a variety of diketide analogues and, in some cases, from the alternative acyl-CoA thioester substrates. RESULTS: Chain initiation in vitro by DEBS 1-TE module 2 using a synthetic diketide analogue as a substrate was tolerant of significant structural variation in the starter unit of the synthetic diketide, but other changes completely abolished activity. Interestingly, a racemic beta-keto diketide was found to be reduced in situ on the PKS and utilised in place of its more complex hydroxy analogue as a substrate for chain extension. The presence of a diketide analogue strongly inhibited chain initiation via the loading module. Significantly higher concentrations of diketide N-acetylcysteamine analogues than their corresponding acyl-CoA thioesters are required to achieve comparable yields of triketide lactones. CONCLUSIONS: Although a broad range of variation in the starter residue is acceptable, the substrate specificity of module 2 of a typical modular PKS in vitro is relatively intolerant of changes at C-2 and C-3. This will restrict the usefulness of approaches to synthesise novel erythromycins using synthetic diketides in vivo. The use of synthetic beta-keto diketides in vivo deserves to be explored.

Relationship of compliance with hormone replacement therapy to short-term healthcare utilization in a managed care population.

OBJECTIVE: To identify healthcare utilization characteristics that distinguish female members of a managed care organization (MCO) who remained compliant with hormone replacement therapy (HRT) from those who had poor compliance during an 18-month period and to estimate the cost of HRT to an MCO. STUDY DESIGN: A retrospective cohort design in a population of continuously enrolled female members of an MCO. METHODS: All female members of the Lovelace Health Plan 40 years of age or older who began HRT between January 1, 1993, and June 30, 1994 (n = 1158). Compliance was determined by calculating an estrogen medication possession ratio based on pharmacy fills over 18 months. "High compliers" (n = 427) were defined as those purchasing at least 80% of their recommended days supply, and "low compliers" (n = 269) as those purchasing less than 20%. Healthcare encounters and costs of high and low compliers were compared. RESULTS: High compliers were younger (P < 0.01), more likely to be non-Hispanic white than Hispanic (P < 0.0001), and had higher costs for obstetric/gynecologic care (P < 0.0001) and non-HRT prescriptions (P < 0.0001). Low compliers had higher point estimates of costs and encounters for all other categories of care, but differences were statistically significant only for emergency department visits (P < 0.001). CONCLUSIONS: The added cost of HRT did not result in higher total healthcare utilization and costs, as women who complied well with therapy had decreased utilization and costs in other categories of care. Differences in subcategories of healthcare utilization suggest that level of HRT compliance reflects differences in how women access healthcare.