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11.
Knowledge-based design of bimodular and trimodular polyketide synthases based on domain and module swaps: a route to simple statin analogues. 总被引:4,自引:0,他引:4
A Ranganathan M Timoney M Bycroft J Cortés I P Thomas B Wilkinson L Kellenberger U Hanefeld I S Galloway J Staunton P F Leadlay 《Chemistry & biology》1999,6(10):731-741
BACKGROUND: Polyketides are structurally diverse natural products that have a range of medically useful activities. Nonaromatic bacterial polyketides are synthesised on modular polyketide synthase (PKS) multienzymes, in which each cycle of chain extension requires a different 'module' of enzymatic activities. Attempts to design and construct modular PKSs that synthesise specified novel polyketides provide a particularly stringent test of our understanding of PKS structure and function. RESULTS: We have constructed bimodular and trimodular PKSs based on DEBS1-TE, a derivative of the erythromycin PKS that contains only modules 1 and 2 and a thioesterase (TE), by substituting multiple domains with appropriate counterparts derived from the rapamycin PKS. Hybrid PKSs were obtained that synthesised the predicted target triketide lactones, which are simple analogues of cholesterol-lowering statins. In constructing intermodular fusions, whether between modules in the same or in different proteins, it was found advantageous to preserve intact the acyl carrier protein-ketosynthase (ACP-KS) didomain that spans the junction between successive modules. CONCLUSIONS: Relatively simple considerations govern the construction of functional hybrid PKSs. Fusion sites should be chosen either in the surface-accessible linker regions between enzymatic domains, as previously revealed, or just inside the conserved margins of domains. The interaction of an ACP domain with the adjacent KS domain, whether on the same polyketide or not, is of particular importance, both through conservation of appropriate protein-protein interactions, and through optimising molecular recognition of the altered polyketide chain in the key transfer of the acyl chain from the ACP of one module to the KS of the downstream module. 相似文献
12.
1-Hydroxy-2-bromopropane and the corresponding 2-iodo compounds react readily with (1,1,2-trifluoro-2-chloroethyl) diethylamine to give the rearranged 1-halogeno-2-fluoro derivatives, whereas 1-hydroxy-2-chloropropane under the same conditions affords the direct substitution product, 1-fluoro-2-chloropropane. 1-Hydroxy-2-bromopropane reacts readily with anhydrous HF to give exclusively the rearranged fluoro compound. The 19F n.m.r. data of these compounds are reported and the mechanism of the rearrangement discussed. 相似文献
13.
Holzbaur IE Harris RC Bycroft M Cortes J Bisang C Staunton J Rudd BA Leadlay PF 《Chemistry & biology》1999,6(4):189-195
BACKGROUND: Polyketides are compounds that possess medically significant activities. The modular nature of the polyketide synthase (PKS) multienzymes has generated interest in bioengineering new PKSs. Rational design of novel PKSs, however, requires a greater understanding of the stereocontrol mechanisms that operate in natural PKS modules. RESULTS: The N-acetyl cysteamine (NAC) thioester derivative of the natural beta-keto diketide intermediate was incubated with DEBS1-TE, a derivative of the erythromycin PKS that contains only modules 1 and 2. The reduction products of the two ketoreductase (KR) domains of DEBS1-TE were a mixture of the (2S, 3R) and (2R,3S) isomers of the corresponding beta-hydroxy diketide NAC thioesters. Repeating the incubation using a DEBS1-TE mutant that only contains KR1 produced only the (2S,3R) isomer. CONCLUSIONS: In contrast with earlier results, KR1 selects only the (2S) isomer and reduces it stereospecifically to the (2S, 3R)-3-hydroxy-2-methyl acyl product. The KR domain of module 1 controls the stereochemical outcome at both methyl-and hydroxyl-bearing chiral centres in the hydroxy diketide intermediate. Earlier work showed that the normal enzyme-bound ketoester generated in module 2 is not epimerised, however. The stereochemistry at C-2 is therefore established by a condensation reaction that exclusively gives the (2R)-ketoester, and the stereo-chemistry at C-3 by reduction of the keto group. Two different mechanisms of stereochemical control, therefore, operate in modules 1 and 2 of the erythromycin PKS. These results should provide a more rational basis for designing hybrid PKSs to generate altered stereochemistry in polyketide products. 相似文献
14.
Kira J. Weissman Cameron J. Smith Ulf Hanefeld Ranjana Aggarwal Matthew Bycroft James Staunton Peter F. Leadlay 《Angewandte Chemie (International ed. in English)》1998,37(10):1437-1440
The production of genetically engineered polyketides depends critically on thioesterase activity for product release. In vitro studies with the thioesterase from the erythromycin polyketide synthase (PKS) have demonstrated that the ability of this enzyme to act as a universal decoupler is limited, but stereochemical variation is readily tolerated. Synthetic analogues with all four stereochemical configurations of the natural substrate's 2-methyl-3-hydroxy substitution pattern ( 1 – 4 ; X=p-nitrophenoxy) were substrates for the enzyme. 相似文献