Alteration of the Substrate Specificity of a Modular Polyketide Synthase Acyltransferase Domain through Site-Specific Mutations

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• 作者：Christopher D. Reeves ; Sumati Murli ; Gary W. Ashley ; Misty Piagentini ; C. Richard Hutchinson ; and Robert McDaniel
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：December 25, 2001
• 年：2001
• 卷：40
• 期：51
• 页码：15464 - 15470
• 全文大小：83K
• 年卷期：v.40,no.51(December 25, 2001)
• ISSN：1520-4995

文摘

Cassette replacement of acyltransferase (AT) domains in 6-deoxyerythronolide B synthase(DEBS) with heterologous AT domains with different substrate specificities usually yields the predictedpolyketide analogues. As reported here, however, several AT replacements in module 4 of DEBS failedto produce detectable polyketide under standard conditions, suggesting that module 4 is sensitive toperturbation of the protein structure when the AT is replaced. Alignments between different modularpolyketide synthase AT domains and the Escherichia coli fatty acid synthase transacylase crystal structurewere used to select motifs within the AT domain of module 4 to re-engineer its substrate selectivity andminimize potential alterations to protein folding. Three distinct primary regions of AT4 believed to conferspecificity for methylmalonyl-CoA were mutated into the sequence seen in malonyl-CoA-specific domains.Each individual mutation as well as the three in combination resulted in functional DEBSs that producedmixtures of the natural polyketide, 6-deoxyerythronolide B, and the desired novel analogue, 6-desmethyl-6-deoxyerythronolide B. Production of the latter compound indicates that the identified sequence motifsdo contribute to AT specificity and that DEBS can process a polyketide chain incorporating a malonateunit at module 4. This is the first example in which the extender unit specificity of a PKS module hasbeen altered by site-specific mutation and provides a useful alternate method for engineering AT specificityin the combinatorial biosynthesis of polyketides.