A Model of Structure and Catalysis for Ketoreductase Domains in Modular Polyketide Synthases
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- 作者：Ralph Reid ; Misty Piagentini ; Eduardo Rodriguez ; Gary Ashley ; Nina Viswanathan ; John Carney ; Daniel V. Santi ; C. Richard Hutchinson ; and Robert McDaniel
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：January 14, 2003
- 年：2003
- 卷：42
- 期：1
- 页码：72 - 79
- 全文大小：334K
- 年卷期：v.42,no.1(January 14, 2003)
- ISSN：1520-4995
文摘
A putative catalytic triad consisting of tyrosine, serine, and lysine residues was identified inthe ketoreductase (KR) domains of modular polyketide synthases (PKSs) based on homology modelingto the short chain dehydrogenase/reductase (SDR) superfamily of enzymes. This was tested by constructingpoint mutations for each of these three amino acid residues in the KR domain of module 6 of the6-deoxyerythronolide B synthase (DEBS) and determining the effect on ketoreduction. Experimentsconducted in vitro with the truncated DEBS Module 6+TE (M6+TE) enzyme purified from Escherichiacoli indicated that any of three mutations, Tyr 
Phe, Ser Ala, and Lys Glu, abolish KR activityin formation of the triketide lactone product from a diketide substrate. The same mutations were alsointroduced in module 6 of the full DEBS gene set and expressed in Streptomyces lividans for in vivoanalysis. In this case, the Tyr Phe mutation appeared to completely eliminate KR6 activity, leading tothe 3-keto derivative of 6-deoxyerythronolide B, whereas the other two mutations, Ser Ala and Lys Glu, result in a mixture of both reduced and unreduced compounds at the C-3 position. The results supporta model analogous to SDRs in which the conserved tyrosine serves as a proton donating catalytic residue. In contrast to deletion of the entire KR6 domain of DEBS, which causes a loss in substrate specificity of the adjacent acyltransferase (AT) domain in module 6, these mutations do not affect the AT6specificity and offer a potentially superior approach to KR inactivation for engineered biosynthesis ofnovel polyketides. The homology modeling studies also led to identification of amino acid residuespredictive of the stereochemical nature of KR domains. Finally, a method is described for the rapidpurification of engineered PKS modules that consists of a biotin recognition sequence C-terminal to thethioesterase domain and adsorption of the biotinylated module from crude extracts to immobilizedstreptavidin. Immoblized M6+TE obtained by this method was over 95% pure and as catalytically effectiveas M6+TE in solution.
Phe, Ser Ala, and Lys Glu, abolish KR activityin formation of the triketide lactone product from a diketide substrate. The same mutations were alsointroduced in module 6 of the full DEBS gene set and expressed in Streptomyces lividans for in vivoanalysis. In this case, the Tyr Phe mutation appeared to completely eliminate KR6 activity, leading tothe 3-keto derivative of 6-deoxyerythronolide B, whereas the other two mutations, Ser Ala and Lys Glu, result in a mixture of both reduced and unreduced compounds at the C-3 position. The results supporta model analogous to SDRs in which the conserved tyrosine serves as a proton donating catalytic residue. In contrast to deletion of the entire KR6 domain of DEBS, which causes a loss in substrate specificity of the adjacent acyltransferase (AT) domain in module 6, these mutations do not affect the AT6specificity and offer a potentially superior approach to KR inactivation for engineered biosynthesis ofnovel polyketides. The homology modeling studies also led to identification of amino acid residuespredictive of the stereochemical nature of KR domains. Finally, a method is described for the rapidpurification of engineered PKS modules that consists of a biotin recognition sequence C-terminal to thethioesterase domain and adsorption of the biotinylated module from crude extracts to immobilizedstreptavidin. Immoblized M6+TE obtained by this method was over 95% pure and as catalytically effectiveas M6+TE in solution.