Structural Investigation of the Substrate Specificity of Offloading and Sugar Methylation in Macrolide Biosynthesis.

Polyketide synthase;Methyltransferase;Thioesterase;Macrolide antibiotic;Crystal engineering;Biological Chemistry;Science;Chemical Biology

Bernard, SteffenSherman, David H. ;

University of Michigan

关键词: Polyketide synthase;    Methyltransferase;    Thioesterase;    Macrolide antibiotic;    Crystal engineering;    Biological Chemistry;    Science;    Chemical Biology;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/110364/steffenb_1.pdf?sequence=2&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

The increase in antibiotic resistance over the past century emphasizes the need for new antibiotic therapies.Macrolide antibiotics are commonly used to treat infections in humans and animals.Many macrolides are produced by type I polyketide synthases in actinobacteria.This thesis investigates the structural basis of substrate specificity of macrolactonization and sugar-O-methylation, two key steps in macrolide biosynthesis.These results are presented with the aim of supporting future efforts to design and produce new compounds through biocatalytic and bioengineering routes.Methylation of sugar hydroxyl groups is common in biosynthetic pathways for macrolides and other natural products.Two distinct methyltransferases act sequentially in the late stages of mycinamicin biosynthesis to methylate the 2’ and 3’ hydroxyls of a 6-deoxyallose sugar.Structural and biochemical investigation of the 3’-O-methyltransferase, MycF, from the mycinamicin biosynthetic pathway provided insight into the mechanism and substrate selectivity of this family of methyltransferases.The series of structures presented herein illuminate the mechanisms that underlie sequential methylation by two families of sugar-O-methyltransferases common in natural product biosynthetic pathways.This foundation led to successful enzyme engineering which circumvented the natural order of the pathway to produce a new macrolide. Macrolactonization is another common feature of natural product biosynthesis.The structure of the thioesterase (TE) domain from the tylosin biosynthetic pathway is the first structure of a 16-membered macrolactone forming TE.Similar to the pikromycin TE, the Tyl TE has a hydrophillic barrier between the active site and the exterior of the protein, which is proposed to aid in promoting cyclization.A model of the product complex provides insights into the substrate specificity of the Tyl TE.Biochemical experiments demonstrated the utility of fluorophosphonate affinity labels in future structural characterization of TE domains.