Unusual carbohydrate biosynthesis : mechanistic studies of DesII and the biosynthesis of formycin A




Ko, Yeonjin

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Carbohydrates are essential biomolecules in all living organisms. Besides serving as energy storage and structural building blocks in primary metabolism, carbohydrates represent the building blocks for numerous bioactive natural products. The presence of sugar moieties in secondary metabolites are important for the biological properties of these natural products. Thus, the study of biosynthetic pathways involving carbohydrate secondary metabolism can reveal some intriguing enzymatic transformations that lead to remarkable structural diversity. Understanding these pathways and the enzymes they contain can provide new insights for pathway engineering and the production of novel sugar structures. The radical S-adenosyl-L-methionine (SAM) enzymes are distinguished by their unique chemistry that results in the reductive homolysis of SAM to generate a reactive 5′- deoxyadenosyl radical. Subsequent formation of a substrate radical intermediate by this radical initiator permits a diverse set of biotransformations. DesII belongs to the radical SAM enzyme superfamily, and is involved in the biosynthesis of TDP-desosamine in Streptomyces venezuelae. DesII catalyzes the deamination of its biosynthetic substrate (i.e., TDP-4,6-dideoxy-3-keto-D-glucose), whereas it promotes an oxidative dehydrogenation reaction when the C4 amino group of the substrate is replaced by a hydroxyl group (i.e., TDP-D-quinovose). Control of the radical intermediate resulting in two distinct reaction outcomes has been a primary focus of this research. It has been proposed that the orbital geometry of the radical intermediates is an important factor in determining whether the enzyme functions as a lyase or a dehydrogenase. To investigate this hypothesis, several substrate analogs with altered stereochemistry of ring substituents were tested as potential substrates for DesII and the reaction products were characterized. While DesII deaminates the C4 axial amino substituent as it does with the C4 equatorial amino group, inversion of stereochemistry of a hydroxyl group at C4 allows dehydration to take place. These results support the working hypothesis that the stereochemical configuration of substrate radical in the active site plays an important role in controlling the partitioning into different reaction pathways. Formycin A and coformycin are nucleoside antibiotics produced by Nocardia interforma and Streptomyces kaniharaensis. Their biosynthetic pathways are of particular interest because of their unusual structural features such as the pyrazolopyrimidine nucleobase with a C-glycosidic linkage in formycin A and the 1,3-diazepine ring in coformycin. Genomic analysis of producing strain suggested that the pyrimidine ring of formycin A is formed in a pathway analogous to that for purines, which led to the identification of a potential biosynthetic gene cluster and pathway for formycin A. Conversion of the putative intermediate, carboxyaminopyrazole ribonucleotide, in this pathway to the final product was demonstrated to be catalyzed by enzymes encoded in the for cluster as proposed. It was also shown that one gene adjacent to the formycin A gene cluster encodes a reductase that catalyzes the last step in the biosynthesis of coformycin. This study aims to elucidate the biosynthetic pathways of formycin A and coformycin with an emphasis on the formation of pyrazolopyrimidine moiety and C-glycosidic bond.



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