Deciphering the sugar biosynthetic pathway and tailoring steps of nucleoside antibiotic A201A unveils a GDP- l -galactose mutase

Abstract

Galactose, a monosaccharide capable of assuming two possible configurational isomers (D-/L-), can exist as a six-membered ring, galactopyranose (Galp), or as a five-membered ring, galactofuranose (Galf). UDP-galactopyranose mutase (UGM) mediates the conversion of pyranose to furanose thereby providing a precursor for D-Galf. Moreover, UGM is critical to the virulence of numerous eukaryotic and prokaryotic human pathogens and thus represents an excellent antimicrobial drug target. However, the biosynthetic mechanism and relevant enzymes that drive L-Galf production have not yet been characterized. Herein we report that efforts to decipher the sugar biosynthetic pathway and tailoring steps en route to nucleoside antibiotic A201A led to the discovery of a GDP-L-galactose mutase, MtdL. Systematic inactivation of 18 of the 33 biosynthetic genes in the A201A cluster and elucidation of 10 congeners, coupled with feeding and in vitro biochemical experiments, enabled us to: (i) decipher the unique enzyme, GDP-Lgalactose mutase associated with production of two unique D-mannose-derived sugars, and (ii) assign two glycosyltransferases, four methyltransferases, and one desaturase that regiospecifically tailor the A201A scaffold and display relaxed substrate specificities. Taken together, these data provide important insight into the origin of L-Galf-containing natural product biosynthetic pathways with likely ramifications in other organisms and possible antimicrobial drug targeting strategies.nucleoside antibiotic A201A | biosynthesis | GDP-L-galactose mutase | methyltransferase | desaturase

Characterization of the First Bacterial and Thermostable GDP-Mannose 3,5-Epimerase

“…A BLAST search with the respective sequence (UniProt: Q9FB21) exposed 42% sequence identity with At GM35E, fortifying the assumption that this concerns a GM35E. Furthermore, the activity of GM35E was recently described in the biosynthetic pathway of the nucleoside antibiotic A201A found in Marinactinospora thermotolerans [48]. This antibiotic contains an l -galactose moiety in its furanose configuration which is essential for the bioactivity of this drug.…”

“…Interestingly, the availability of these NDP-sugars forms a gateway to glycorandomization, a process in which the carbohydrate moiety of antibiotics is altered in order to tailor their pharmacological properties and/or biological activity [60,61,62]. As l -galactose and l -gulose are two l -sugars that are known to be present in natural antibiotics, they form an interesting starting point for this technique [4,48]. Moreover, the l -Gul moiety in bleomycin facilitates its uptake by cancer cells, assuming that it might be interesting to link this l -sugar to other anticancer drugs as well [14].…”

Ilamycin E, a natural product of marine actinomycete, inhibits triple-negative breast cancer partially through ER stress-CHOP-Bcl-2

“…The discovery, structural elucidation and biosynthesis of Ilamycin E and Ilamycin F have been described previously 18, 19. All compounds were dissolved in DMSO (Sigma, St Louis, MO, USA).…”

“…As the most abundant resource on the earth, ocean organisms are a very promising source for anti-cancer drug discovery 12, 14. Ilamycins are a series of cyclic peptides with anti-tuberculosis activity that are produced by Streptomyces atratus and Streptomyces islandicus 15-19. The anti-tumor activities of Ilamycins in MCF7 cells have been investigated 18.…”

Amino Acid Metabolism and Transport Mechanisms as Potential Antifungal Targets

“…Galactofuranose (Galf) is the 5-member-ring form of galactose found in the walls of many pathogenic molds, including species Aspergillus , but it is not in mammals [ 54 ]. Galf is found in many medically-relevant mycoses, indicating that Galf is an important component for survival and reproduction [ 55 , 56 ]. Interestingly, Galf has never been found humans, thus Galf-biosynthetic pathways have raised much interest as targets for drug development as they pertain to amino acids and their transport [ 57 , 58 , 59 , 60 , 61 ].…”