Nucleotidylation of unsaturated carbasugar in validamycin biosynthesis

Abstract

Validamycin A is a member of microbial-derived C7N-aminocyclitol family of natural products that is widely used as crop protectant and the precursor of the antidiabetic drug voglibose. Its biosynthetic gene clusters have been identified in several Streptomyces hygroscopicus strains, and a number of genes within the clusters have been functionally analyzed. Of these genes, valB, which encodes a sugar nucleotidyltransferase, was found through inactivation study to be essential for validamycin biosynthesis, but its role was unclear. To characterize the role of ValB in validamycin biosynthesis, four carbasugar phosphate analogues were synthesized and tested as substrate for ValB. The results showed that ValB efficiently catalyzes the conversion of valienol 1-phosphate to its nucleotidyl diphosphate derivatives, whereas other unsaturated carbasugar phosphates were found to be not the preferred substrate. ValB requires Mg2+, Mn2+, or Co2+ for its optimal activity and uses the purine-based nucleotidyltriphosphates (ATP and GTP) more efficiently than the pyrimidine-based NTPs (CTP, dTTP, and UTP) as nucleotidyl donor. ValB represents the first member of unsaturated carbasugar nucleotidyltransferases involved in natural products biosynthesis. Its characterization not only expands our understanding of aminocyclitol-derived natural products biosynthesis, but may also facilitate the development of new tools for chemoenzymatic synthesis of carbohydrate mimetics.

Distinct Substrate Specificity and Catalytic Activity of the Pseudoglycosyltransferase VldE

“…No other NDP-valienols have previously been tested, because the corresponding nucleotidyltransferase enzyme VldB appeared to prefer GTP as substrate, and produces GDP-valienol (Yang et al, 2011). The preference of VldE toward GDP-valienol was also supported by its X-ray crystal structures (Cavalier et al, 2012).…”

“…Therefore, we prepared all five possible NDP-valienols and evaluated the substrate preference of VldE – whether or not it can process other NDP-valienols. GDP-, ADP-, and CDP-valienols were prepared from valienol 1-phosphate and their corresponding nucleotidyl triphosphates (GTP, ATP, or CTP), using VldB (Yang et al, 2011), whereas UDP- and dTDP-valienol were prepared from valienol 1-phosphate and UTP and dTTP, respectively, using RmlA-L89T, an engineered broad spectrum nucleotidyltransferase (Moretti et al, 2011), kindly provided by Prof. Jon Thorson at the University of Kentucky (Figures S2A and S2B). Although RmlA-L89T has been known to have relaxed substrate specificity towards various sugar phosphates (Moretti et al, 2011), the present study showed that RmlA-L89T is also able to process an unsaturated carbasugar phosphate.…”

“…NDP-valienol was prepared according to a reported procedure (Yang et al, 2011). Briefly, valienol 1-phosphate (6 mM) was incubated with ATP, GTP, CTP, UTP or dTTP (6 mM) in the presence of VldB or RmlA-L89T (10 µM) and MgCl 2 (10 mM) in Tris-HCl buffer (50 mM, pH 7.5) at 30 °C and the progress of the reactions was monitored by ESI-MS. After 4 h, the reactions were quenched with an equal volume of MeOH and centrifuged (14,500 rpm, 5 min, 4 °C).…”

Biosynthesis and Biological Activity of Carbasugars

“…A preparative glycosylation reaction was performed using 189 as the acceptor with the cloned rat kidney enzyme. A tetrasaccharide formed by the addition of a Glc pNAc residue (190) was the sole product detected (Scheme 22).…”

“…Compound 102 is then epimerized at C2 to give 5-epi-valiolone-7-phosphate (103). Further steps involving dehydration (104), carbonyl reduction and phosphorylations (105), nucleotidylation [190] (106), and final nucleophilic displacement by the nitrogen atom of 95 would afford compound 93 [191][192][193][194]. In Scheme 15, the transformation 105-106 constitutes a nucleotidylation reaction: the transfer of an entire nucleotidyl unit, rather than just a phospho group [195].…”