Identification of a Pyrrole Intermediate Which Undergoes C‐Glycosidation and Autoxidation to Yield the Final Product in Showdomycin Biosynthesis

Abstract

Showdomycin is aC -nucleoside bearing an electrophilic maleimide base.H erein, the biosynthetic pathwayo f showdomycin is presented. The initial stages of the pathway involve non-ribosomal peptide synthetase (NRPS) mediated assembly of a2 -amino-1H-pyrrole-5-carboxylic acid intermediate.This intermediate is prone to air oxidation whereupon it undergoes oxidative decarboxylation to yield an imine of maleimide,which in turn yields the maleimide upon acidification. It is also shown that this pyrrole intermediate serves as the substrate for the C-glycosidase SdmA in the pathway.A fter coupling with ribose 5-phosphate,t he resulting C-nucleoside undergoes as imilar sequence of oxidation, decarboxylation and deamination to affordshowdomcyin after exposure to air. These results suggest that showdomycin could be an artifact due to aerobic isolation;h owever,t he autoxidation maya lso serve to convert an otherwise inert product of the biosynthetic pathway to an electrophilic C-nucleotide thereby endowing showdomycin with its observed bioactivities. Results and DiscussionTo address these unresolved questions,t he sdm gene cluster was analyzed, and acassette consisting of sdmC, D, E, Figure 1. C-Ribosylnucleoside antibiotics.

Characterization of the Oxazinomycin Biosynthetic Pathway Revealing the Key Role of a Nonheme Iron-Dependent Mono-oxygenase

“…In the biosynthesis of pyrazofurin ( 6 ), the glycosidase PyfQ catalyzes C–C bond formation between phosphoribosyl pyrophosphate (PRPP, 13 ) and pyrazole dicarboxylic acid ( 10 ) via a decarboxylative electrophilic aromatic substitution to afford 14 (Figure ). ,, In contrast, C -glycoside formation catalyzed by YeiN and SdmA during biosynthesis of pseudouridine ( 3 ) and showdomycin ( 5 ), respectively, involves activation of ribose 5-phosphate (R5P, 15 ) by a catalytic lysine residue followed by a Knoevenagel–Mannich-type condensation with the nucleophilic heterocycle such as 11 to yield the C -nucleoside product 12 . ,, Sequence alignment of OzmB with homologous enzymes SdmA and YeiN from showdomycin and pseudouridine biosynthesis, respectively, revealed that the catalytic lysine residue in SdmA and YeiN is also conserved in OzmB (Figure S9). Therefore, OzmB catalysis may follow the same mechanism as for YeiN and SdmA, which starts with Schiff base formation ( 26 ) between the conserved lysine residue and the linearized R5P ( 15 ). ,, Subsequent nucleophilic attack by 16 followed by the collapse of the protein-tethered intermediate 27 would yield the C -glycosidation product 18 and regenerate the lysine residue (Figure B).…”

“… ,, In contrast, C -glycoside formation catalyzed by YeiN and SdmA during biosynthesis of pseudouridine ( 3 ) and showdomycin ( 5 ), respectively, involves activation of ribose 5-phosphate (R5P, 15 ) by a catalytic lysine residue followed by a Knoevenagel–Mannich-type condensation with the nucleophilic heterocycle such as 11 to yield the C -nucleoside product 12 . ,, Sequence alignment of OzmB with homologous enzymes SdmA and YeiN from showdomycin and pseudouridine biosynthesis, respectively, revealed that the catalytic lysine residue in SdmA and YeiN is also conserved in OzmB (Figure S9). Therefore, OzmB catalysis may follow the same mechanism as for YeiN and SdmA, which starts with Schiff base formation ( 26 ) between the conserved lysine residue and the linearized R5P ( 15 ). ,, Subsequent nucleophilic attack by 16 followed by the collapse of the protein-tethered intermediate 27 would yield the C -glycosidation product 18 and regenerate the lysine residue (Figure B). Indeed, R5P ( 15 ) rather than PRPP ( 13 ) is required for the formation of 19 as indicated by the dimerized product 22 , and mutation of K168 of OzmB to alanine resulted in complete loss of its C -glycosidase activity (Figure S9).…”

“…In showdomycin biosynthesis, the nucleobase 2-aminopyrrole-4-carboxylate ( 11 ) is derived from l -glutamine ( 8 ) via the catalytic actions of SdmECDFG. Oxidation of the resulting C -glycosidation product 12 to showdomycin ( 5 ) can then occur nonenzymatically …”

“…Oxidation of the resulting C-glycosidation product 12 to showdomycin (5) can then occur nonenzymatically. 16 Despite the significant progress made in understanding the biosynthesis of several C-nucleosides, information regarding the biosynthesis of oxazinomycin (1) has been scarce. Oxazinomycin, which carries an unusual 1,3-oxazine aglycone, was first isolated from Streptomyces sp.…”

Identification of the Early Steps in Herbicidin Biosynthesis Reveals an Atypical Mechanism of C-Glycosylation

“… The biosynthesis of C -glycosylated natural products follows a similar paradigm requiring activation of a nucleophilic carbon on the acceptor fragment before addition to the electrophilic NDP–sugar donor. For example, aromatic aglycones often possess a phenolic hydroxyl group o rth o or para to the nucleophilic carbon facilitating electrophilic aromatic substitution of the aglycone by the sugar donor. , An analogous mechanism has also been noted to construct the C -glycosidic bond in the biosynthesis of pyrazofurin and several other C -nucleosides. − In contrast, the structures of herbicidins show little resemblance to the typical C -glycosylated natural products. Hence, ring B assembly in herbicidin biosynthesis may proceed along a unique course of C–C bond formation.…”