Divergent Biosynthesis of C-Nucleoside Minimycin and Indigoidine in Bacteria

Abstract

SummaryMinimycin (MIN) is a C-nucleoside antibiotic structurally related to pseudouridine, and indigoidine is a naturally occurring blue pigment produced by diverse bacteria. Although MIN and indigoidine have been known for decades, the logic underlying the divergent biosynthesis of these interesting molecules has been obscure. Here, we report the identification of a minimal 5-gene cluster (min) essential for MIN biosynthesis. We demonstrated that a non-ribosomal peptide synthetase (MinA) governs “the switch” for the divergent biosynthesis of MIN and the cryptic indigoidine. We also demonstrated that MinCN (the N-terminal phosphatase domain of MinC), MinD (uracil phosphoribosyltransferase), and MinT (transporter) function together as the safeguard enzymes, which collaboratively constitute an unusual self-resistance system. Finally, we provided evidence that MinD, utilizing an unprecedented substrate-competition strategy for self-resistance of the producer cell, maintains competition advantage over the active molecule MIN-5′-monophosphate by increasing the UMP pool in vivo. These findings greatly expand our knowledge regarding natural product biosynthesis.

Reverse C-glycosidase reaction provides C-nucleotide building blocks of xenobiotic nucleic acids

“…Other C-nucleosides, such as showdomycin, formycin, pseudouridimycin and minimycin ( Supplementary Fig. 1), constitute an emerging class of microbial natural products that exhibit unusual structures and diverse biological activities, including antibiotic efficacy [15][16][17][18][19][20][21] .…”

“…A synthesis that exploits the high stereo-and regiocontrol of enzymatic reactions for installing the C-glycosidic linkage would be highly desirable. Natural enzymes of C-glycoside biosynthesis, such as tRNA pseudouridine synthase 27,28,51,52 and C-glycosynthase 15,[18][19][20][21]53,54 have complex substrate requirements, leading to perceived limitations on their applicability. Therefore, unlike N-nucleosides for which N-nucleoside phosphorylases present excellent tools of glycoside synthesis [55][56][57][58][59][60] , biocatalytic methods are lacking for C-nucleoside synthetic chemistry.…”

Investigation of Indigoidine Synthetase Reveals a Conserved Active-Site Base Residue of Nonribosomal Peptide Synthetase Oxidases

“…11,12 These NRPSs having a 4'-phosphopantetheine (Ppant) modification on the PCP domain (holo form) convert L-glutamine to 5-amino-3H-pyridine-2,6-dione (1), a putative product that is readily oxidized to a 3,3'-bipyridyl natural product, indigoidine (2), which as a bright blue color. [12][13][14][15] Indigoidine synthetase genes have been engineered and introduced into living systems to construct a cross-kingdom reporter system 16 , generate blue rose 17 , and produce 2 as a promising water-insoluble dye 18,19 . Interestingly, indigoidine synthetase genes are widely distributed in bacteria, including Actinobacteria and Proteobacteria ( Figure S1), and frequently colocalized with genes encoding enzymes that glycosylate C-5 of 1, leading to the water-soluble blue pigment indochrome 20,21 , and the C-nucleoside antibiotic, minimycin 13 (Figure 1b).…”

“…[12][13][14][15] Indigoidine synthetase genes have been engineered and introduced into living systems to construct a cross-kingdom reporter system 16 , generate blue rose 17 , and produce 2 as a promising water-insoluble dye 18,19 . Interestingly, indigoidine synthetase genes are widely distributed in bacteria, including Actinobacteria and Proteobacteria ( Figure S1), and frequently colocalized with genes encoding enzymes that glycosylate C-5 of 1, leading to the water-soluble blue pigment indochrome 20,21 , and the C-nucleoside antibiotic, minimycin 13 (Figure 1b). As such, indigoidine synthetases are involved in biosynthesis of a broad range of bacterial secondary metabolites.…”

“…11 The conversion of 3 to 1 by indigoidine synthetase is a black box (Figure 1b) : 3 can be either first cyclized by the TE domain to α-amino glutarimide (4) and then oxidized by the Ox domain to form 1 (path a), 11,12 or 3 can be first oxidized to dehydroglutaminyl-S-PCP (5) and then cyclized to 1 (path b). 13 Here, by monitoring the acylation changes on the PCP domain, we resolved the biosynthesis by indigoidine synthetase, which reveals the catalytic mechanism of NRPS Ox domains.…”