Publications

• The biosynthesis of capuramycin-type antibiotics: identification of the A-102395 biosynthetic gene cluster, mechanism of self-resistance, and formation of uridine-5'-carboxamide.

  Cai W., Goswami A., Yang Z., Liu X., Green K.D., Barnard-Britson S., Baba S., Funabashi M., Nonaka K., Sunkara M., Morris A.J., Spork A.P., Ducho C., Garneau-Tsodikova S., Thorson J.S., Van Lanen S.G.

  A-500359s, A-503083s, and A-102395 are capuramycin-type nucleoside antibiotics that were discovered using a screen to identify inhibitors of bacterial translocase I, an essential enzyme in peptidoglycan cell wall biosynthesis. Like the parent capuramycin, A-500359s and A-503083s consist of three s ... >> More

  A-500359s, A-503083s, and A-102395 are capuramycin-type nucleoside antibiotics that were discovered using a screen to identify inhibitors of bacterial translocase I, an essential enzyme in peptidoglycan cell wall biosynthesis. Like the parent capuramycin, A-500359s and A-503083s consist of three structural components: a uridine-5'-carboxamide (CarU), a rare unsaturated hexuronic acid, and an aminocaprolactam, the last of which is substituted by an unusual arylamine-containing polyamide in A-102395. The biosynthetic gene clusters for A-500359s and A-503083s have been reported, and two genes encoding a putative non-heme Fe(II)-dependent α-ketoglutarate:UMP dioxygenase and an l-Thr:uridine-5'-aldehyde transaldolase were uncovered, suggesting that C-C bond formation during assembly of the high carbon (C6) sugar backbone of CarU proceeds from the precursors UMP and l-Thr to form 5'-C-glycyluridine (C7) as a biosynthetic intermediate. Here, isotopic enrichment studies with the producer of A-503083s were used to indeed establish l-Thr as the direct source of the carboxamide of CarU. With this knowledge, the A-102395 gene cluster was subsequently cloned and characterized. A genetic system in the A-102395-producing strain was developed, permitting the inactivation of several genes, including those encoding the dioxygenase (cpr19) and transaldolase (cpr25), which abolished the production of A-102395, thus confirming their role in biosynthesis. Heterologous production of recombinant Cpr19 and CapK, the transaldolase homolog involved in A-503083 biosynthesis, confirmed their expected function. Finally, a phosphotransferase (Cpr17) conferring self-resistance was functionally characterized. The results provide the opportunity to use comparative genomics along with in vivo and in vitro approaches to probe the biosynthetic mechanism of these intriguing structures. << Less

  J. Biol. Chem. 290:13710-13724(2015) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Pyridoxal-5'-phosphate as an oxygenase cofactor: Discovery of a carboxamide-forming, alpha-amino acid monooxygenase-decarboxylase.

  Huang Y., Liu X., Cui Z., Wiegmann D., Niro G., Ducho C., Song Y., Yang Z., Van Lanen S.G.

  Capuramycins are antimycobacterial antibiotics that consist of a modified nucleoside named uridine-5'-carboxamide (CarU). Previous biochemical studies have revealed that CarU is derived from UMP, which is first converted to uridine-5'-aldehyde in a reaction catalyzed by the dioxygenase CapA and su ... >> More

  Capuramycins are antimycobacterial antibiotics that consist of a modified nucleoside named uridine-5'-carboxamide (CarU). Previous biochemical studies have revealed that CarU is derived from UMP, which is first converted to uridine-5'-aldehyde in a reaction catalyzed by the dioxygenase CapA and subsequently to 5'-C-glycyluridine (GlyU), an unusual β-hydroxy-α-amino acid, in a reaction catalyzed by the pyridoxal-5'-phosphate (PLP)-dependent transaldolase CapH. The remaining steps that are necessary to furnish CarU include decarboxylation, O atom insertion, and oxidation. We demonstrate that Cap15, which has sequence similarity to proteins annotated as bacterial, PLP-dependent l-seryl-tRNA(Sec) selenium transferases, is the sole catalyst responsible for complete conversion of GlyU to CarU. Using a complementary panel of in vitro assays, Cap15 is shown to be dependent upon substrates O₂ and (5'<i>S</i>,6'<i>R</i>)-GlyU, the latter of which was unexpected given that (5'<i>S</i>,6'<i>S</i>)-GlyU is the isomeric product of the transaldolase CapH. The two products of Cap15 are identified as the carboxamide-containing CarU and CO₂ While known enzymes that catalyze this type of chemistry, namely α-amino acid 2-monooxygenase, utilize flavin adenine dinucleotide as the redox cofactor, Cap15 remarkably requires only PLP. Furthermore, Cap15 does not produce hydrogen peroxide and is shown to directly incorporate a single O atom from O₂ into the product CarU and thus is an authentic PLP-dependent monooxygenase. In addition to these unusual discoveries, Cap15 activity is revealed to be dependent upon the inclusion of phosphate. The biochemical characteristics along with initiatory mechanistic studies of Cap15 are reported, which has allowed us to assign Cap15 as a PLP-dependent (5'<i>S</i>,6'<i>R</i>)-GlyU:O₂ monooxygenase-decarboxylase. << Less

  Proc. Natl. Acad. Sci. U.S.A. 115:974-979(2018) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Amalgamation of nucleosides and amino acids in antibiotic biosynthesis: discovery of an L-threonine:uridine-5'-aldehyde transaldolase.

  Barnard-Britson S., Chi X., Nonaka K., Spork A.P., Tibrewal N., Goswami A., Pahari P., Ducho C., Rohr J., Van Lanen S.G.

  The lipopeptidyl nucleoside antibiotics represented by A-90289, caprazamycin, and muraymycin are structurally highlighted by a nucleoside core that contains a nonproteinogenic β-hydroxy-α-amino acid named 5'-C-glycyluridine (GlyU). Bioinformatic analysis of the biosynthetic gene clusters revealed ... >> More

  The lipopeptidyl nucleoside antibiotics represented by A-90289, caprazamycin, and muraymycin are structurally highlighted by a nucleoside core that contains a nonproteinogenic β-hydroxy-α-amino acid named 5'-C-glycyluridine (GlyU). Bioinformatic analysis of the biosynthetic gene clusters revealed a shared open reading frame encoding a protein with sequence similarity to serine hydroxymethyltransferases, resulting in the proposal that this shared enzyme catalyzes an aldol-type condensation with glycine and uridine-5'-aldehyde to furnish GlyU. Using LipK involved in A-90289 biosynthesis as a model, we now functionally assign and characterize the enzyme responsible for the C-C bond-forming event during GlyU biosynthesis as an l-threonine:uridine-5'-aldehyde transaldolase. Biochemical analysis revealed this transformation is dependent upon pyridoxal-5'-phosphate, the enzyme has no activity with alternative amino acids, such as glycine or serine, as aldol donors, and acetaldehyde is a coproduct. Structural characterization of the enzyme product is consistent with stereochemical assignment as the threo diastereomer (5'S,6'S)-GlyU. Thus this enzyme orchestrates C-C bond breaking and formation with concomitant installation of two stereocenters to make a new l-α-amino acid with a nucleoside side chain. << Less

  J. Am. Chem. Soc. 134:18514-18517(2012) [PubMed] [EuropePMC]