Publications

• Fe(II)-dependent, uridine-5'-monophosphate alpha-ketoglutarate dioxygenases in the synthesis of 5'-modified nucleosides.

  Yang Z., Unrine J., Nonaka K., Van Lanen S.G.

  Several nucleoside antibiotics from various actinomycetes contain a high-carbon sugar nucleoside that is putatively derived via C-5'-modification of the canonical nucleoside. Two prominent examples are the 5'-C-carbamoyluridine- and 5'-C-glycyluridine-containing nucleosides, both families of which ... >> More

  Several nucleoside antibiotics from various actinomycetes contain a high-carbon sugar nucleoside that is putatively derived via C-5'-modification of the canonical nucleoside. Two prominent examples are the 5'-C-carbamoyluridine- and 5'-C-glycyluridine-containing nucleosides, both families of which were discovered using screens aimed at finding inhibitors of bacterial translocase I involved in the assembly of the bacterial peptidoglycan cell wall. A shared open reading frame was identified whose gene product is similar to enzymes of the nonheme, Fe(II)-, and α-ketoglutarate-dependent dioxygenases. The enzyme LipL from the biosynthetic pathway for A-90289, a 5'-C-glycyluridine-containing nucleoside, was functionally characterized as an UMP:α-ketoglutarate dioxygenase, providing the enzymatic imperative for the generation of a nucleoside-5'-aldehdye that serves as a downstream substrate for an aldol or aldol-type reaction leading to the high-carbon sugar scaffold. The functional assignment of LipL and the homologous enzymes-including bioinformatic analysis, iron detection and quantification, and assay development for biochemical characterization-is presented herein. << Less

  Methods Enzymol. 516:153-168(2012) [PubMed] [EuropePMC]

• 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.

• Evidence that oxidative dephosphorylation by the nonheme Fe(II), alpha-ketoglutarate:UMP oxygenase occurs by stereospecific hydroxylation.

  Goswami A., Liu X., Cai W., Wyche T.P., Bugni T.S., Meurillon M., Peyrottes S., Perigaud C., Nonaka K., Rohr J., Van Lanen S.G.

  LipL and Cpr19 are nonheme, mononuclear Fe(II)-dependent, α-ketoglutarate (αKG):UMP oxygenases that catalyze the formation of CO₂ , succinate, phosphate, and uridine-5'-aldehyde, the last of which is a biosynthetic precursor for several nucleoside antibiotics that inhibit bacterial tran ... >> More

  LipL and Cpr19 are nonheme, mononuclear Fe(II)-dependent, α-ketoglutarate (αKG):UMP oxygenases that catalyze the formation of CO₂ , succinate, phosphate, and uridine-5'-aldehyde, the last of which is a biosynthetic precursor for several nucleoside antibiotics that inhibit bacterial translocase I (MraY). To better understand the chemistry underlying this unusual oxidative dephosphorylation and establish a mechanistic framework for LipL and Cpr19, we report herein the synthesis of two biochemical probes-[1',3',4',5',5'-² H]UMP and the phosphonate derivative of UMP-and their activity with both enzymes. The results are consistent with a reaction coordinate that proceeds through the loss of one ² H atom of [1',3',4',5',5'-² H]UMP and stereospecific hydroxylation geminal to the phosphoester to form a cryptic intermediate, (5'R)-5'-hydroxy-UMP. Thus, these enzyme catalysts can additionally be assigned as UMP hydroxylase-phospholyases. << Less

  FEBS Lett 591:468-478(2017) [PubMed] [EuropePMC]

• Characterization of LipL as a non-heme, Fe(II)-dependent alpha-ketoglutarate:UMP dioxygenase that generates uridine-5'-aldehyde during A-90289 biosynthesis.

  Yang Z., Chi X., Funabashi M., Baba S., Nonaka K., Pahari P., Unrine J., Jacobsen J.M., Elliott G.I., Rohr J., Van Lanen S.G.

  Fe(II)- and α-ketoglutarate (α-KG)-dependent dioxygenases are a large and diverse superfamily of mononuclear, non-heme enzymes that perform a variety of oxidative transformations typically coupling oxidative decarboxylation of α-KG with hydroxylation of a prime substrate. The biosynthetic gene clu ... >> More

  Fe(II)- and α-ketoglutarate (α-KG)-dependent dioxygenases are a large and diverse superfamily of mononuclear, non-heme enzymes that perform a variety of oxidative transformations typically coupling oxidative decarboxylation of α-KG with hydroxylation of a prime substrate. The biosynthetic gene clusters for several nucleoside antibiotics that contain a modified uridine component, including the lipopeptidyl nucleoside A-90289 from Streptomyces sp. SANK 60405, have recently been reported, revealing a shared open reading frame with sequence similarity to proteins annotated as α-KG:taurine dioxygenases (TauD), a well characterized member of this dioxygenase superfamily. We now provide in vitro data to support the functional assignment of LipL, the putative TauD enzyme from the A-90289 gene cluster, as a non-heme, Fe(II)-dependent α-KG:UMP dioxygenase that produces uridine-5'-aldehyde to initiate the biosynthesis of the modified uridine component of A-90289. The activity of LipL is shown to be dependent on Fe(II), α-KG, and O(2), stimulated by ascorbic acid, and inhibited by several divalent metals. In the absence of the prime substrate UMP, LipL is able to catalyze oxidative decarboxylation of α-KG, although at a significantly reduced rate. The steady-state kinetic parameters using optimized conditions were determined to be K(m)(α-KG) = 7.5 μM, K(m)(UMP) = 14 μM, and k(cat) ≈ 80 min(-1). The discovery of this new activity not only sets the stage to explore the mechanism of LipL and related dioxygenases further but also has critical implications for delineating the biosynthetic pathway of several related nucleoside antibiotics. << Less

  J. Biol. Chem. 286:7885-7892(2011) [PubMed] [EuropePMC]