title: Characterization of LipL as a non-heme, Fe(II)-dependent α-ketoglutarate:UMP dioxygenase that generates uridine-5'-aldehyde during A-90289 biosynthesis.
abstract: 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.
authors.affiliation: From the Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536.
authors.forename: Zhaoyong
authors.initials: Z
authors.lastname: Yang
authors.affiliation: From the Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536.
authors.forename: Xiuling
authors.initials: X
authors.lastname: Chi
authors.affiliation: Biopharmaceutical Research Group I, Biopharmaceutical Technology Research Laboratories, Pharmaceutical Technology Division, Daiichi Sankyo Co., Ltd., 389-4, Aza-ohtsurugi, Shimokawa, Izumi-machi, Iwaki-shi, Fukushima 971-8183, Japan, and.
authors.forename: Masanori
authors.initials: M
authors.lastname: Funabashi
authors.affiliation: Biopharmaceutical Research Group I, Biopharmaceutical Technology Research Laboratories, Pharmaceutical Technology Division, Daiichi Sankyo Co., Ltd., 389-4, Aza-ohtsurugi, Shimokawa, Izumi-machi, Iwaki-shi, Fukushima 971-8183, Japan, and.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Baba
authors.affiliation: Biopharmaceutical Research Group I, Biopharmaceutical Technology Research Laboratories, Pharmaceutical Technology Division, Daiichi Sankyo Co., Ltd., 389-4, Aza-ohtsurugi, Shimokawa, Izumi-machi, Iwaki-shi, Fukushima 971-8183, Japan, and.
authors.forename: Koichi
authors.initials: K
authors.lastname: Nonaka
authors.affiliation: From the Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536.
authors.forename: Pallab
authors.initials: P
authors.lastname: Pahari
authors.affiliation: the Department of Plant and Soil Sciences, College of Agriculture, University of Kentucky, Lexington, Kentucky 40536.
authors.forename: Jason
authors.initials: J
authors.lastname: Unrine
authors.affiliation: From the Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536.
authors.forename: Jesse M
authors.initials: JM
authors.lastname: Jacobsen
authors.affiliation: From the Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536.
authors.forename: Gregory I
authors.initials: GI
authors.lastname: Elliott
authors.affiliation: From the Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Rohr
authors.affiliation: From the Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40536,. Electronic address: svanlanen@uky.edu.
authors.forename: Steven G
authors.initials: SG
authors.lastname: Van Lanen
mesh_terms: D001381:Azepines / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007501:Iron / Q000737:chemistry / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000737:chemistry / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005810:Multigene Family / Q000502:physiology; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology*; D014498:Uracil / Q000031:analogs & derivatives / Q000096:biosynthesis* / Q000737:chemistry
chemical_list: D001381:Azepines; D001426:Bacterial Proteins; D007656:Ketoglutaric Acids; D014498:Uracil; D007501:Iron; D006899:Mixed Function Oxygenases; D010100:Oxygen