RESUMEN
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.
Asunto(s)
Azepinas/metabolismo , Proteínas Bacterianas/metabolismo , Oxigenasas de Función Mixta/metabolismo , Streptomyces/enzimología , Uracilo/biosíntesis , Azepinas/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Catálisis , Escherichia coli/enzimología , Escherichia coli/genética , Hierro/química , Hierro/metabolismo , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/genética , Familia de Multigenes/fisiología , Oxígeno/química , Oxígeno/metabolismo , Uracilo/análogos & derivados , Uracilo/químicaRESUMEN
The iodocyclization of gem-difluorohomoallenyl and gem-difluorohomopropargyl alcohols with I2 and ICl, respectively, produced the corresponding fluorinated iodofuran analogues in good yields. The iodo substituent in fluorinated 4-iodofurans was utilized as a synthetic handle to prepare multi-substituted 3-fluorofurans using a Suzuki cross-coupling reaction. The yields of both iodocyclization of gem-difluorohomopropargyl alcohol and subsequent Suzuki coupling were dramatically enhanced by microwave irradiation.