Fe(II)-Dependent, Uridine-5′-Monophosphate α-Ketoglutarate Dioxygenases in the Synthesis of 5′-Modified Nucleosides

Abstract

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-containing 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 non-haem, Fe(II)- and a-ketoglutarate-dependent dioxygenases. The enzyme LipL from the biosynthetic pathway for A-90289, a 5′-C-glycyluridine-containing nucleoside, was functionally characterized as 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.

Unusual α-Carbon Hydroxylation of Proline Promotes Active-Site Maturation

“…Therefore, we used, as described in the Experimental Section, SEC online with ICP-MS for metal detection in Bc 1960 (Figure S3). The method has been used successful in various studies for the detection of Fe and other metals in similar proteins. , The enzyme-bound metals were detected at around 445 s elution time, whereas unbound metals were detected at around 540 s elution time. The analysis revealed the presence of enzyme-bound Zn 2+ in Bc 1960, which can be removed by EDTA.…”

Study the effect of antioxidants and ROS levels on antibacterial activity of some of modified pyrimidine derivatives

“…These microorganisms are able to thrive in places that would normally be considered unfriendly, such as the intestines of mammals, thanks to this capacity. LPS has previously been demonstrated to operate as a physical or chemical barrier that prevents ROS produced outside of cells from interacting with a critical target, together with membrane or cytoplasmic additions [20] . As a result, certain lines that are unable to deliver a significant portion of the LPS have shown more susceptibility to exogenous ROS than lines that are still able to do so.…”

Evidence that oxidative dephosphorylation by the nonheme Fe(II), α‐ketoglutarate:UMP oxygenase occurs by stereospecific hydroxylation

“…Cloned DNA was sequenced to confirm PCR fidelity. Protein production, purification, and assessment were performed using routine conditions as previously described . His 6 ‐tagged proteins were utilized without further modifications.…”

“…The cloning of lipL and cpr19 and the production of purified proteins were previously reported . Routine reactions with LipL consisted of 50 m m Tris‐HCl (pH 7.5), 1 m m UMP, 1.25 m m αKG, 200 μ m ascorbate, 100 μ m FeCl 2 , and 100 n m LipL at 30 °C.…”

“…LipL and Cpr19 are relatively new members of the superfamily that catalyze the O 2 , Fe(II), and αKG‐dependent conversion of UMP to uridine‐5′‐aldehyde (U5′A; Fig. A) , the latter of which is a biosynthetic precursor for several highly modified uridine‐containing antibiotics including the 5′‐ C ‐glycyluridine‐containing liponucleosides represented by A‐90289 A, caprazamycins, and liposidomycins (Fig. B) and the uridine‐5′‐carboxamide‐containing monosaccharidyl nucleosides represented by A‐102395, A‐503083 B, and capuramycin (Fig.…”