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.

FIGURE 1.

Structures of representative nucleoside antibiotics that inhibit bacterial translocase I. Highlighted in blue is the unusual C-C bond resulting in the classification as high carbon nucleosides.

FIGURE 2.

Identification of the LipL product. HPLC analysis of the LipL reaction using authentic UMP without α-KG (I), a 15-min reaction containing all of the necessary components (II), the 15-min reaction mixture spiked with synthetic uridine-5′-aldehyde (III), and synthetic uridine-5′-aldehdye (IV) are shown. A₂₆₀, absorbance at 260 nm.

FIGURE 3.

Activity optimization and kinetic analysis of LipL using the malachite green binding assay. A, optimal activity of LipL with respect to varied FeCl₂ in reactions containing 100 μm ascorbate and 40 nm LipL. B, optimal activity of LipL with respect to varied ascorbate in reactions containing of 20 μm FeCl₂ and 30 nm LipL. C, single-substrate kinetic analysis using variable α-KG (2 μm–3 mm), near saturating UMP (2 mm) and 34 nm LipL. D, single-substrate kinetic analysis using variable UMP (2 μm–1 mm), near saturating α-KG (1 mm) and 34 nm LipL. Data represent the average of minimally three independent replicates obtained using end point assays under initial velocity conditions; S.E. was <15% in all cases.

FIGURE 4.

Proposed biochemical pathway leading to several high carbon nucleosides inhibiting bacterial translocase I. Highlighted in bold is the enzyme characterized in this study; enzymes with likely identical function as LipL include ORF7 from the A-500359 gene cluster, CapA from the A-503083 gene cluster, LpmM from the liposidomycin gene cluster, Cprz15 from the caprazamycin gene cluster, and PacF/PacX from the pacidamycin gene cluster. AMT, aminotransferase; GTase, glycosyltransferase.