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

Abstract

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.

Keywords: antibiotic; biosynthesis; nonheme iron; nucleoside; oxygenase; translocase I.

Fig. 1

Function of LipL and Cpr19. (A) The oxidative dephosphorylation reaction catalyzed by LipL and Cpr19. (B) Structures of representative liponucleoside antibiotics containing a 5′-C-glycyluridine core (GlyU, highlighted in red). (C) Structures of representative monosaccharidyl nucleoside antibiotics containing a uridine-5′-carboxamide core (CarU, highlighted in blue).

Fig. 2

Enzymatic synthesis and analytical analysis of [1′,3′,4′,5′,5′-²H]UMP. (A) One-pot, total enzymatic synthesis of UMP from D-[U-²H]glucose. The primary metabolic pathway for highlighted enzymes are ^aglycolysis, ^bpentose phosphate, and ^cnucleotide metabolism. Abbreviations are G6PDH, glucose-6-phosphate dehydrogenase; PGDH, 6-phosphogluconate dehydrogenase; RPI, ribose-5-phosphate isomerase; PRPS, phosphoribosylpyrophosphate synthetase; UPRT, uracil phosphoribosyltransferase; PK, pyruvate kinase; GD, glutamate dehydrogenase; AK, adenylate kinase (myokinase); IP inorganic pyrophosphatase; PEP, phosphoenolpyruvate; and αKG, α-ketoglutarate. (B) HPLC analysis of the one-pot enzymatic synthesis of UMP. A₂₆₀, absorbance at 260 nm. (C) Negative mass spectra for the peak corresponding to UMP starting with unlabeled glucose (left) or D-[U-²H]glucose (right).

Fig. 3

Monitoring the loss of H atoms for the Cpr19-catalyzed reaction. The expected reaction catalyzed by Cpr19, and mass spectra (positive-ion mode) for the peak corresponding to U5ʹA starting with unlabeled UMP or D-[1ʹ,3ʹ,4ʹ,5ʹ,5ʹ-²H]UMP.

Fig. 4

Reaction of Cpr19 and LipL with the phosphonate derivative of UMP. (A) Proposed outcome for the reaction of 5′-deoxyuridine-5′-methylphosphonate (UMcP) with LipL and Cpr19. Amino acid ligands denote protein side chains used to bind the iron and co-product succinate. (B) Lineweaver-Burk plot of UMcP inhibition with respect to variable UMP with LipL. (C) HPLC analysis of the Cpr19-catalyzed reaction starting with UMcP in comparison to synthetic standards (5′S)-hydroxy-UMcP and (5′R)-hydroxy-UMcP and control reactions without enzyme. *denotes an unidentified contaminant of synthetic 5′-deoxyuridine-5′-methylphosphonate with a distinct UV-VIS profile compared to uracil-containing compounds; U denotes uracil. A₂₆₀, absorbance at 260 nm.

Fig. 5

Proposed mechanism following the αKG-dependent formation of the Fe(IV)-oxo species for UMP:α-ketoglutarate dioxygenases using Cpr19 as a model. ps, primary substrate.