Identification of the Glucosyltransferase That Converts Hydroxymethyluracil to Base J in the Trypanosomatid Genome

Bullard, Whitney; Rosa-Spiegler, Jessica Lopes da; Liu, Shuo; Wang, Yinsheng; Sabatini, Robert

doi:10.1074/jbc.m114.579821

Cited by 59 publications

(61 citation statements)

References 47 publications

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“…After submission of this manuscript, a study published elsewhere [21] confirmed the complete loss of J after knockout of Tb927.10.6900 in T. brucei and that reintroduction of the gene into Tb927.10.6900 -null T. brucei restored J synthesis. That study also showed that reduction of HmdUGT mRNA by RNAi causes reduced J and increased HOMedU levels and that the glucosyltransferase uses uridine diphosphoglucose to transfer glucose to HOMedU.…”

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confidence: 65%

Tb927.10.6900 encodes the glucosyltransferase involved in synthesis of base J in Trypanosoma brucei

Sekar

Merritt

Baugh

et al. 2014

Molecular and Biochemical Parasitology

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Base J is a DNA modification found in the genome of Trypanosoma brucei and all other kinetoplastids analyzed, where it replaces a small fraction of Ts, mainly in telomeric and chromosome-internal transcription initiation and termination regions. The synthesis of base J is a two-step process whereby a specific T is converted to HOMedU (hydroxymethyldeoxyuridine) and subsequently glucosylated to generate J. The thymidine hydroxylases (JPB1 and JBP2) that catalyze the first step have been characterized, but the identity of the glucosyltransferase catalyzing the second step has proven elusive. Recent bioinformatic analysis by Iyer et al. [Nucleic Acids Res. 2013; 41, 7635] suggested that Tb927.10.6900 encodes the glucosyltransferase (HmdUGT) responsible for converting HOMedU to J in T. brucei. We now present experimental evidence to validate this hypothesis; null mutants of Tb927.10.6900 are unable to synthesize base J. Orthologues from related kinetoplastids show only modest conservation, with several insertion sequences found in those from Leishmania and related genera.

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confidence: 65%

Tb927.10.6900 encodes the glucosyltransferase involved in synthesis of base J in Trypanosoma brucei

Sekar

Merritt

Baugh

et al. 2014

Molecular and Biochemical Parasitology

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“…1c). JBP1 and 2 are capable of oxidizing thymine to both 5-hydroxymethyluracil (hmU) and 5-formyluracil, though a glucosyltransferase normally diverts hmU to form base J as part of the trypanosome’s mechanism for immune evasion 26,27 .…”

Section: Discussionmentioning

confidence: 99%

Mutations along a TET2 active site scaffold stall oxidation at 5-hydroxymethylcytosine

et al. 2016

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Ten-eleven translocation (TET) enzymes catalyze stepwise oxidation of 5-methylcytosine (mC) to yield 5-hydroxymethylcytosine (hmC) and the rarer bases 5-formylcytosine (fC) and 5-carboxylcytosine (caC). Stepwise oxidation obscures how each individual base forms and functions in epigenetic regulation and prompts the question of whether TET enzymes primarily serve to generate hmC, or whether they are adapted to produce fC and caC as well. By mutating a single, conserved active site residue in human TET2, Thr1372, we uncovered enzyme variants that permit oxidation to hmC but largely eliminate fC/caC. Biochemical analyses, combined with molecular dynamics simulations, elucidated an active site scaffold that is required for WT stepwise oxidation and that, when perturbed, explains the mutants’ hmC-stalling phenotype. Our results suggest that the TET2 active site is shaped to enable higher-order oxidation and provide the first TET variants that could be used to probe the biological functions of hmC separately from fC and caC.

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“…In trypanosomatid parasites such as Leishmania and Trypanosoma , sugar-activating enzymes - including UGP - represent attractive drug targets since many vital processes within the parasites utilize UDP-Glc and UDP-Gal38394041. However, drugs targeting structurally conserved elements (i.e.…”

Section: Discussionmentioning

confidence: 99%

A Quaternary Mechanism Enables the Complex Biological Functions of Octameric Human UDP-glucose Pyrophosphorylase, a Key Enzyme in Cell Metabolism

Führing

Cramer

Schneider

et al. 2015

Sci Rep

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In mammals, UDP-glucose pyrophosphorylase (UGP) is the only enzyme capable of activating glucose-1-phosphate (Glc-1-P) to UDP-glucose (UDP-Glc), a metabolite located at the intersection of virtually all metabolic pathways in the mammalian cell. Despite the essential role of its product, the molecular basis of UGP function is poorly understood. Here we report the crystal structure of human UGP in complex with its product UDP-Glc. Beyond providing first insight into the active site architecture, we describe the substrate binding mode and intermolecular interactions in the octameric enzyme that are crucial to its activity. Importantly, the quaternary mechanism identified for human UGP in this study may be common for oligomeric sugar-activating nucleotidyltransferases. Elucidating such mechanisms is essential for understanding nucleotide sugar metabolism and opens the perspective for the development of drugs that specifically inhibit simpler organized nucleotidyltransferases in pathogens.

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Identification of the Glucosyltransferase That Converts Hydroxymethyluracil to Base J in the Trypanosomatid Genome

Cited by 59 publications

References 47 publications

Tb927.10.6900 encodes the glucosyltransferase involved in synthesis of base J in Trypanosoma brucei

Tb927.10.6900 encodes the glucosyltransferase involved in synthesis of base J in Trypanosoma brucei

Mutations along a TET2 active site scaffold stall oxidation at 5-hydroxymethylcytosine

A Quaternary Mechanism Enables the Complex Biological Functions of Octameric Human UDP-glucose Pyrophosphorylase, a Key Enzyme in Cell Metabolism

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