Abstract
To protect viral DNA against the host bacterial restriction system, bacterio-phages utilize a special modification system - hydroxymethylation - in which dCMP hydroxymethylase (dCH) converts dCMP to 5-hydroxymethyl-dCMP (5hm-dCMP) using N5,N10-methylenetetrahydrofolate as a cofactor. Despite shared similarity with thymidylate synthase (TS), dCH catalyzes hydroxylation through an exocyclic methylene intermediate during the last step, which is different from the hydride transfer that occurs with TS. In contrast to the extensively studied TS, the hydroxymethylation mechanism of a cytosine base is not well understood due to the lack of a ternary complex structure of dCH in the presence of both its substrate and cofactor. This paper reports the crystal structure of the ternary complex of dCH from bacteriophage T4 (T4dCH) with dCMP and tetrahydrofolate at 1.9 Å resolution. The authors found key residues of T4dCH for accommodating the cofactor without a C-terminal tail, an optimized network of ordered water molecules and a hydrophobic gating mechanism for cofactor regulation. In combination with biochemical data on structure-based mutants, key residues within T4dCH and a substrate water molecule for hydroxymethylation were identified. Based on these results, a complete enzyme mechanism of dCH and signature residues that can identify dCH enzymes within the TS family have been proposed. These findings provide a fundamental basis for understanding the pyrimidine modification system.
Highlights
Our high-resolution substrate–cofactor ternary complex structure of T4dCH in combination with mutational and biochemical data has provided an understanding of the mechanism of pyrimidine hydroxymethylation and insights into identifying dCMP hydroxymethylase (dCH) proteins from various species within the CMP hydroxymethylase (CH) and thymidylate synthase (TS) groups. The former includes how dCH recognizes THF – despite the lack of the C-terminal tail that is characteristic of TS – and which water molecules are critical for hyroxymethylation
We propose an updated mechanism of dCMP hydroxymethylation based on our ternary complex structure of T4dCH and previous studies of TS (Hardy et al, 1995; Graves et al, 1992; Butler et al, 1994; Graves & Hardy, 1994; Carreras & Santi, 1995; Kamb et al, 1992; Fritz et al, 2002; Finer-Moore et al, 2003; Perry et al, 1993; Agarwalla et al, 1997)
For a single methyl group donation, mTHF has to be converted into mTHF+; this step is of the protonated mTHF at the C5 position of intermediate 1
Summary
Hydroxymethylation of the pyrimidine bases of DNA is associated with diverse cellular processes including epigenetic gene regulation, embryonic development and host immune escape (Weigele & Raleigh, 2016; Gommers-Ampt & Borst, 1995; Pastor et al, 2013; Borst & Sabatini, 2008; Warren, 1980; Olinski et al, 2016). 5-Hydroxymethylcytosine (5hmC) was first identified in T-even phages (Wyatt & Cohen, 1952) and was found to protect the viral genome from the restrictionmodification system of the bacterial host (Labrie et al, 2010; Samson et al, 2013). Hydroxymethylation of the pyrimidine bases of DNA is associated with diverse cellular processes including epigenetic gene regulation, embryonic development and host immune escape (Weigele & Raleigh, 2016; Gommers-Ampt & Borst, 1995; Pastor et al, 2013; Borst & Sabatini, 2008; Warren, 1980; Olinski et al, 2016). To generate a modified cytosine in T4 bacteriophage, a multi-enzyme cascade is involved (Miller et al, 2003; Warren, 1980). 5hm-dCMP is phosphorylated to 5hm-dCTP by T4 deoxynucleoside monophosphate kinase from T4 bacteriophage and nucleoside diphosphate kinase from the host bacteria. T4 DNA polymerase incorporates the modified base at the position of the cytosine within
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