A cytosine modification mechanism revealed by the structure of a ternary complex of deoxycytidylate hydroxymethylase from bacteriophage T4 with its cofactor and substrate

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.

Keywords: bacteriophage T4; cytosine modification; deoxycytidylate hydroxy­methylase; thymidylate synthase.

Figure 1

Ternary complex structure of T4dCH with dCMP and THF. (a) Ribbon diagram showing the ternary complex structure of dimeric T4dCH viewed along the crystallographic twofold symmetry axis. N- and C-terminal ends are colored red and labeled as Nt and Ct, respectively, Secondary structural elements are colored green (α-helix and 3₁₀-helix), purple (β-strand) and cyan (loops). The bound dCMP (yellow) and THF (orange) are drawn using the ball and stick model. The bound iodide is also shown as a gray ball. (b) Electron-density maps showing the bound dCMP (yellow) and THF (orange) in the active site of T4dCH. The 2F _o − F _c map (blue) is contoured at 1.0σ. (c) Ball-and-stick model of dCMP (yellow) and THF (orange) in the deep binding pocket, which is represented as an electrostatic surface. The positively and negatively charged areas are blue and red, respectively. Each subpart of THF is labeled with black dotted circles. (d) B factor diagram of the apo (green), binary (cyan) and ternary complex (blue) forms. The thickness of the tube reflects the value of the B factor, i.e. the higher the B factor, the thicker the tube.

Figure 2

Structure and ligand geometry of THF in the active site of T4dCH. (a) Chemical formula of THF. The subparts are named as pterin, PABA and the glutamate tail. The methyl group is linked to the N5 and N10 positions in mTHF. (b) Stereo diagram showing the overall interaction of THF in the binding site of T4dCH. THF and dCMP are drawn as stick models (orange). Oxygen and nitrogen atoms are colored red and blue, respectively. The THF-recognizing residues are drawn as thin stick models (purple) with the cartoon representation of T4dCH (off-white). Water molecules are depicted as red balls. (c) Interaction of the pterin core in the active site. Hydrogen bonds are represented by dash lines. (d) Recognition of the PABA ring and the hydrogen-bonding interaction at the N10 position with Glu60. (e) Hydrophilic interaction of the glutamate tail with charged residues on the surface of T4dCH. Ionic and hydrogen interactions are drawn by dashed lines.

Figure 3

Structural comparison between the ternary complexes of T4dCH and EcTS. (a) Structural superposition of T4dCH (white) and EcTS (cyan). (b) Differences in the THF ligand geometries in the active sites of T4dCH and EcTS. This orientation was obtained from the optimal superposition of T4dCH and EcTS protein as shown in (a). Carbon atoms of THF bound to T4dCH and EcTS are colored white and cyan, respectively. (c) Schematic plot of the interaction between T4dCH and THF. (d) Schematic plot of the interaction between EcTS and THF. The dotted lines and numbers indicate the hydrogen bonds and their distances in Å, respectively. The starburst indicates the hydrophobic interactions. Water molecules are labeled as ‘w’ for clarity. Nitrogen and oxygen atoms are colored blue and red, respectively, for all panels.

Figure 4

Reaction mechanism of T4dCH. (a) Schematic diagram showing the proposed mechanism of cofactor activation. For methyl-group transfer, the mTHF is converted to protonated mTHF⁺ by a water (Wat10) that had been activated by Glu60. The electron flows are indicated by arrows. (b) Schematic diagram showing the proposed mechanism of hydroxy­methylation by T4dCH. The intermediates 1, 2, 3 and 4 are shown. The hydrogen-bonding interactions are represented as dotted lines.