doi:10.1016/j.jmb.2007.12.064 
Copyright © 2007 Elsevier Ltd All rights reserved.
Crystal Structures of Streptococcus mutans 2′-Deoxycytidylate Deaminase and Its Complex with Substrate Analog and Allosteric Regulator dCTP·Mg2 +
Hai-Feng Hou1, 2, Yu-He Liang3, Lan-Fen Li3, Xiao-Dong Su3 and Yu-Hui Dong1, 
, 
1Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China
2Graduate School, Chinese Academy of Sciences, Beijing 100049, China
3National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Science, Peking University, Beijing 100871, China
Received 29 July 2007;
revised 13 December 2007;
accepted 23 December 2007.
Edited by M. Guss.
Available online 5 January 2008.
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Abstract
2′-Deoxycytidylate deaminase [or deoxycytidine-5′-monophosphate (dCMP) deaminase, dCD] catalyzes the deamination of dCMP to deoxyuridine-5′-monophosphate to provide the main nucleotide substrate for thymidylate synthase, which is important in DNA synthesis. The activity of this homohexameric enzyme is allosterically regulated by deoxycytidine-5′-triphosphate (dCTP) as an activator and by deoxythymidine-5′-triphosphate as an inhibitor. In this article, we report the crystal structures of dCMP deaminase from Streptococcus mutans and its complex with dCTP and an intermediate analog at resolutions of 3.0 and 1.66 Å. The protein forms a hexamer composed of subunits adopting a three-layer α/β/α sandwich fold. The positive allosteric regulator dCTP mainly binds at the interface between two monomers in a molar ratio of 1:1 and rearranges the neighboring interaction networks. Structural comparisons and sequence alignments revealed that dCMP deaminase from Streptococcus mutans belongs to the cytidine deaminase superfamily, wherein the proteins exhibit a similar catalytic mechanism. In addition to the two conserved motifs involved in the binding of Zn2 +, a new conserved motif, (G43YNG46), related to the binding of dCTP was also identified. N-terminal Arg4, a key residue located between two monomers, binds strongly to the γ phosphate group of dCTP. The regulation signal was transmitted by Arg4 from the allosteric site to the active site via modifications in the interactions at the interface where the substrate-binding pocket was involved and the relocations of Arg26, His65, Tyr120, and Arg121 to envelope the active site in order to stabilize substrate binding in the complex. Based on the enzyme–regulator complex structure observed in this study, we propose an allosteric mechanism for dCD regulation.
Keywords: crystal structure; 2′-deoxycytidylate deaminase; enzyme complex; allosteric regulation; deoxycytidine-5′-triphosphate
Abbreviations: dCMP, deoxycytidine-5′-monophosphate; dCD, 2′-deoxycytidylate deaminase; dCTP, deoxycytidine-5′-triphosphate; dTTP, deoxythymidine-5′-triphosphate; dUMP, deoxyuridine-5′-monophosphate; dUTP, deoxyuridine-5′-triphosphate; T4, T4 bacteriophage; CDA, cytidine deaminase; WT, wild type; Sm-dCD, Streptococcus mutans dCD; DHOMP, 3,4-dihydrouridine-5′-monophosphate; PdR, pyrimidine-2-one deoxyriboside; MAD, multiwavelength anomalous dispersion; PDB, Protein Data Bank; RMSD, root-mean-square deviation
Fig. 1. Schematic diagram of the substrate analog (PdR) and its hydrate derivative (DHOMP).
Fig. 2. Kinetic assay for Sm-dCD. One unit of activity refers to the deamination of 1 μM dCMP per minute under the condition of the assay. (a) Activation of Sm-dCD by dCTP at increasing concentrations of dCMP. (b) Regulation of Sm-dCD by different ratios of dCTP to dTTP at increasing concentrations of dCMP.
Fig. 3. Stereoview of the complex structure of Sm-dCD with PdR and dCTP. (a) Overall structure of Sm-dCD monomer with the substrate analog and allosteric regulator bound. The substrate analog PdR and allosteric regulator dCTP are shown in stick style. The catalytically essential zinc ion is denoted as Zn2. (b) Hexamer generated by crystallographic symmetry. Molecules A, B, and C, which are referenced in the text, are shown in magenta, cyan, and yellow, respectively.
 |
Fig. 4. (a) Structure-based sequence alignment of the dCD family. Eight aligned sequences are from six bacteria: Sm, T4, B. subtilis (Bs), Magnetospirillum magnetotacticum (Mm), Mycoplasma pulmonis (Mp), Spiroplasma citri (Sc), and the eukaryotes Homo sapiens (Hs) and Caenorhabditis elegans (Ce). The secondary structure elements of Sm-dCD protein are placed above the sequences, with α-helices depicted as cylinders, β-strands as arrows, and three helices as coils. Every tenth residue is labeled for the Sm sequence. Residues interacting with zinc ions, DHOMP, and dCTP are annotated as indicated in the legend. Completely conserved residues among these sequences are shaded in dark green, and at least 60% conserved residues are shown in light green. Numbers before sequences indicate starting residues of each block; those after sequences, ending residues. Numbers in parentheses indicate the length of omitted residues. The alignment was prepared by CLUSTALW.15 (b) Stereoview of the structural superposition of Sm-dCD (2HVW; red), T4-dCD (1VQ2; bright orange), yeast cytosine deaminase (1UAQ; yellow), and CDA from B. subtilis (1JTK; purple).
Fig. 5. The Fobs − Fcalc omitted maps of substrate and allosteric regulator-binding sites; the density maps were drawn at 4σ. The substrate analog PdR, the allosteric regulator dCTP, and the residues surrounding them are shown in stick style. (a) Substrate-binding site. The substrate analog PdR was omitted from map calculating; the zinc ions are represented with a gray sphere; (b) Allosteric regulator-binding site. The dCTP, Mg2 +, and three ligated water molecules were omitted from map calculating; the Mg2 + and ligated water molecules are represented with green and red spheres, respectively. The labels before the residues are identical with chains A–C in Fig. 1.
Fig. 6. Schematic presentation of dCTP–protein interaction produced by the program LIGPLOT.21 Highly conserved residues for binding dCTP: Trp7, Tyr44, and Asn75 are emphasized by a cyan background.
Fig. 7. The superposition of the free-state enzyme structure (blue) and the complex structure (red) on the residues coordinated with catalytic zinc ion. It is shown that the substrate-binding pocket of the free-state enzyme served as an “open state,” while the complex served as a “closed state.” The conformation of the labeled residues changed significantly to make the substrate analog binding more stable in the complex structure.
Fig. 8. Quaternary structural changes caused by dCTP binding depicted in a dimer. The free-state enzyme structure (blue) and the complex structure (red) were superposed on Cα atoms of one monomer; the γ phosphate of the dCTP contacts tightly with N-terminal Arg4 of the neighboring monomer and thus pulls the α1 helix to form a tightly packing form in interface AB.
Table 1.
Requirement of Sm-dCD for metal ion
The reaction conditions were those used in the assay with metal ion additions as indicated.
Table 2.
Crystallographic data and structure refinement statistics
a Values for the data in the highest-resolution shell are shown in parentheses.
b Rfree = ∑
Test||
Fobs| − |
Fcalc||/∑
Test|
Fobs|, where “Test” is a test set of about 10% and 5% of the total reflections randomly chosen and set aside prior to refinement for the free-state enzyme and the complex, respectively.
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