Structure of the putative dihydroorotate dehydrogenase from Streptococcus mutans

Abstract

Streptococcus mutans is one of the pathogenic species involved in dental caries, especially in the initiation and development stages. Here, the crystal structure of SMU.595, a putative dihydroorotate dehydrogenase (DHOD) from S. mutans, is reported at 2.4 Å resolution. DHOD is a flavin mononucleotide-containing enzyme which catalyzes the oxidation of L-dihydroorotate to orotate, which is the fourth step and the only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. The reductive lysine-methylation procedure was applied in order to improve the diffraction qualities of the crystals. Analysis of the S. mutans DHOD crystal structure shows that this enzyme is a class 1A DHOD and also suggests potential sites that could be exploited for the design of highly specific inhibitors using the structure-based chemotherapeutic design technique.

Figure 1

Multiple alignment of amino-acid sequences of DHODs. The alignment was produced for S. mutans SMU.595, L. lactis DHOD (LlDHOD), T. brucei DHOD (TbDHOD) and Homo sapiens DHOD (HuDHOD). Strictly conserved residues are boxed in red and similar residues are represented by red letters. The alignment was performed using the programs ClustalX (Larkin et al., 2007 ▶) and ESPript v.2.2 (Gouet et al., 1999 ▶).

Figure 2

Structure of SMU.595. (a) The overall crystal structure of SMU.595 is shown as a ribbon diagram with secondary-structure elements labelled in cyan (helices), violet (strands) and pink (loops). The FMN cofactor is represented as green sticks. (b) The crystal structure of the SMU.595 tetramer is shown as a ribbon diagram. The FMN cofactor is represented as orange sticks. (c) The dimer structure viewed from above the twofold axis. The β6–α6 loop and helices α6 and α7, which participate in dimer-interface interactions, are shown in pink and deep blue, respectively. The FMN cofactor is represented as orange sticks. (d) Left, structural overlay of the homodimers of SMU.595 (wheat yellow), TbDHOD (light blue) and LlDHOD (light pink). Right, the secondary structures (including the β2–α2, β4–αA and β6–α6 loops) surrounding the FMN cofactor. FMNs are shown in green, blue and pink. The amino-acid residues that are well conserved in the three sequences and that participate in interactions with FMN cofactors are shown as orange, blue and pink sticks, respectively. The methylated lysines are shown in yellow.

Figure 3

Crystals of S. mutans DHOD as grown after lysine methylation.

Figure 4

Dimethylated lysines (orange) are shown in stick representation. The homodimer of SMU.595 is shown as ribbon models in cyan and green.

Figure 5

Structural overlay of residues interacting with FMN and orotate. The residues, FMN and orotate are shown as stick models. SMU.595 is shown in pale green and TbDHOD is shown in light pink. SMU.595 residues are labelled in parentheseses. The hydrogen bonds between FMN (or orotate) and side chains of residues are shown as dotted lines. (a) The FMNs of SMU.595 and TbDHOD are shown in green and pink, respectively. (b) The orotate of TbDHOD is shown in orange. Residues 123–146 are shown as a ribbon model. Figs. 2, 3, 4 and 5 were generated using PyMOL (DeLano, 2002 ▶).