Figure 1. TbMDD and SaMDD expression complements the temperature-sensitive phenotype of MN19-34ts yeast. (a) MN19-34ts yeast grown at 28 °C transformed with: 1, no plasmid; 2, pPA295 containing the ScMDD ORF; 3, pPA295 containing the TbMDD ORF. (b) Replica plate of (a), grown at 36 °C. 1, No colony of untransformed yeast grows; 2, but MN19-34ts yeast transformed with copies of ScMDD; or 3, TbMDD can grow at 36 °C. (c) MN19-34ts yeast grown at 36 °C (control). (d) MN19-34ts yeast transformed with a copy of SaMDD can grow at 36 °C.

Figure 2. The architecture of MDD. Left: A TbMDD monomer. α-Helices are colored purple, β-strands are colored cyan and the connecting loops are colored gray. The P-loop is arrowed, and the ordered sulfate in the active site is depicted as yellow (S atom) and red (O atoms) spheres. Right: The SaMDD monomer in a similar orientation.

Figure 3. An alignment of the amino acid sequences of TbMDD, SaMDD and ScMDD. The secondary structure of TbMDD is shown where, α is α-helix; β is β-strand, η is 3₁₀ helix, and TT is β-turn. Residues are numbered according to the TbMDD sequence. Red boxes mark residues strictly conserved in all three sequences. Cyan stars indicate residues proposed to be involved in substrate binding. Yellow diamonds indicate residues that are likely involved in ATP binding.

Figure 4. The asymmetric units of (a) TbMDD and (b) SaMDD. The color scheme is the same as that used in Figure 2. A black line marks the interface between molecules in the asymmetric units of the structures.

Figure 5. Stereoviews of the TbMDD active site. (a) Amino acids and the sulfate ion are shown as sticks colored according to atom type: C, wheat; N, blue; O, red; and S yellow. Water molecules are depicted as blue spheres. The omit difference density map (F_o–F_c, α_calc, where F_o and F_c are the observed and calculated structure factors, respectively, and α_calc is the calculated phases without the scattering contributions of the sulfate ion) is shown at the 3σ level as purple chicken wire. (b) The proposed ternary complex of TbMDD:ATP:MDP. The TbMDD structure (as in (a)), with ATP (anti-conformation) and MDP modeled in the active site. The terminal phosphate group of MDP is positioned where an ordered sulfate ion is observed. ATP and MDP are shown as transparent van der Waals spheres: C, green; N, blue; O, red; and P, yellow.

Figure 6. The position of selected residues in the MDD active site. The enzyme is depicted as a semi-transparent van der Waals surface colored according to charge with blue representing basic areas, red representing acidic and grey representing neutral or hydrophobic. The substrates ATP and MDP together with Lys18, Tyr19, Trp20, Arg77, Ser146, Arg149 and Asp293 are shown as sticks colored according to atom type.

Figure 7. A plausible mechanism for MDD and the chemical structures of compounds used to investigate specificity. (a) A schematic of the proposed reaction mechanism for TbMDD together with residues implicated in creating the active site and/or binding MDP. (b) The structure of 3-hydroxy-3-ethyl 5-diphosphopentanoate, a non-physiological substrate. (c) Structure of 2-fluoromevalonate 5-diphosphate, a time-dependent irreversible inhibitor of MDD.

Crystallographic statistics for TbMDD and SaMDD

Values in parentheses are for the highest resolution bin: 1.97–1.8 Å for TbMDD, and 2.42–2.30 Å for SaMDD.