Mevalonate 5-diphosphate mediates ATP binding to the mevalonate diphosphate decarboxylase from the bacterial pathogen Enterococcus faecalis

Abstract

The mevalonate pathway produces isopentenyl diphosphate (IPP), a building block for polyisoprenoid synthesis, and is a crucial pathway for growth of the human bacterial pathogen Enterococcus faecalis The final enzyme in this pathway, mevalonate diphosphate decarboxylase (MDD), acts on mevalonate diphosphate (MVAPP) to produce IPP while consuming ATP. This essential enzyme has been suggested as a therapeutic target for the treatment of drug-resistant bacterial infections. Here, we report functional and structural studies on the mevalonate diphosphate decarboxylase from E. faecalis (MDD_EF). The MDD_EF crystal structure in complex with ATP (MDD_EF-ATP) revealed that the phosphate-binding loop (amino acids 97-105) is not involved in ATP binding and that the phosphate tail of ATP in this structure is in an outward-facing position pointing away from the active site. This suggested that binding of MDD_EF to MVAPP is necessary to guide ATP into a catalytically favorable position. Enzymology experiments show that the MDD_EF performs a sequential ordered bi-substrate reaction with MVAPP as the first substrate, consistent with the isothermal titration calorimetry (ITC) experiments. On the basis of ITC results, we propose that this initial prerequisite binding of MVAPP enhances ATP binding. In summary, our findings reveal a substrate-induced substrate-binding event that occurs during the MDD_EF-catalyzed reaction. The disengagement of the phosphate-binding loop concomitant with the alternative ATP-binding configuration may provide the structural basis for antimicrobial design against these pathogenic enterococci.

Keywords: Enterococcus; crystal structure; decarboxylase; drug resistance; enzyme kinetics; enzyme mechanism; isothermal titration calorimetry (ITC); mevalonate pathway.

Figure 1.

Decarboxylation reaction of MVAPP to IPP by MDD. MVAPP and MgATP are the two substrates of MDD enzymes. MgADP, CO₂, phosphate, and IPP are the products.

Figure 2.

Protein purification and structure determination of MDD_EF. A, purification profile of MDD_EF from a nickel affinity column. The detailed procedures of protein purification are discussed under “Experimental procedures.” mAU, milli-absorbance unit. B, SDS-polyacrylamide gel for the MDD_EF protein purification. M, protein marker; Pellet, sample in the pellet after French press; Flow through, an aliquot of flow-through containing proteins that cannot be trapped on the Ni-NTA column; fractions from 12 to 18 were combined. C, SDS-polyacrylamide gel for the TEV-protease treatment profile of MDD_EF. The lane 0 hr shows the His-tagged MDD_EF without TEV treatment. The TEV treatment process is done in 2 days (Day1 and Day2; 4 and *16 hrs indicate the TEV treatment at different time courses; *, dialysis against buffer without DTT and EDTA; RT, treatment at room temperature; Flow through, an aliquot containing MDD_EF proteins without a His tag which cannot be trapped on a Ni-NTA column; Strip, an aliquot after 1 column volume of strip buffer (containing 50 mm EDTA) flowing through the column. D, superposition of MDD_EF and the MDD_EF–ATP complex. Two structures of MDD_EF (gray) and MDD_EF–ATP (pink) were Cα-aligned (r.m.s.d. = 0.174 Å). The position of the phosphate-binding loop (97–104) can be determined in MDD_EF–ATP but not MDD_EF. The missing residues (dashed line) in MDD_EF range from 183 to 192; in MDD_EF–ATP, residues from 184 to 189 are missing.

Figure 3.

Superimposition of complex structures of MDD_EF–ATP and MDD from S. epidermidis bound with FMVAPP and ATPγS (MDD_S.E.–FMVAPP–ATPγS). A, ribbon model of the crystal structure of MDD_EF–ATP is shown in pink with the bound ATP molecule as a stick model. B, ATP molecule is surrounded by the SA-omit map (mF_o − DF_c at a contour of 3σ, cropped at 5 Å from ATP). Residues (Gln-68, Ser-93, Asn-95, and Ser-105) that form hydrogen bonds with ATP are shown as stick models. The hydrogen bonding partners (Gln-68–O and ATP–O2′, 3.3 Å; Ser-93–Oγ and ATP–N₆, 3.0 Å; Asn-95–Oδ and ATP–N₆, 2.9 Å; Asn-95–Nδ and ATP–N₇, 3.3 Å; Ser-105–N and ATP–Oα, 2.9 Å) are connected by dashed lines. C, overlay of the models of MDD_SE–FMVAPP–ATPγS (PDB code 4DPT, green) and MDD_EF–ATP (pink) (r.m.s.d. = 0.66 Å) is depicted with the phosphate-binding loops emphasized. D, ligands from two structures are shown as stick models (FMVAPP and ATPγS from MDD_SE–FMVAPP–ATPγS; ATP from MDD_EF–ATP). The arrows in black indicate the distinct orientations of the phosphate tails of ATPγS and ATP from these two structures.

Figure 4.

Kinetic analysis of MDD_EF. A, set of kinetic data at varying concentrations of MVAPP and several fixed concentrations of MgATP ((●) 50, (○) 100, (▾) 200, (▿) 400, (■) 600, (□) 800, and (♦) 1000 μm) is shown as a Michaelis-Menten plot. B, same data set from A is shown as a Lineweaver-Burk plot. C, data set at varying concentrations of MgATP and several fixed concentrations of MVAPP ((●) 10, (○) 15, (▾) 25, (▿) 50, (■) 100, (□)150, (♦) 200, and (♢) 300 μm) is shown as a Michaelis-Menten plot. D, same data set from C is shown as a Lineweaver-Burk plot. Kinetic data are represented by Equation 1 and used for a sequential bi-substrate model. Each data point represents independent triplicate results, and the error bar for each point indicates standard deviations.

Figure 5.

Inhibitory assays of MDD_EF. A, different fixed concentrations of ATPγS ((●) 0, (○) 100, (▾) 200, or (▿) 400 μm) were added to the reactions at a fixed concentration of MVAPP (40 μm) and varying concentrations of MgATP (100, 150, 200, or 400 μm). The set of inhibitory data is shown as a Michaelis-Menten plot. B, same set of data from A is shown as a Lineweaver-Burk plot. C, different fixed concentrations of ATPγS ((●) 0, (○) 100, (▾) 200, and (▿) 400 μm) were added to the reactions at a fixed concentration of MgATP (200 μm) and varying concentrations of MVAPP (10, 20, 40, and 80 μm). The set of inhibitory data is shown as a Michaelis-Menten plot. D, same set of data from C is shown as a Lineweaver-Burk plot. A and B can be analyzed as a competitive inhibition model; C and D can be analyzed as an uncompetitive inhibition model. The means and the standard deviation of each data point were obtained from a triplicate test. Each data point represents independent triplicate results, and the error bar for each point indicates standard deviations.

Figure 6.

Original titration curves from ITC experiments with MDD_EF. A, MDD_EF (100 μm) titrated with MVAPP (2 mm). B, MDD_EF titrated with ATP (3 mm). C, MDD_EF titrated with ATPγS (3 mm). D, MDD_EF pre-incubated with MVAPP (1 mm) and then titrated with ATPγS (2 mm). The protein concentration is adjusted to 100 μm, and all the protein and titrants are dissolved in the buffer containing 100 mm HEPES, pH 7, 100 mm KCl, and 10 mm MgCl₂.

Figure 7.

Induced substrate-binding mechanism of MDD proteins. Left panel (green), apo-form of MDD in which two pockets for substrate binding are empty; middle panel (orange), the MVAPP-bound MDD in which the binding of MVAPP (shown in a red triangle) triggers conformational changes of the enzyme and reshapes the ATP-binding pocket, which allows the binding of ATP (shown in a blue arrow) to its catalytically favored position; right panel (purple), two-substrate-bound MDD. Step 1, the binding of MVAPP; step 2, the binding of ATP; step 3, enzyme catalysis and product release. Products in different shapes are shown in gray.