Figure 1.
Analytical gel filtration chromatography of Thermus thermophilus MDH (TtMDH).
TtMDH (0.1 ml with 8.8 mg/ml) was subjected to Superdex 200 HR and the elution profile was compared with high-molecular-weight (HMW) standard proteins (thyroglobulin, 669 kDa; ferritin, 440 kDa; aldolase, 158 kDa; conalbumin, 75 kDa; and ovalbumin, 43 kDa, obtained from GE Healthcare). TtMDH showed a peak with estimated molecular mass 61 kDa. Inset, elution volumes of peaks of HMW standards (•) and peak TtMDH (▴) in the logarithmic scale.
Figure 2.
(A) Effect of pH on TtMDH enzymatic activity measured with 100 mM glycine-NaOH buffer of pH 8.5–11.0 under standard assay conditions. All TtMDH activity values were obtained from the means of independent experiments. Specific activity is expressed as units per mg protein; 1 unit is defined as 1 μmole NADH comsumed per minute. (B) Kinetic analysis of TtMDH. The initial enzymatic reaction velocity was measured at 0.15 mM NADH with oxaloacetate concentration from 2–0.015 mM in glycine-NaOH buffer (pH 10.0) at 37°C. The KM, Vmax and kcat values were calculated from Lineweaver-Burk plots (the double-reciprocal plot showed in the inset). (C) Temperature profile of TtMDH. The optimum temperature for enzyme activity was determined by measuring TtMDH activity at various temperatures from 37°C to 95°C under standard assay condition. All values of TtMDH activity were obtained from the means of three independent experiments. Specific activity is expressed as units per mg protein; 1 unit is defined as 1 μmole NADH consumed per minute.
Figure 3.
Spectroscopy features and thermal stability of TtMDH.
Far-UV circular dichroism (CD) spectra (195–260 nm) for TtMDH at different (A) pH values and (B) temperature. CD spectra were measured from 260–190 nm at different pH values (pH 8.5–11.0) at 25°C and at various temperatures from 25–95°C at pH 7.0.
Figure 4.
(A) Ribbon drawing shows 2 monomers related by a pseudo-two-fold. Each monomer consists of 12 helices and 11 strands. (B) Subunit A structure of TtMDH shown as a ribbon representation by 90° rotation around the Z-axis. Secondary structure elements are numbered sequentially as α1–α12 and β1–β11 for the common α-helices and β-strands, respectively. N and C refer to N- and C-terminal regions, respectively. (C) Sausage representation colored by B-factor. The color range bar represents the B-factor scale from 20 (blue) to 60 (orange) Å2. (D) Representative 2Fo-Fc electron-density maps contoured at 1σ.
Table 1.
Data collection and refinement statistics for apo and NAD-bound forms of Thermus thermophilus MDH (TtMDH).
Figure 5.
Root mean square fluctuation of Cα atoms of TtMDH after fitting the corresponding atom positions from molecular dynamics (MD) trajectory to the initial (X-ray) coordinates.
Results from different trajectories (different temperatures) are indicated with different-colored lines and compared with the trajectory at 298 K (black line).
Figure 6.
Ribbon diagrams of crystallographic and high-temperature–simulated TtMDH as well as cytoplasmic MDH (cMDH) in complex with NAD showing the structural similarity of the enzymes and the large conformational differences of the co-substrate NAD binding loop.
For TtMDH (cyan) the loop region is open, whereas for cMDH (pink), the loop adopts a compact conformation. For the high-temperature–simulated TtMDH, the loop is in a more closed position as for cMDH.
Table 2.
Thermodynamic parameters for NADH binding to TtMDH.