Structure prediction, molecular dynamics simulation and docking studies of D-specific dehalogenase from Rhizobium sp. RC1

Abstract

Currently, there is no three-dimensional structure of D-specific dehalogenase (DehD) in the protein database. We modeled DehD using ab initio technique, performed molecular dynamics (MD) simulation and docking of D-2-chloropropionate (D-2CP), D-2-bromopropionate (D-2BP), monochloroacetate (MCA), monobromoacetate (MBA), 2,2-dichloropropionate (2,2-DCP), d,l-2,3-dichloropropionate (d,l-2,3-DCP), and 3-chloropropionate (3-CP) into the DehD active site. The sequences of DehD and D-2-haloacid dehalogenase (HadD) from Pseudomonas putida AJ1 have 15% sequence similarity. The model had 80% of the amino acid residues in the most favored region when compared to the crystal structure of DehI from Pseudomonas putida PP3. Docking analysis revealed that Arg107, Arg134 and Tyr135 interacted with D-2CP, and Glu20 activated the water molecule for hydrolytic dehalogenation. Single residue substitutions at 25-30 °C showed that polar residues of DehD were stable when substituted with nonpolar residues and showed a decrease in activity within the same temperature range. The molecular dynamics simulation of DehD and its variants showed that in R134A variant, Arg107 interacted with D-2CP, while in Y135A, Gln221 and Arg231 interacted with D-2CP. It is our emphatic belief that the new model will be useful for the rational design of DehDs with enhanced potentials.

Figure 1

Secondary structural elements (SSEs) of DehD. Letters h stands for helix, c for coil and e for strand.

Figure 2

Secondary structure of DehD. N-terminal is shown in blue and C-terminal in red.

Figure 3

DehD Structure showing the accessible surface colored according to electrostatic potential (ESP). (A) Shows graphical rotation of front side view of the accessible surface. (B) Shows graphical rotation of back side view of the accessible surface.

Figure 4

Total energy for the molecular dynamics simulation of DehD.

Figure 5

RMSD of DehD stable conformation observed after the extension of a simulation run from 5000 ps to 20,000 ps. Extension from 5000 ps to 20,000 ps shown in black color.

Figure 6

RMS fluctuations of amino acid residues of DehD compared to its variants. (i) black is wild-type DehD. (ii) blue is the R134A mutant. (iii) green is the Y135A mutant. (iv) red is the double substitution R134A/Y135A mutant.

Figure 7

The radius of gyration of DehD compared with its variants. (i) black is DehD wild-type (DehD WT). (ii) red is the R134A mutant. (iii) green is the Y135A mutant. (iv) blue is the double substitution R134A/Y135A mutant.

Figure 8

Ramachandran plot of the 3D structure of DehD evaluated by the PROCHECK program. (A) Model before refinement. (B) Model after MD simulation. The most favored and favored region are indicated with red and yellow colors, respectively. The generously allowed region is shown in pale yellow and the disallowed region is in white color.

Figure 9

The active site of DehD. The Asp and Glu binding sites are colored blue; the Phe and Tyr binding sites are colored yellow; the Val, Ile and Leu binding sites are colored purple; the other residues (Ala, Gly, Met, Trp, Pro, Ser, Thr, Cys, Asn, Gln, Lys, Arg, and His are colored green).

Figure 10

The structure of DehD showing active site amino acid residues.

Figure 11

The active site of DehD superimposed onto that of DehI from Pseudomonas putida strain PP3. The catalytic residues of DehI from Pseudomonas putida strain PP3 are shown with blue sticks, and the catalytic residues of DehD with red sticks.

Figure 12

The interacting residues of DehD protein with D-2CP after 2000 ps MD simulation showing hydrogen bonding. Hydrogen bond distances are in Å.

Figure 13

The interacting residues of DehD protein with D-2CP after 5000 ps MD simulation showing hydrogen bonding. Hydrogen bond distances are in Å.

Figure 14

The interacting residues of DehD variant R134A with D-2CP. Hydrogen bond distances are in Å.

Figure 15

The interacting residues of DehD variant Y135A with D-2CP. Hydrogen bond distances are in Å.

Figure 16

The interacting residues of DehD variant Y107A with D-2CP. Hydrogen bond distances are in Å.

Figure 17

The interacting residues of the double substitution variant of DehD (R134A and Y135A) with D-2CP. Hydrogen bond distances are in Å.

Figure 18

The interaction of D-2BP with catalytic amino acid residues of DehD enzyme. Hydrogen bond distances are in Å.

Figure 19

The interacting residues of DehD with (A) MCA (B) MBA. Hydrogen bond distances are in Å.

Figure 20

The interacting residues of DehD with D,L-2,3-DCP. Hydrogen bond distances are in Å.

Figure 21

The interacting residues of DehD with 2,2-DCP. Hydrogen bond distances are in Å.

Figure 22

A proposed reaction mechanism for DehD, showing Glu20 activating the water molecule to attack the carbon–halogen bond and displace chloride without the formation of an ester intermediate.