Evidence of the participation of remote residues in the catalytic activity of Co-type nitrile hydratase from Pseudomonas putida

Abstract

Active sites may be regarded as layers of residues, whereby the residues that interact directly with substrate also interact with residues in a second shell and these in turn interact with residues in a third shell. These residues in the second and third layers may have distinct roles in maintaining the essential chemical properties of the first-shell catalytic residues, particularly their spatial arrangement relative to the substrate binding pocket, and their electrostatic and dynamic properties. The extent to which these remote residues participate in catalysis and precisely how they affect first-shell residues remains unexplored. To improve our understanding of the roles of second- and third-shell residues in catalysis, we used THEMATICS to identify residues in the second and third shells of the Co-type nitrile hydratase from Pseudomonas putida (ppNHase) that may be important for catalysis. Five of these predicted residues, and three additional, conserved residues that were not predicted, have been conservatively mutated, and their effects have been studied both kinetically and structurally. The eight residues have no direct contact with the active site metal ion or bound substrate. These results demonstrate that three of the predicted second-shell residues (α-Asp164, β-Glu56, and β-His147) and one predicted third-shell residue (β-His71) have significant effects on the catalytic efficiency of the enzyme. One of the predicted residues (α-Glu168) and the three residues not predicted (α-Arg170, α-Tyr171, and β-Tyr215) do not have any significant effects on the catalytic efficiency of the enzyme.

Figure 1

Superposition of wild type ppNHase (PDB ID: 3QXE, this work) and wild type ptNHase (PDB ID: 1IRE (30)) structures. ppNHase and ptNHase α-subunits are in yellow and red and β-subunits are in green and blue, respectively. RMSD for the α subunits is 0.7 Å over 177 residues and 0.9 Å over 183 residues for the β subunits. The active site cobalt is enlarged and shown in pink. The two glycerol molecules associated with each dimer are rendered as ball and stick and shown in CPK coloring.

Figure 2

Active site of Co-type nitrile hydratase from Pseudomonas putida shown in stereo-view. Shown are first-, second- and third-shell residues. (purple sphere = cobalt, red spheres = water)

Figure 3

Comparison of the active site of wild-type ppNHase (A) and the active site of the second-shell R-Glu168Gln mutant ppNHase (PDB entry 3QZ5, this work) (B). In the mutant structure, residue 168 has flipped out of being within salt bridge distance of β-Arg52 and forms an H-bond with the backbone oxygen atom of β-Val169 (purple sphere for cobalt).

Figure 4

Comparison of the active site of wild type ppNHase (A) and the active site of the second-shell β-Glu56Gln mutant ppNHase (PDB ID: 3QYG, this work) (B). Wild type and mutant structures are essentially the same. (purple sphere = cobalt, red sphere = water).

Figure 5

Comparison of the active site of wild type ppNHase (A) and the active site of the third-shell β-His71Leu mutant ppNHase (PDB ID: 3QYH, this work) (B). Wild type and mutant structures are essentially the same, with a slight movement in one of the waters (*). (purple sphere = cobalt, red sphere = water)

Figure 6

Comparison of the active site of wild type ppNHase (A), (C) and the active site of the third-shell β-Tyr215Phe mutant ppNHase (PDB ID: 3QZ9, this work) (B), (D). Wild type and mutant structures are essentially the same in panels A and B. However, panels C and D show a lengthening in the salt bridge distance between α-Glu168 and β-Arg52, shown as red dotted lines. (purple sphere = cobalt).