Journal:JBIC:32

Analyzing the Catalytic Role of Active Site Residues in the Fe-Type Nitrile Hydratase from Comamonas testosteroni Ni1

Salette Martinez, Rui Wu, Karoline Krzywda, Veronika Opalka, Hei Chan, Dali Liu, and Richard C. Holz ^[1]

Molecular Tour
from Comamonas testosteroni Ni1 (CtNHase, PDB ID: 4fm4) in ball-and-stick form is shown. A strictly conserved active site arginine residue (αR157) and two histidine residues (αH80 and αH81) located near the active site of the CtNHase, . The wild-type is in dark magenta, αH80A/αH81A mutant is in light green, αH80W/αH81W is in orange, and the αR157A mutant is in light blue. . These mutant enzymes were examined for their ability to bind iron and hydrate acrylonitrile. For the αR157A mutant, the residual activity (k_cat = 10 ± 2 s−1) accounts for less than 1 % of the wild-type activity (k_cat = 1100 ± 30 s^-1) while the K_m value is nearly unchanged at 205 ± 10 mM. On the other hand, mutation of the active site pocket αH80 and αH81 residues to alanine resulted in enzymes with k_cat values of 220 ± 40 and 77 ± 13 s^-1, respectively, and K_m values of 187 ± 11 and 179 ± 18 mM. The double mutant (αH80A/αH81A) was also prepared and provided an enzyme with a k_cat value of 132 ± 3 s^-1 and a K_m value of 213 ± 61 mM. These data indicate that all three residues are catalytically important, but not essential. X-ray crystal structures of the αH80A/αH81A, αH80W/αH81W, and αR157A mutant CtNHase enzymes were solved to 2.0, 2.8, and 2.5 Å resolutions, respectively. In each mutant enzyme, hydrogen-bonding interactions crucial for the catalytic function of the αCys104-SOH ligand are disrupted. Disruption of these hydrogen bonding interactions likely alters the nucleophilicity of the sulfenic acid oxygen and the Lewis acidity of the active site Fe(III) ion. , the regions with pronounced difference between the wild-type and the mutant enzymes are indicated. .