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 green, αH80W/αH81W is in orange, and the αR157A mutant is in deep sky blue. . These mutant enzymes were examined for their ability to bind iron and hydrate acrylonitrile. For the αR157A mutant, the residual activity (kcat = 10 ± 2 s−1) accounts for less than 1 % of the wild-type activity (kcat = 1100 ± 30 s-1) while the Km 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 kcat values of 220 ± 40 and 77 ± 13 s-1, respectively, and Km values of 187 ± 11 and 179 ± 18 mM. The double mutant (αH80A/αH81A) was also prepared and provided an enzyme with a kcat value of 132 ± 3 s-1 and a Km value of 213 ± 61 mM. These data indicate that all three residues are catalytically important, but not essential. X-ray crystal structures of the , , and mutant CtNHase enzymes were solved to 2.0, 2.8, and 2.5 Å resolutions, respectively. In each mutant enzyme, . 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. .
PDB references: Mutant R157A of Fe-Type Nitrile Hydratase from Comamonas testosteroni Ni1, 4zgd; Double Mutant H80W/H81W of Fe-Type Nitrile Hydratase from Comamonas testosteroni Ni1, 4zge; Double Mutant H80A/H81A of Fe-Type Nitrile Hydratase from Comamonas testosteroni Ni1, 4zgj.
