Nitrilases are enzymes that convert nitriles to the corresponding carboxylic acids and ammonia or to the corresponding amides. They have substantial sequence homology to the cysteine-dependent amidases, which convert amides to the corresponding carboxylic acids and ammonia, but the nitrilases do not convert amides. Unlike the amidases, for which there are many crystal structures, nitrilases form spiral assemblies that have not been crystallized. The atomic resolution visualization of the nitrilases that I have used in my study has been made possible by cryo-electron microscopy. Whereas previous attempts at defining the nitrilase mechanism have been unsatisfactory because they have not utilized structural information, my study provides detailed explanation of most aspects of the reaction and identify the active site residues that differentiate nitrilases from amidases.
Sequence and structural homology with other superfamily members has identified a conserved active site grouping comprising a cysteine, two glutamates, and a lysine. Furthermore, mass spectroscopy has provided evidence for the existence of two covalent intermediates: a thioimidate and a thioester. The covalent linkage of the substrate to the active site cysteine restricts the possible geometries of the substrate in the active site and enables reliable quantum mechanical modelling of the reaction pathway.
However, current generation modelling tools do not allow for the facile modelling of such covalently modified residues. To address this shortcoming, we have developed a procedure that enables the geometric optimization of covalent intermediates using the ISOLDE package incorporated in UCSF ChimeraX. In addition to coordinates that conventional docking programs rely on, ISOLDE employs electron density to minimize protein structures.
In this study, guided the new structural information, we used molecular dynamics simulations and natural bond orbital analysis to describe a unified mechanism operating in the catalysis propagation for both nitrilases and amidases. This approach relies on the arming/disarming of the glutamate/lysine catalytic dyad to drive the hydrolysis. Our analysis shows in detail how geometric considerations determine the progression of the reaction at each stage.
