The structure, mechanism and specificity of amidases

The structure, mechanism and specificity of amidases

Nitrilases are enzymes that convert nitriles into the corresponding carboxylic acid and ammonia. This activity makes the enzyme class particularly useful and it has been used in a variety of contexts in living systems. The mechanism of the nitrilases is not understood. It is widely stated that a cysteine, a lysine and a glutamate lie in the active site and that the cysteine forms a covalent thioimidate intermediate with the nitrile substrate. Our published work has demonstrated that they form homo-oligomeric spirals and that spiral formation is essential for activity. They were the first enzyme class in which these properties were discovered. We have visualized the spiral of the cyanide dihydratase at near atomic resolution by electron microscopy and are now close to understanding some of the properties of this enzyme.  High resolution crystallographic studies by ourselves and others have  suggested a mechanism for the closely related amidases that involves the use of not one, but two glutamates in addition to the cysteine and lysine. It has also cast doubt on the plausibility of the existence of the thioimidate in the case of the nitrilases. Our approach utilizes structural bioinformatics, biochemical and biophysical analysis, quantum mechanical molecular modeling, site directed mutagenesis, cross-linking, mass spectroscopy, X-ray crystallography and three-dimensional electron microscopy to probe the structure, mechanism and specificity of the nitrilases and related enzymes. It is our intention to develop a novel, experimentally validated model during the course of this grant that will provide a sound basis for the design of these enzymes for the production of multiple synthetic products.