The catalytic mechanism of the nitrilase enzyme from Synechocystis PCC6803 (NIT6803).

The proposed experiment aims to investigate the catalytic mechanism of the nitrilase Synechocystis PCC6803 (NIT6803) enzyme, using cryo-electron microscopy (Cryo-EM). NIT6803 is of particular interest as it can convert a nitrile to the corresponding acid and ammonia (nitrilase activity) or to an amide (nitrile hydratase activity). This feature is termed catalytic promiscuity. Extensive studies have demonstrated how the acid/amide ratio can be altered by site-directed mutagenesis. Furthermore, there is a crystal structure of most of the enzyme (PDB:3wuy) (Zhang et al, 2014), which is an excellent start for our studies.

We wish to visualise nitrile and amide substrates in the active, wild-type, nitrilase enzyme – with a view to visualizing a trapped intermediate (Aguirre-Sampieri et al, 2024) as well as in an inactive variant of the enzyme in which the active site cysteine is substituted by an alanine (C169A).