The Fe-type Nitrile Hydratase from Comamonas testosteroni Ni1 Does Not Require an Activator Accessory Protein for Expression in Escherichia coli

We report herein the functional expression of an Fe-type nitrile hydratase (NHase) without the co-expression of an activator protein or the Escherichia coli chaperone proteins GroES/EL. Soluble protein was obtained when the α- and β-subunit genes of the Fe-type NHase Comamonas testosteroni Ni1 ( Ct NHase) were synthesized with optimized E. coli codon usage and co-expressed. As a control, the Fe-type NHase from Rhodococcus equi TG328–2 ( Re NHase) was expressed with ( Re NHase ^+Act ) and without ( Re NHase ^−Act ) its activator protein, establishing that expression of a fully functional, metallated Re NHase enzyme requires the co-expression of its activator protein, similar to all other Fe-type NHase enzymes reported to date, whereas the Ct NHase does not. The X-ray crystal structure of Ct NHase was determined to 2.4 Å resolution revealing an αβ heterodimer, similar to other Fe-type NHase enzymes, except for two important differences. First, two His residues reside in the Ct NHase active site that are not observed in other Fe-type NHase enzymes and second, the active site Fe(III) ion resides at the bottom of a wide solvent exposed channel. The solvent exposed active site, along with the two active site histidine residues, are hypothesized to play a role in iron incorporation in the absence of an activator protein.