Four types of reaction carried out by nitrilase superfamily members. (a) The nitrilase reaction is performed by branch 1 enzymes. In plants, the substrate is indole-3-acetonitrile and the product is indole-3-acetic acid. (b) The amidase reaction is the most frequently observed activity in the superfamily. Branch 2-4 enzymes are amidases and nitrilase-related domains of branch 7 and 8 enzymes are proposed to be amidases specific for glutamine. (c) The carbamylase reaction is a special case of the amidase reaction, carried out by branch 5 and 6 enzymes. (d) Branch 9 N-acyltransferases perform the amidase reaction in reverse, transferring a fatty acid from phospholipid (not shown) to a polypeptide amino terminus. The polypeptide acceptor usually contains an amino-terminal diacylglyceride-modified cysteine (not shown). All nitrilase-related reactions are thought to proceed through acylenzyme intermediates.

The nitrilase superfamily catalytic triad motifs. Consensus sequences flanking the invariant catalytic triad residues, glutamate, lysine and cysteine, were obtained by doing multiple sequence alignments within each branch [54]. Red letters on a yellow background indicate the same residue is conserved in all branches. Dark blue letters on light blue background indicate the residue is conserved in nine or more branches. Green background shows positions in which the conserved amino acid is found in six to eight of the branches. Upper case letters indicate 90% or greater consensus levels within a branch, whereas lower case are 50% or greater. Residue numbers are shown for the prototypical members of branches 1 to 12 and for the first listed member of branch 13.

Domain structures for 13 branches of the nitrilase superfamily. Additional domains are found in members of seven branches. Parentheses denote domains found in only some members of the branch. In branch 4, vanins and biotinidases have carboxy-terminal domains unique to these two sub-branches and one vanin has additional full and partial nitrilase-related domains. The NAD synthetase domains of eukaryotes are always fused with a nitrilase-related domain. In contrast, only some prokaryotic NAD synthetases are fusion proteins with a nitrilase-related domain. This led to the prediction that branch 7 and 8 nitrilase domains are glutamine amidotransferases for the associated NAD synthetases (see text for details). Apolipoprotein N-acyltransferases (branch 9) always have a hydrophobic amino-terminal domain and one member is fused to an apparent dolichol phosphate mannose synthetase, which underscores the proposed function of branch 9 enzymes in post-translational modification. Nit proteins, branch 10, are found as fused Rosetta Stone proteins with Fhit in invertebrates and are coordinately expressed with separate Fhit proteins in mammals. Branch 12 enzymes are predicted to have protein substrates as they are fused to a homolog of an amino-terminal acetyltransferase.

Nitrilase-related active site of C. elegans NitFhit. Stereoview of sidechains of invariant and highly conserved residues from the crystal structure of NitFhit [12].