Nitrilases features

ExPASy (Expert Protein Analysis System, www.expasy.ch) or the National Centre for Biotechnology Information (NCBI, www.ncbi.nlm.gov) websites. Both websites provide bioinformatics tools, links to sequence databases and extensive bibliographic resources. As an example of the wealth of information available on individual enzymes, at the time of writing a search based on nitrilase in the Entrez protein section of NCBI will recover more than 10000 references to nitrilase enzyme amino acid sequences. These can be rapidly screened online by organism, and the individual entries will have links to amino acid and gene sequence, relevant literature and information on protein features (such as conserved domains). 

The a-chloroacetamide group has features that are beneficial for undirected ABPP. Its small size does not bias binding elements towards a specific class of enzyme, and it possesses reactivity towards a broad variety of nucleophilic amino acid residues. A library of a-chloroacetamide-based probes were synthesized by Cravatt s group. The binding element in these probes was a dipeptide that was varied with small, large, hydrophobic, and charged side chains, and a biotin or rhodamine tag was appended as a reporter tag. Upon screening of eukaryotic proteomes with this library, many enzymes previously unaddressed by directed ABPP probes were uncovered. These included fatty acid synthase, hydro-xypyruvate reductase, malic enzyme, and the nitrilase superfamily [163, 164]. In contrast to the sulfonate esters, a-chloroacetamides react preferentially with cysteine residues in the proteome. 

For the adiponitrile, the nitrilase activity was independant of pH and for ammonium 5-cyanovalerate, the higher its ionic feature the worse the nitrilase activity. Therefore, the chemo-selectivity of the nitrilase between adiponitrile and ammonium 5-cyanovalerate was 64 % at pH 4 and 97 % at pH 7. 

Further, E. coli cells expressing the cyanide hydratases genes from A. niger and P, chrysogenum were found to exhibit activities for both HCN and some nitrile compounds, preferably 2-cyanopyridine (1-3.6% relative activity compared to HCN) [6]. Dual nitrilase/cyanide hydratase activities were also described for the enzymes in Fusarium oxysporum and Fusarium lateritium [36, 37]. It is possible that this dual activity is a general feature of cyanide hydratases but has largely gone unnoticed. 

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