The Nitrile-Forming Enzyme 7-Cyano-7-Deazaguanine Synthase from Geobacillus kaustophilus: A Reverse Nitrilase?

7-Cyano-7-deazaguanine synthase (E.C. 6.3.4.20) is an enzyme that catalyzes the formation of a nitrile from a carboxylic acid and ammonia at the expense of ATP. The protein from G.kaustophilus was heterologously expressed, and its biochemical characteristics were explored by using a newly developed HPLC-MS based assay, (PNMR)-P-31, and a fluorescence-based thermal-shift assay. The protein showed the expected high thermostability, had a pH optimum at pH9.5, and an apparent temperature optimum at 60 degrees C. We observed strict substrate specificity of QueC for the natural substrate 7-carboxy-7-deazaguanine, and determined AMP and pyrophosphate as co-products of preQ(0).