Cloning and expression of the phenazine-1-carboxamide hydrolysis gene pzcH and the identification of the key amino acids necessary for its activity

Phenazine-1-carboxamide (PCN), a phenazine derivative, can cause toxicity risks to non target organisms. In this study, the Gram-positive bacteria Rhodococcus equi WH99 was found to have the ability to degrade PCN. PzcH, a novel amidase belonging to amidase signature (AS) family, responsible for hydrolyzing PCN to PCA was identified from strain WH99. PzcH shared no similarity with amidase PcnH which can also hydrolyze PCN and belong to the isochorismatase superfamily from Gram-negative bacteria Sphingomonas histidinilytica DS-9. PzcH also showed low similarity (˂ 39%) with other reported amidases. The optimal catalysis temperature and pH of PzcH was 30 °C and 9.0, respectively. The Km and kcat values of PzcH for PCN were 43.52 ± 4.82 μM and 17.028 ± 0.57 s-1, respectively. The molecular docking and point mutation experiment demonstrated that catalytic triad Lys80-Ser155-Ser179 are essential for PzcH to hydrolyze PCN. Strain WH99 can degrade PCN and PCA to reduce their toxicity against the sensitive organisms. This study enhances our understanding of the molecular mechanism of PCN degradation, presents the first report on the key amino acids in PzcH from the Gram-positive bacteria and provides an effective strain in the bioremediation PCN and PCA contaminated environments.