An Amidase Gene, ipaH, Is Responsible for the Initial Step in the Iprodione Degradation Pathway of Paenarthrobacter sp. Strain YJN-5.

Iprodione [3-(3,5-dichlorophenyl) N-isopropyl-2,4-dioxoimidazolidine-1-carboxamide] is a highly effective broad-spectrum dicarboxamide fungicide. Several bacteria with iprodione-degrading capabilities have been reported; however, the enzymes and genes involved in this process have not been characterized. In this study, an iprodione-degrading strain, Paenarthrobacter sp. strain YJN-5, was isolated and characterized. Strain YJN-5 degraded iprodione through the typical pathway, with hydrolysis of its N-1 amide bond to N-(3,5-dichlorophenyl)-2,4-dioxoimidazolidine as the initial step. The ipaH gene, encoding a novel amidase responsible for this step, was cloned from strain YJN-5 by the shotgun method. IpaH shares the highest similarity (40%) with an indoleacetamide hydrolase (IAHH) from Bradyrhizobium diazoefficiens USDA 110. IpaH displayed maximal enzymatic activity at 35°C and pH 7.5, and it was not a metalloamidase. The kcat and Km of IpaH against iprodione were 22.42 s-1 and 7.33 μM, respectively, and the catalytic efficiency value (kcat/Km ) was 3.09 μM-1 s-1 IpaH has a Ser-Ser-Lys motif, which is conserved among members of the amidase signature family. The replacement of Lys82, Ser157, and Ser181 with alanine in IpaH led to the complete loss of enzymatic activity. Furthermore, strain YJN-5M lost the ability to degrade iprodione, suggesting that ipaH is the only gene responsible for the initial iprodione degradation step. The ipaH gene could also be amplified from another previously reported iprodione-degrading strain, Microbacterium sp. strain YJN-G. The sequence similarity between the two IpaHs at the amino acid level was 98%, indicating that conservation of IpaH exists in different strains.IMPORTANCE Iprodione is a widely used dicarboxamide fungicide, and its residue has been frequently detected in the environment. The U.S. Environmental Protection Agency has classified iprodione as moderately toxic to small animals and a probable carcinogen to humans. Bacterial degradation of iprodione has been widely investigated. Previous studies demonstrate that hydrolysis of its N-1 amide bond is the initial step in the typical bacterial degradation pathway of iprodione; however, enzymes or genes involved in iprodione degradation have yet to be reported. In this study, a novel ipaH gene encoding an amidase responsible for the initial degradation step of iprodione in Paenarthrobacter sp. strain YJN-5 was cloned. In addition, the characteristics and key amino acid sites of IpaH were investigated. These findings enhance our understanding of the microbial degradation mechanism of iprodione.