A Novel Trifunctional Hydrolase Cleaving C−O, C−N, and C−C Bonds and Its Widespread Distribution in Proteobacteria and Actinobacteria

The extensive application of cyhalofop-butyl, an aryloxyphenoxypropionate (AOPP) herbicide, for weed control in rice fields inevitably results in environmental pollution and ecological toxicity. Microbial degradation of cyhalofop-butyl is garnering immense research attention. This study revealed that Sphingobium yanoikuyae B1 can efficiently degrade the cyhalofop-butyl, completely breaking down 30   mg/L within 12   h via ester bond hydrolysis, forming cyhalofop acid. A novel hydrolase gene cbeH (37.39% identity) was identified in B1. CbeH exhibited catalytic activity by hydrolyzing the ester bond (C-O) in cyhalofop-butyl and other AOPP herbicides, yielding AOPP acids. Additionally, it catalyzed the cleavage of the C-N bond in amide herbicides propanil and diuron, as well as the C-C bond in the aromatic compound 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid. Sequence alignments and site-directed mutagenesis indicated the catalytic triad Ser-His-Asp in CbeH serves as active center cleaving C-O, C-C, and C-N bonds. Comparative analysis revealed the gene cbeH , highly conserved within Sphingomonads , was located within multiple insertion sequences on the plasmid of Rhodococcus strains. CbeH was widespread in Proteobacteria and Actinobacteria, with its host strains prevalent in aromatic-polluted environments. This study provides novel enzyme resources for remediating combined pollution and enhances our understanding of the genetic organization and environmental distribution of trifunctional pollutant-degrading genes.