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.