JadH is a bifunctional hydoxylase/dehydrase involved in jadomycin biosynthesis; it catalyzes a post-PKS modification reaction to convert 2,3-dehydro-UWM6 to dehydrorabelomycin. To identify the key residues involved in substrate-binding and catalysis, structural modeling and multiple sequence alignments of JadH homologs were performed to predict nine residues at the proximity of substrate. Site-directed mutagenesis of the corresponding residues and in vitro evaluation of the activities of the mutant enzymes, indicate these mutations severely reduced JadH activity. Our results indicate these residues are specifically involved in substrate-binding or catalysis in JadH.