DEVELOPMENT AND APPLICATION OF HIGH THROUGHPUT ASSAY SYSTEMS FOR THE DETECTION OF RIESKE DIOXYGENASE ACTIVITY

Rieske dioxygenases are a class of enzymes commonly found in soil bacteria, that are known to catalyze the oxidative dearomatization of aromatic compounds via dihydroxylation to form enantiopure cis-diol metabolites. The regio-/stereoselective nature of enzymes such as Rieske dioxygenases make them a valuable tool for performing synthetic chemistry. 

 

Enzymes can be an environmentally friendly alternative to traditional catalysts by eliminating the need for petroleum-based solvents, as enzymes can operate in aqueous solutions, as well as eliminating the need for toxic heavy metals. However, natural enzymes are limited by their substrate scope and strict selectivity. In order to overcome this, protein engineering has been employed to expand the reactivity of enzymes. 

 

To detect the activity of engineered Rieske dioxygenases, a novel assay system for the detection of aromatic substrate metabolites was developed. Here, the cis-diol metabolites were oxidized to form dialdehydes. These aldehydes were then conjugated to a fluorescent probe to give a strong fluorescent signal. However, this metaperiodate fluorescein cis-diol assay (MPFCD) was shown not give a strong fluorescent signal with aliphatic substrates. Thus, the development of an assay system to detect the metabolites produced from the dihydroxylation of aliphatic olefins was investigated. These high-throughput assays can allow for the identification of engineered Rieske dioxygenase variants that show improved reactivity towards substrates not native to the enzyme.