Abstract:
The chemical industry currently accounts for 14% of all greenhouse gas emissions and is
projected to become the largest global consumer of oil by 2030. In the Ball State Laboratory for
Biocatalysis Research, we strive to address this problem through the development and application
of novel enzymatic catalysts. Enzymatic catalysts are environmentally benign because they can be
used in aqueous solutions and are fully biodegradable. As such, they can play a role in eliminating
the need for petroleum-based solvents and toxic heavy-metal catalysts in the production of fine
chemicals.
Rieske dioxygenases (RDO) are a class of enzymes known for their ability to perform the
cis-dihydroxylation of aromatic compounds. They have been widely applied in synthetic chemistry
due to their ability to provide enantiopure metabolites that can be used in the synthesis of highvalue
compounds. Yet, the utility of these enzymes has been limited by their substrate scope and
strict selectivity.
Here, we seek to develop novel RDO catalysts with improved or expanded reactivity
through directed evolution. In order to detect the relative cis-dihydroxylation activity of engineered
Rieske dioxygenase variants, a novel high throughput assay system for the detection of their cisdiol
metabolites was developed. Here, the cis-diol metabolites produced by active dioxygenases
in aqueous fermentation broths are oxidized using sodium (meta)periodate to produce dienedialdehydes.
These dialdehydes are then conjugated with a reactive fluorescent probe to give a
strong, concentration-dependent fluorescent signal. In this way, this metaperiodate fluorescein cisdiol
assay (MPFCD) detects the relative activity of RDOs with different aromatic substrates. This
novel high throughput assay system has allowed for the activity of rationally engineered Rieske
dioxygenase variants, produced through saturation mutagenesis, to be determined, and for the
identification of variants that demonstrate significantly altered reactivity profiles in comparison to
the parent enzyme.
