Directed evolution of dehalogenase activity in the short-chain dehydrogenase/reductase family

Abstract

Directed evolution is promising technique to improve enzyme properties, but the successful evolution of a novel enzyme activity has not been reported. From an evolutionary point of view, bacterial carbon-halogen bond-cleaving enzymes that convert anthropogenic halogenated hydrocarbons are especially intriguing, as some of them may have only recently emerged in response to environmental pollution. Although the evolutionary origin of most dehalogenases remains elusive, structural characterization of a haloalcohol dehalogenase clearly showed that these enzymes have evolved from at least two different NAD-dependent short-chain dehydrogenases/reductases (SDRs). Whereas their precursors facilitate NAD-dependent REDOX reactions, the dehalogenases catalyze an one-step intramolecular substitution reaction. They have retained the SDR catalytic triad, but contain a variable halide-binding site that replaces the SDR cofactor-binding site. Several important NAD-binding residues are still present in the dehalogenases, suggesting that adaptation of a selected SDR enzyme to a dehalogenase could be within reach of directed evolution experiments. This conclusion inspired me to explore the possibilities of directed evolution to evolve a dehalogenation activity in the NAD-dependent 7alpha-hydroxysteriod dehydrogenase from Escherichia coli. Selection assays will be based on selective growth advantages of transformants on haloalcohol-containing minimal media in the absence of sugars. This research will complement my background in structural biology, and broaden my research expertise with novel experimental approaches to study structure-function relationships in an evolutionary context.