X-ray crystallographic studies of therapeutic enzymes: nitroreductase and AKR1C3

Lovering, Andrew (2003). X-ray crystallographic studies of therapeutic enzymes: nitroreductase and AKR1C3. University of Birmingham. Ph.D.

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Abstract

The \(Escherichia\) \(coli\) enzyme nitroreductase has been proposed as a candidate for the Gene-Directed Enzyme Prodrug Therapy approach in treating cancer. Structural studies on the enzyme were instigated in a first step towards improving enzyme activity. The enzyme was crystallized with the substrate analogue, nicotinic acid, and the structures of three crystal forms obtained. The fold has a mixed a/P structure, with a molecule of nicotinic acid bound next to the FMN cofactor. Several active site residues were identified as candidates for mutation. This procedure produced many mutant enzymes with increased catalytic activity. One double and four single mutants were chosen from these and crystal structures determined. The resulting information from this, and the establishment of a proof of principle, provides the basis for iterative cycles of enzyme improvement.

The human hydroxysteroid dehydrogenase AKR1C3 has been proposed to play a role in prostaglandin metabolism. Its inhibition by non-steroidal anti-inflammatory drugs may be important in a tumour differentiation strategy. AKR1C3 was crystallized, and the structure solved with bound nucleotide cofactor and several inhibitors, including the drugs indomethacin and flufenamic acid. Having obtained information on drug binding to AKR1C3, selective inhibitors can be designed, avoiding inhibition of "housekeeping" enzymes such as cyclo-oxygenases.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

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