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De novo protein design of a molten globule enzyme

Final Activity Report Summary - CM REDESIGN (De novo protein design of a molten globule enzyme)

Proteins are macromolecules with wide variation of functions. One very important group of proteins, called enzymes, act as biological catalysts. These molecules are made of small units called amino acids that are linked like beads on a string. The amino acid sequence for each protein is encoded in DNA molecules as a gene. The string in turn has to assume a 3-dimensional structure in order to function. While some proteins are functional with one unit (monomers) others need two or more units that interact with each other and make a more complex structure (oligomers). How enzymes have evolved to catalyse many types of biochemical reactions is an intriguing question? Today there is no doubt that proteins are products of a Darwinian evolutionary process. Many attempts have been made to accomplish in the test tube what nature has done since the dawn of life. By mimicking evolution, one hopes to draw general conclusions that help researchers to find new drugs with no side effects or to achieve more environmentally friendly chemical production.

This postdoctoral research project was focused on engineering new enzymes from a different and so far unexplored angle, namely to use a minimized monomeric mutant enzyme with a flexible structure (a molten globule) as the starting material. The targeted reaction was conversion of prephenate to phenylpyruvate. This reaction is an essential step in the biosynthesis of phenylalanine. Our hypothesis was that the plasticity of the molten globule would improve the odds of designing new functional properties. We used a natural chorismate mutase and the corresponding mutant molten globule variant of this enzyme that catalyse the prior step in this metabolic pathway. Our strategy was to mutate the active site of the chorismate mutase randomly to generate a population of different mutated enzymes and use an in vivo selection assay to search for functional variants in the pool chorismate mutase variants.

In the funding period a powerful phenylalanine auxotroph selection strain was developed. The utility of the selection system was verified by growth studies of the bacteria expressing prephenate dehydrates with high and low activity. The sensitivity of this selection procedure is highly tune-able which was suited for search for low activity. This selection system was applied to six different chorismate mutase libraries none of which yielded in catalysts for the targeted reaction. The results indicate that our original goal was surprisingly challenging. We believe the first and recent x-ray structure of prephenate dehydrates together with mechanistic information of the natural enzyme is needed for designing the proposed functionality.

I have also started exploring periplasmic amino acid binding proteins as scaffold to engineer prephenate dehydrates activity. These proteins are highly similar to cyclohexadienyl dehydratase which is a prephenate dehydratase analog. As the first step the active site of cyclohexadienyl dehydratase is transplanted into the binding site of a His binding protein as well as LAO binding protein. This work is now in progress.