The objective of this project is to learn how enzymes evolve to recognize new substrates. A better understanding of adaptive evolution will explain how the complex molecular machinery of life arose, and enable the artificial selection of novel catalysts for industrial or medical applications. Enzymes could theoretically adapt to new substrates by the following ordered mechanism: 1) mutations that destabilize the conformation of the active-site initially broaden substrate specificity; 2) active-site flexibility enables the occurrence of mutations that introduce novel interactions with the new substrate; 3) mutations that stabilize the new productive active-site conformation further improve catalytic activity. This hypothesis will be tested by directing the evolution of a model enzyme called beta-glucuronidase (GUS). The gene encoding GUS will be randomly mutated. The resulting library will be expressed in populations of Escherichia coli, and screened for variant enzymes exhibiting increased catalytic activity in reactions with a novel substrate analogue. The corresponding alleles will be isolated and randomly mutated for another round of screening. After three rounds of evolution, the beta- galactosidase activity of GUS has already increased 500-fold, with a 52,000,000-fold inversion of specificity. The process will be continued until the catalytic efficiency and specificity of the wild-type lacZ beta-galactosidase has been matched. The entire evolutionary process will also be repeated with different substrate analogues. The structural and functional changes that occur along each evolutionary pathway will be studied and compared, and the rules governing adaptation will be inferred.
