Enzymes are natural protein molecules that convert one biological substance into another. Enzymes often have extraordinary selectivity properties in that they are able to recognize and convert a specific substrate molecule from a sea of other molecules. Because enzymes are encoded by genes in living organisms, their chemical properties of substrate recognition and conversion are optimized and refined by evolution. This research project is designed to study the evolutionary optimization of an interesting enzyme from soil bacteria that mediates the destruction of a family of pesticides and chemical warfare agents called phosphotriesters. The enzyme, called Phosphotriesterase, is highly efficient at carrying out this task and this is unusual because it's phosphotriester substrates are synthetic chemicals that were first prepared about forty years ago. This suggests that the evolutionary process for optimizing this enzyme happened at an unprecedented rate. We have discovered an enzyme from a related bacteria, E. coli, that is highly similar in structure to the Phosphotriesterase enzyme, and we believe that the E. coli homolog represents a member of a new family of enzymes from which Phosphotriesterase arose. Thus, we have the starting point and ending point for a pair of evolutionarily related enzymes, and we will carry out experiments designed to provide useful structure-function clues to this evolutionary process. Scanlan, Thomas S . Research Plan The research plan for this proposal focuses on a newly identified open reading frame (ORF) from Escherichia coli which appears to have been a starting point for rapid evolution of a new enzyme. The well-characterized enzyme Phosphotriesterase from soil bacteria catalyzes the hydrolytic cleavage of synthetic phosphotriester insecticides and chemical warfare agents in a hlghly efficient manner. The enzyme-mediated cleavage reaction occurs at the limit of diffusion control, suggesting that Phosphotriesterase has reached its evolutionary endpoint in catalytic optimization. The suggestion that this enzyme has achieved evolutionary perfection is amazing considering that its phosphotriester substrates were first synthesized in the 1940's and 50's, thus providing a fifty year time window for evolution of maximal catalytic power. We have recently discovered that a newly identified chromosomal ORF in E. coli has high sequence similarity to Phosphotriesterase, and many of the important active site residues are conserved between the two enzymes. This E. coli ORF, which we have named Phosphotriesterase Homolog Protein (PHP), therefore appears to be a member of the subfamily from which Phosphotriesterase evolved, and provides the basis for an interesting research project in molecular evolution and protein engineering that may have practical value in producing new organophosphate detoxification agents. This research proposal describes our plans to characterize the biophysical and enzymatic properties of PHP, and elucidate the natural function that this enzyme performs in E. coli. In addition, we plan structure-guided mutagenesis studies aimed at retracing Phosphotriesterase's evolutionary path to catalytic perfection using PHP as a starting point. Finally, laboratory evolution experiments are described, with the aim of developing new organophosphate detoxification enzymes using the principles of natural selection. Teachin~ Plan The teaching plan for this proposal seizes upon the opportunity of creating a strong graduate education program in bioorganic chemistry at UCSF. Several important strategies are discussed, including plans to develop new chemistry-based graduate courses, methods of integrating students from the various biology-centered departments at UCSF into these courses, and plans to introduce minority undergraduate students to research opportunities at the chemistry/biology interface. Teaching responsibilities anticipated during the award period are discussed, and previous accomplishments and awards in tea ching excellence are presented. NSF FORM 1358 (1/94) 2
