Mechanism and Structure in an Enzyme Superfamily

Portland State University, Portland OR

Investigators

Abstract

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Dirk Iwata-Reuyl of Portland State University to investigate the molecular/structural basis for the divergent chemistries exhibited by enzymes belonging to the Tunneling-fold superfamily (T-fold). Members of the T-fold superfamily catalyze a remarkable diversity of chemistries, and while T-fold proteins typically exhibit very low sequence homology, their topological homology is extremely high. Specifically, this project addresses two T-fold enzymes involved in the biosynthesis of the modified tRNA nucleosides queuosine and archaeosine, the enzymes QueF and QueF-L. QueF catalyzes the reduction of a nitrile precursor to an amine in the queuosine pathway in Bacteria, the only example of a nitrile reduction known in biology. QueF-L exhibits high sequence homology to QueF by catalyzes the chemically distinct conversion of a nitrile to an amidino group in the archaeosine pathway in Archaea. These systems will be studied via the application of kinetic (steady state and pre-steady state), mechanistic, and X-ray structural studies of the wild-type and mutant enzymes to elucidate the chemical mechanisms, as well as the structural basis for substrate/cofactor binding and catalysis. The large number of protein structures elucidated through X-ray crystallography over the past several decades whose coordinates are available through the Protein Data Bank has revealed that the number of basic protein scaffolds responsible for the myriad proteins in biology is surprisingly small. Elucidating the molecular basis for the different functions exhibited by members in a given structural family is one of the next challenges facing structural and mechanistic biology, impacting our ability to correctly identify the function of new proteins identified by whole genome sequencing, to engineer proteins for new functions, as well as our understanding of how proteins evolve. This project focuses on elucidating the mechanistic chemistry of several members of the so-called Tunneling-fold family and identifying the protein structural elements responsible for that chemistry. The work is multidisciplinary, approaching the problem with tools from chemistry, biochemistry, and structural and molecular biology to provide an understanding of the molecular basis for the biological function of this functionally diverse family of proteins. In addition to supporting the work of graduate and undergraduate students, the project also incorporates the efforts of high school teachers through a summer outreach program that engages K12 teachers in research.