CAREER: Structural Basis of Specificity for Gramicidin A Synthetase

Dartmouth College, Hanover NH

Investigators

Abstract

The objective of this project is to determine the structural basis of amino acid specificity for the non-ribosomal peptide synthesis (NRPS) enzyme, gramicidin A synthetase (GrsA). Different NRPS enzymes incorporate amino acid substrates to produce a variety of peptides; the long-term goal of this research is to alter the specificity of a particular NRPS enzyme to incorporate new functional groups into novel peptides. In order to accomplish this goal, the following issues will be addressed: 1) The physical arrangement and interactions between the three domains (with three concomitant enzymatic functions) of GrsA synthetase. In preliminary studies, crystals of the entire GrsA synthetase, bound to its substrates, have been grown and they show diffraction amplitudes to beyond 3 A resolution; 2) Whether the substrate is transferred between domains via a covalently bound cofactor at the second domain. Crystal structures of the cofactor-bound and substrate-bound GrsA synthetase will be determined; 3) The rules of recognition for acceptance of the substrate amino acid, since the initial aminoacyl adenylation domain is the primary determinant for specificity. The crystal structures of three adenylation domains that bind distinctly different amino acids, D-alanine, L-proline, and a-aminoadipine will be solved and compared with the structure of the previously determined L-phenylalanine adenylation domain. Novel computational algorithms will be used to search mutation space and conformation space to propose possible mutants that will reprogram the L-Phe adenylation domain to accept a new substrate amino acid. Biochemical assays, along with crystal structure determination of the modified A domain, will be part of an iterative cycle to incorporate a non-native amino acid in an NRPS peptide product. Reflecting a strong movement toward multi-disciplinary research, today's chemistry education should also be multi-disciplinary, incorporating fields such as biology, physics and computer science. An integral interdisciplinary approach will be applied to newly developed courses in biophysical chemistry bringing structural biology to the forefront of the biophysical chemistry curriculum at Dartmouth College. It is also important to attract younger children to science. Through a partnership with a K-12 outreach program at a neighboring science museum, young children are learning the relationship of basic science principles to current research. Using interactive demonstrations of the properties of light with matter, children learn that the three-dimensional structures of proteins can be determined.