Investigating Structure and Substrate Selectivity of NphT7, Acetoacetyl-CoA Synthase

Living organisms use enzymes to catalyze a multitude of chemical reactions. Due to their speed and selectivity, many enzymes are used in biomedical and environmental settings to generate synthetic molecules. Enzymes that generate carbon-carbon bonds are especially important since these reactions are the backbone of organic synthesis. However, enzymes often have limited substrate scope. To overcome this challenge, structural information and directed evolution can be combined to broaden the substrate range of naturally occurring enzymes. We focused on the enzyme NphT7 from Streptomyces sp. CL190, which performs one such reaction, a decarboxylative Claisen condensation of malonyl-CoA and acetyl-CoA to form acetoacetyl-CoA. To understand substrate recognition and protein-protein interactions in this recently discovered enzyme, we sought to determine its atomic structure by X-ray crystallography. We recombinantly expressed NphT7 in E. coli and are performing X-ray diffraction analysis on crystals of purified, active NphT7. As a complementary approach, we generated an in silico structural model of NphT7 based on E. coli FabH, a related enzyme. The model allowed us to identify residues potentially critical for substrate specificity. To test this, we generated three mutant libraries containing degenerate codons at these sites and are screening for mutants with altered substrate preference. The results from these experiments will further our understanding of carbon-carbon bond forming enzymes and provide fresh insights into how to adapt NphT7 and related enzymes for the production of new and beneficial molecules.