Engineering promiscuous enzymes for synthesis of biological building blocks

The use of enzymes in synthesis has had an enormous impact on the development of bioactive compounds, as they can perform transformations with unparalleled selectivity at a low cost without using toxic solvents. However, there is a dearth of enzymes that catalyze C-C bond formation on preparative scales. Many common enzymes from central metabolism have exquisite substrate selectivity or rely on coupling to downstream reactions as a thermodynamic driving force, limiting their utility. We have identified a suit of pyridoxal-phosphate (PLP) dependent enzymes that catalyze stand-alone C-C bond forming reactions that are mechanistically distinct from their counterparts in central metabolism. We propose mechanistic analysis of these enzymes, encompassing structural, kinetic, and spectroscopic studies, that will reveal how these enzymes form high-energy intermediates that are shielded from destructive interactions with solvent. This information will enable hypothesis-driven strategies to alter and improve enzyme function. In an allied effort, we are exploring new strategies to increase the efficiency of screening-based directed evolution. Assaying mixtures of substrates in direct competition, followed by resolution and quantitation of the products contain a wealth of un-tapped information. We will explore how to maximize the information present in substrate mixtures and how to use multiplexed data to guide evolutionary steps that are driven by either changes in total activity or by changes in specificity. These advances in engineering will synergize with our practical efforts to evolve C-C bond forming enzymes to perform new catalytic reactions. This research will have immediate impacts because the enzymes will produce non-canonical amino acids (ncAAs). Nature often uses ncAAs bearing side chain stereocenters to tune bioactivity, but the structural complexity of these molecules makes many out of reach for standard organic chemistry. The ncAAs made here will add new and valuable diversity to the medicinal chemistry repertoire.