The origins of amino acid selectivity in the homologation pathway

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PROJECT SUMMARY The objective of this proposed project is to understand the fundamental mechanisms and functions of enzymes that catalyze homologation (insertion of a methylene group) of an amino acid side chain in natural product (NP) biosynthesis. The homologation pathway is proposed to be comprised of four enzymes, one known and three novel ones, and has a narrow substrate scope. The project is focused on determining the origins of substrate specificity in the pathway for L-Phe and L-Tyr homologation in the biosynthesis of cyanobacterium NPs anabaenopeptins. The following Aims are designed to achieve this objective: Aim 1: determination of the origins of specificity (among novel enzymes, HphA, HphB, or HphCD) in the homologation pathway of L-Phe and L-Tyr and Aim 2: rational homology-based mutagenesis to alter the substrate scope of specific homologation enzyme(s) and determination of crystal structure of these enzymes. By biochemical characterization of each enzyme in Aim 1a and establishment of the substrate profile in Aim 1b, the enzyme(s) contributing to the specificity of homologation will be identified. The substrate selective enzyme(s) will be mutated to alter or expand the substrate scope of the enzyme(s) so that nonproteinogenic or other proteinogenic amino acids can be homologated in Aim 2a. These enzymes will be characterized structurally by X-ray crystallography to further guide the mutagenesis studies in Aim 2b. NPs are one of the major sources of biological probes and medicines. Modification of known bioactive compounds is a cost- and time-effective way to discover and expand the chemical diversity of bioactive compounds to alter or improve their properties. However, the complex structure of NPs makes the specific modification of the molecules by organic chemistry challenging. Therefore, combinatorial biosynthesis that uses the recombinant biosynthetic pathway to produce “unnatural” natural products is highly attractive. Homologation of amino acid moieties is a rare modification in peptide NPs, termed nonribosomal peptides (NRPs) which are one of the major targets of NP engineering due to the flexibility of their biosynthetic pathways. Since this modification is observed only in cyanobacterial and fungal species, it has great potential to derivatize NRPs produced by these and other species, which has never been performed. The knowledge gained by this project will ultimately lead to an enzymatic and genetic tool that can derivatize a variety of NPs.