Grants and Contracts Details
Project summary: Natural products (NPs) have been and continue to be a source and an inspiration for many of the most effective therapeutics used today. A major class of NPs is comprised of nonribosomal peptides (NRPs), which are synthesized using proteinogenic and non-proteinogenic amino acids in an assembly-like fashion by multimodular enzymes termed nonribosomal peptide synthetases (NRPSs). Three core catalytic domains are found within each NRPS module: adenylation (A), condensation (C), and thiolation (T) domains. A domains are key determinants that control the diversity of the NRP product as they dictate the amino acid incorporated into the final NRP. Further enhancing the diversity of the NPs, additional chemical modifications can be done by auxiliary domains such as methyltransferase (M), ketoreductase (KR), glycosyltransferase (GT), and oxidase (Ox). These auxiliary domains serve to further diversify and decorate NRPs. Remarkably, in Nature, there are examples of bifunctional A domains. These bifunctional A domains have auxiliary domains inserted into an A domain. They are capable of performing adenylation and the function of the auxiliary domain such as methylation, monooxygenation, or ketoreduction. The most common interrupted A domains contain the catalytic region of an M domain. These interrupted A domains were previously thought to be inactive and therefore overlooked, which has resulted in an information deficit. This presents the opportunity to explore these unique bifunctional enzymes, including enzyme engineering to generate unnatural bifunctional enzymes. To accomplish this, I have developed three research aims. Aim 1 is to engineer novel multifunctional enzymes for NRPS biosynthesis. Drawing inspiration from naturally occurring bifunctional enzymes, I will expand the diversity and add auxiliary domains to monofunctional A domains. This will not only provide a proof-of-principle platform for other NRPS systems, but also allow the incorporation of alternative functional groups into existing molecules to provide site and stereo specific modification of complex molecules. Aim 2 is to structurally study engineered and natural multifunctional enzymes. Structural insight into how these proteins work is a great asset to determine modifications and catalytic activity, to date there is no published structure of an interrupted A domain. Aim 3 is to explore and expand the promiscuity of native and engineered A domains. This would increase diversity in existing NRPS systems by incorporation of non-native units. I expect this work to (i) increase the overall understanding of these unique bifunctional enzymes (biochemically and structurally), (ii) provide an exploratory platform to expand these bifunctional enzymes to more systems, and (iii) expand the biosynthetic arsenal to incorporate unnatural substrates thus increasing the diversity potential for biosynthetic machinery.
|Effective start/end date
|9/1/19 → 8/31/20
- American Foundation for Pharmaceutical Education: $10,000.00
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