Author ORCID Identifier
Year of Publication
Doctor of Philosophy (PhD)
Dr. Sylvie Garneau-Tsodikova
Nature has historically served as a prolific source of biologically active molecules produced by plants, fungi, and bacteria termed natural products (NPs). These NPs often serve as therapeutic leads due to their structural diversity and unique mechanisms of action. However, many need modifications to make them more effective or safer for human use. This can be a daunting and complicated task to do via traditional organic chemistry because of their complexity, copious stereocenters, and a multitude of reactive functional groups. For this reason, enzymatic modification of these NP during their biosynthesis is an appealing option. One major class of NPs is nonribosomal peptides (NRPs) synthesized by nonribosomal peptide synthetases (NRPSs) mega-enzymes, which use natural and unnatural amino acid building blocks to yield a final NRP. NRPSs can be subdivided into individual domains that synthesize NRP in an assembly-line fashion, with each domain carrying out a particular function. The final structure of the NRP is dictated by the arrangement of the domains and their selectivity, not by an mRNA template. The adenylation (A) domain is responsible for activating a specific amino acid, via adenylation, and loading it onto the thiolation (T) domain. The condensation (C) domain can then catalyze formation of the peptide bond. In this process, the A domain is considered the gatekeeper of diversity in NRPs because it dictates the amino acids that are incorporated into the final structure. In addition to the core A, T, and C domains, there can be auxiliary domains that carry out additional chemistry, one of the most common being a methylation (M) domain. Methylation is an important feature in many NPs, for example, it has been shown to increase the oral bioavailability of NRPs or improve the stability of NRPs. Interestingly, a single M domain can be embedded within an A domain, termed an interrupted A domain. These M domains can catalyze either side chain or backbone methylation of amino acids and can occur between a2-a3 or a8-a9 of the 10 conserved motifs (a1-a10) of A domains. Therefore, to better understand these multifunctional enzymes, we demonstrated that (i) through enzyme engineering, interrupted A domains can be created de novo from uninterrupted A domains, (ii) one A domain can in fact support two different interruptions in different locations within the A domain, (iii) there is a new naturally occurring type of dimethylating interrupted A domain, which contains two back-to- back M domains within the same interruption site rather than the typical single M iii domain, and (iv) there are more varieties of interrupted A domains than previously reported and they can be divided into different classes, each class with distinct defining characteristics. Together these insights lay the foundation for future engineering studies and combinatorial biosynthesis of site-specifically methylated NRPs. More broadly, these studies will pave the way for the design of complex NPs that may become therapeutic interventions in a broad range of human diseases.
Digital Object Identifier (DOI)
University of Kentucky College of Pharmacy 2018-2019 Pharmaceutical Sciences Excellence in Graduate Achievement Fellowship to Taylor A. Lundy 2018-2019
University of Kentucky College of Pharmacy 2019- 2020 Pharmaceutical Sciences Excellence in Graduate Achievement Fellowship to Taylor A. Lundy 2019-2020
2019-2020 Pre-doctoral fellowship in Pharmaceutical Sciences from the American Foundation of Pharmaceutical Education to Taylor A. Lundy 2019-2020
Lundy, Taylor A., "Characterization and Engineering of Interrupted Adenylation Domains" (2020). Theses and Dissertations--Pharmacy. 118.