UC Irvine

Enzymes in the acetate and non-ribosomal peptide pathways generate chemically diverse and complex bioactive molecules, with the intermediates being chauffeured between catalytic partners via a carrier protein. Recent efforts have been made to engineer these systems to expand their product diversity. A major stumbling block is our poor understanding of the transient protein-protein and protein-substrate interactions between the carrier protein and its many catalytic partner domains. The innate reactivity of pathway intermediates has obfuscated our mechanistic understanding of these interactions during the biosynthesis of these natural products, ultimately impeding the engineering of these systems for the generation of “unnatural” natural products.

Molecular dynamics can be used to provide models of these key interactions that are difficult to capture experimentally, providing the potential to expand the diversity in these systems. Current force fields support basic biochemical building blocks, and specialized force fields support post-translational modifications and non-canonical amino acids, yet none currently exist that are capable of modeling bound intermediates from these systems.

The objective of this dissertation is to address this gap in knowledge and available technology and to present the description of the development of a force field that can be used with MD and other computational techniques to provide models of these experimentally intractable transient interactions. This objective will be divided into three aims.

Aim 1: The development and validation of a force field for use in investigations and engineering efforts involving these pathways. A fragmentation approach was used, providing a degree of modularity to the force field while at the same time reducing the size, complexity and degrees of freedom during the charge fitting and parameterization steps. Tutorials and a web interface were also developed to provide end users access to this tool.

Aim 2: The application of the force field towards understanding dynamics and interactions in these pathways. Regulation of the transient protein-protein interactions and discrete steps in fatty acid biosynthesis remain poorly understood. MD was able to show that specific interactions with its partner are either strengthened or weakened depending on the loaded state of the carrier protein. The force field was also used to model a plant-based polyketide synthase at different pHs, resulting in the identification of an allosterically modulated pH sensor on the surface of the polyketide synthase.

Aim 3: The application of the force field in engineering efforts in these systems to produce biofuels. MD simulations were employed to provide a deeper understanding of protein-substrate interactions in a reductase domain from a non-ribosomal peptide megasynthase. This led to a deeper understanding of this domain, and further identified residues critical for structure integrity and substrate binding, leading to a rationally altered variant with improved activity toward highly reduced substrates.