Date of Award
Campus Access Dissertation
Doctor of Philosophy (PhD)
Daniel P. Dowling
Jason R. Green
In one use, “modularity” refers to properties of the components of a system that permit them to be separated and preserve functionality upon recombination in various ways. Constraints are often placed on modularity of natural biosynthetic pathways so that a limited set of products is selectively produced. Control is largely achieved by two means: 1) enzyme specificity for substrate features other than the moiety acted on by catalysis and 2) spatiotemporal separation of enzyme activities. Classic examples of modular enzyme systems are found in the megaenzymes of nonribosomal peptide and polyketide biosynthesis. Even in those systems, however, there are substrate and protein-protein interaction specificities such that attempts to mix and match domains have typically resulted in reduced activity for the products targeted by engineering relative to the activities of wild-type systems for their products. The work presented here examines properties that permit and control modularity in the activities of enzymes in two systems. The first body of work describes using experimental phasing techniques to solve the crystal structures of the first two enzymes of DNA hypermodification in Pseudomonas phage M6: the first representative of a family of kinases with the remarkable activity of phosphorylating the major-groove edge of a 5-hydroxymethyldeoxyuridine (5-hmdU) base in double-stranded DNA, and the first representative of a family of transferases that exchange the phosphate group on the DNA base for an amino acid. These structures have identified substrate ATP and metal cofactor binding sites and reveal how evolution has repurposed two common enzyme folds to achieve modification of a nucleobase in the DNA fiber. The second body of work describes computational studies of a heterocyclization domain from the nonribosomal peptide synthetase (NRPS) that produces the plague virulence factor and siderophore yersiniabactin. This work explores substrate binding and the potential roles of domain flexibility in traversing the reaction coordinate as well as in allosteric communication of interdomain binding events to the active site for control of the catalytic cycle. Both projects will be discussed in relation to each system’s modularity and the potential for enhancement and use of that modularity in protein engineering efforts.
Gnann, Andrew D. II, "Toward Modular Enzyme Systems for Modifying DNA and Peptides: First Structures of Phage DNA Hypermodification Enzymes and Computational Analysis of NRPS Heterocyclization Domains" (2023). Graduate Doctoral Dissertations. 871.
Available for download on Monday, September 01, 2025