The cyclic-di-GMP receptors of S. Typhimurium: testing their signaling specificity through second messenger affinity and their use as biosensors

Thesis (Ph.D.)--University of Washington, 2012c-di-GMP is a second messenger that regulates motility and the production of adhesive factors in many bacterial species. Enzymes containing specific c-di-GMP metabolizing domains integrate information about the environment into an intracellular level of c-di-GMP that then binds to specific downstream receptors, including proteins that contain the PilZ domain. Many bacterial species encode dozens of c-di-GMP metabolizing enzymes in their genomes. Although each of these enzymes metabolizes the same small, diffusible second messenger molecule, many of these proteins can be specifically linked to downstream c-di-GMP-regulated processes. The mechanisms involved in achieving this signaling specificity between c-di-GMP metabolizing enzymes and their downstream receptors are not known. Here, we provide evidence that c-di-GMP signaling specificity is achieved through differences in the binding affinities of downstream receptors. Salmonella Typhimurium harbors two PilZ domain proteins: YcgR, which controls flagellar-based motility, and BcsA, an enzyme that produces cellulose. Using a Forster resonance energy transfer (FRET)-based method, we measured the binding affinities of these PilZ domain proteins and found that they span a 43-fold range. Increasing the binding affinity of BcsA for c-di-GMP increased the amount of cellulose that this enzyme produced at lower levels of c-di-GMP. Decreasing the affinity of YcgR for c-di-GMP increased the amount of this second messenger needed for YcgR to inhibit motility. In addition, we found that mutation in yhjH, which encodes a predicted c-di-GMP-degrading enzyme, increased the fraction of the cellular population that demonstrated c-di-GMP levels high enough to bind to the higher-affinity YcgR protein, but did not enough to bind to the lower-affinity BcsA protein and stimulate cellulose production. Thus, the specific affinities of these proteins for c-di-GMP are important for their biological functions. Additionally, the binding affinities of the eight PilZ domain proteins in Pseudomonas aeruginosa were measured and found to span a 145-fold range, implying that regulation by binding affinity of downstream receptors for c-di-GMP may be a common theme in c-di-GMP signaling. Finally, we generated a panel of FRET-based c-di-GMP biosensors which will allow for the accurate measurement of the free c-di-GMP level in individual cells from the nanomolar to the micromolar range

Computational Design of an α‑Gliadin Peptidase

The ability to rationally modify enzymes to perform novel chemical transformations is essential for the rapid production of next-generation protein therapeutics. Here we describe the use of chemical principles to identify a naturally occurring acid-active peptidase, and the subsequent use of computational protein design tools to reengineer its specificity toward immunogenic elements found in gluten that are the proposed cause of celiac disease. The engineered enzyme exhibits a <i>k</i>_cat/<i>K</i>_M of 568 M^–1 s^–1, representing a 116-fold greater proteolytic activity for a model gluten tetrapeptide than the native template enzyme, as well as an over 800-fold switch in substrate specificity toward immunogenic portions of gluten peptides. The computationally engineered enzyme is resistant to proteolysis by digestive proteases and degrades over 95% of an immunogenic peptide implicated in celiac disease in under an hour. Thus, through identification of a natural enzyme with the pre-existing qualities relevant to an ultimate goal and redefinition of its substrate specificity using computational modeling, we were able to generate an enzyme with potential as a therapeutic for celiac disease

Expanding the Product Profile of a Microbial Alkane Biosynthetic Pathway

Microbially produced alkanes are a new class of biofuels that closely match the chemical composition of petroleum-based fuels. Alkanes can be generated from the fatty acid biosynthetic pathway by the reduction of acyl-ACPs followed by decarbonylation of the resulting aldehydes. A current limitation of this pathway is the restricted product profile, which consists of <i>n</i>-alkanes of 13, 15, and 17 carbons in length. To expand the product profile, we incorporated a new part, FabH2 from <i>Bacillus subtilis</i>, an enzyme known to have a broader specificity profile for fatty acid initiation than the native FabH of <i>Escherichia coli</i>. When provided with the appropriate substrate, the addition of FabH2 resulted in an altered alkane product profile in which significant levels of <i>n</i>-alkanes of 14 and 16 carbons in length are produced. The production of even chain length alkanes represents initial steps toward the expansion of this recently discovered microbial alkane production pathway to synthesize complex fuels. This work was conceived and performed as part of the 2011 University of Washington international Genetically Engineered Machines (iGEM) project