STRUCTURAL AND THERMODYNAMIC CHARACTERIZATION OF SPERMIDINE AND SPERMINE SYNTHASES
Open Access
- Author:
- Nadaraia-Hoke, Shorena
- Graduate Program:
- Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- August 21, 2009
- Committee Members:
- Dr Flanagan, Dissertation Advisor/Co-Advisor
John Michael Flanagan Jr., Committee Chair/Co-Chair
Anthony Edward Pegg, Committee Member
Kent Eugene Vrana, Committee Member
Ira Joseph Ropson, Committee Member
Thomas E Spratt, Committee Member - Keywords:
- polyamines
spermidine synthase
spermine synthase
isothermal titration calorimetry
snyder-robinson syndrome - Abstract:
- Polyamines are ubiquitous, physiological cations that are essential for a wide range of functions in normal cells, including cell proliferation, differentiation, signal transduction, and apoptosis. Polyamines interact and stabilize various negatively charged macromolecules such as cellular nucleic acids, alter membrane structure and act as modulators of ion channels. Cells utilize complex metabolic systems and multiple compensatory mechanisms to ensure polyamine homeostasis and, even though their functions at the molecular level are not completely understood, polyamines are critical to cell survival. In mammals, the polyamines, spermidine and spermine, are synthesized by two aminopropyltransferases, spermidine- and spermine-synthases, respectively. Both enzymes utilize decarboxylated S-adenosylmethionine and produce a second product, 5’-methylthioadenosine. Recent structural studies have revealed that the catalytic domains of these enzymes share a common fold. However, potentially, they are regulated in different ways. Spermidine synthase has a gatekeeping loop that is thought to control access to the active site, whereas spermine synthase does not have a gatekeeping loop but contains an additional N-terminal domain that may have a regulatory role. In the presented studies, several biophysical methods were used to begin to understand how these enzymes are regulated. Thermotoga maritima spermidine synthase and human spermine synthase were used as model systems. Specifically, the effect of substrates, products, and inhibitors on spermidine synthase were examined using high-throughput high-resolution deuterium exchange mass spectrometry and isothermal titration calorimetry. In addition, N-terminal deletions of human spermidine synthase and single point variants, which are found in Snyder Robinson Syndrome, were characterized to investigate their effect on the tertiary structure and domain organization. The results of these studies point towards a critical role of oligomerization in each enzyme and suggest the existence of novel regulatory mechanisms controlling their activities in the polyamine biosynthetic pathway.