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
  • John Michael Flanagan Jr., Committee 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.