Structure and function of eukaryotic phosphatidylserine decarboxylase

Structure and function of eukaryotic phosphatidylserine decarboxylase

Objectives

  • Examine the regulation of the enzyme phosphatidylserine decarboxylase (PSD), which produces the essential membrane component phosphatidylethanolamine
  • Obtain specific biochemical information about new ways to selectively block the activity of the enzyme in pathogens including bacteria, fungi and malarial parasites
  • Test the hypothesis that the Plasmodium knowlesi PSD (PkPSD) proenzyme is initially a serine protease that undergoes a molecular metamorphosis to become a decarboxylase
  • Examine the lipid regulation of the PkPSD by testing for the presence of specific phospholipid binding sites
  • Investigate the hypothesis that lipid regulation of PkPSD is mechanistically coupled to proenzyme processing by inducing conformational changes to the enzyme that either activate or inhibit the protease function

PI Institution(s)

Principal Investigator (PI)

Funding source(s)

Abstract

Phosphatidylethanolamine (PE) is an essential lipid in organisms ranging from bacteria to humans and a pivotal enzyme in the production of this phospholipid is phosphatidylserine decarboxylase (PSD). Although the deduced primary structure of eukaryotic PSD has been known for more than a decade, the details about how the activity of this enzyme is regulated have been elusive. In addition, PSD belongs to an unusual family of enzymes that contain a pyruvoyl prosthetic group. Progress in understanding eukaryotic PSD enzymes has been hampered by its integral membrane structure and relative lability in the presence of detergents. Recently, we cloned a cDNA encoding PSD from the parasite Plasmodium knowlesi (PkPSD). The PkPSD exists in both soluble and membrane bound forms. The availability of soluble forms of PkPSD has now enabled new lines of inquiry into the structure and function of this enzyme. Using coupled in vitro transcription/ translation systems we have begun to dissect the early events that regulate the conversion of nascent proenzyme to the mature enzyme, consisting of a small subunit containing the pyruvoyl prosthetic group, and a large subunit. We have now devised a system for examining the in vitro processing of the proenzyme to the mature enzyme, and have succeeded in expressing high levels of the proenzyme in bacteria. Utilizing these systems we now plan to conduct experimentation to elucidate the mechanisms of proenzyme processing and post translational regulation of catalytic activity. From these studies, we will obtain a comprehensive view of how the lipid composition of cell membranes allosterically influences the activation of an essential enzyme in phospholipid synthesis. Understanding this aspect of PSD regulation coupled to membrane lipid composition will have important consequences for intervening in phospholipid metabolism of pathogens and mammalian cells with unregulated growth.

https://goo.gl/PywZQK

Key facts

  • Dates
    Aug 2013 to Jul 2017
    Funding amount
    $1,217,280
    Country
    • United States

MESA tags

  • Methodology
    Basic science
    Theme(s)
    Other