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. This work will be undertaken in a research plan containing three Specific Aims. The first Specific Aim will test the hypothesis that the PkPSD proenzyme is initially a serine protease that undergoes a molecular metamorphosis to become a decarboxylase. The protease activity of the proenzyme is proposed to be activated by phosphatidylserine and inhibited by phosphatidylglycerol, phosphatidylinositol and phosphatidic acid. The second Specific Aim will examine the lipid regulation of the PkPSD by testing for the presence of specific phospholipid binding sites. The third Specific Aim will 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. 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.

Public Health Relevance

This proposal examines the regulation of the enzyme phosphatidylserine decarboxylase, which produces the essential membrane component phosphatidylethanolamine. The work will provide specific biochemical information about new ways to selectively block the activity of the enzyme in pathogens including bacteria, fungi and malarial parasites.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM104485-02
Application #
8706914
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Chin, Jean
Project Start
2013-08-01
Project End
2017-07-31
Budget Start
2014-08-01
Budget End
2015-07-31
Support Year
2
Fiscal Year
2014
Total Cost
$304,320
Indirect Cost
$112,320
Name
National Jewish Health
Department
Type
DUNS #
076443019
City
Denver
State
CO
Country
United States
Zip Code
80206