Event Title

Exploring a Possible Moonlighting Role for Global Phosphatase in S. pneumonia

Session Title

Cancer and Biomedical Research

Faculty Mentor

Nicholas Grossoehme, Ph.D.

College

College of Arts and Sciences

Department

Department of Chemistry, Physics, and Geology

Location

WEST 219

Start Date

12-4-2019 1:45 PM

Description

Iron is essential to an overwhelming majority of life on Earth; however, in aerobic conditions, it can take on multiple oxidation states and create harmful oxidative species that must be regulated to maintain the health of the cell. Many bacteria use one of the common metal regulatory proteins (e.g., FUR) to maintain safe levels of iron in the cell, but genome analysis of S. pneumonia indicates that it lacks any of the standard sensors. Interestingly, the presence of extracellular iron triggers an intracellular uptake response; this process involves three proteins: StkP (membranous kinase), RitR (transcription factor), and PhpP (phosphatase). It is likely that the intracellular iron sensor is linked to this uptake system; in fact, we hypothesize that the intracellular sensor is built directly into this system. Noting that PhpP is a magnesium-dependent enzyme, we hypothesize that perhaps PhpP is activated by intracellular iron in S. pneumonia, thus providing the intracellular iron sensor that it needs. Using a combination of UV-visible and fluorescence spectroscopic methods, we tested this hypothesis. Using para-nitrophenylphosphate assays (PNPP, a surrogate for phosphorylated RitR) along with manganese as an aerobic-friendly surrogate, we demonstrated that PhpP is activated by manganese. Using fluorescence competition experiments with the metal binding fluorophore Mag-Fura-2, we quantified the affinity of PhpP for manganese (Kd = 2.16 mM) and magnesium (Kd = 185.1 mM). Comparison of Mn2+ and Fe2+ activation of PhpP yielded values that suggest a decrease in affinity for PNPP; however, it shows a 6-fold increase in rate.

Previously Presented/Performed?

American Chemical Society (ACS) National Meeting and Exposition, Boston, Massachusetts, August 2018

Grant Support?

Supported by an REU grant from the South Carolina EPSCoR/IDeA Program, and by an SC INBRE grant from the National Institute of General Medical Sciences (NIH-NIGMS)

Course Assignment

CHEM 551, 552 – Hanna

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Apr 12th, 1:45 PM

Exploring a Possible Moonlighting Role for Global Phosphatase in S. pneumonia

WEST 219

Iron is essential to an overwhelming majority of life on Earth; however, in aerobic conditions, it can take on multiple oxidation states and create harmful oxidative species that must be regulated to maintain the health of the cell. Many bacteria use one of the common metal regulatory proteins (e.g., FUR) to maintain safe levels of iron in the cell, but genome analysis of S. pneumonia indicates that it lacks any of the standard sensors. Interestingly, the presence of extracellular iron triggers an intracellular uptake response; this process involves three proteins: StkP (membranous kinase), RitR (transcription factor), and PhpP (phosphatase). It is likely that the intracellular iron sensor is linked to this uptake system; in fact, we hypothesize that the intracellular sensor is built directly into this system. Noting that PhpP is a magnesium-dependent enzyme, we hypothesize that perhaps PhpP is activated by intracellular iron in S. pneumonia, thus providing the intracellular iron sensor that it needs. Using a combination of UV-visible and fluorescence spectroscopic methods, we tested this hypothesis. Using para-nitrophenylphosphate assays (PNPP, a surrogate for phosphorylated RitR) along with manganese as an aerobic-friendly surrogate, we demonstrated that PhpP is activated by manganese. Using fluorescence competition experiments with the metal binding fluorophore Mag-Fura-2, we quantified the affinity of PhpP for manganese (Kd = 2.16 mM) and magnesium (Kd = 185.1 mM). Comparison of Mn2+ and Fe2+ activation of PhpP yielded values that suggest a decrease in affinity for PNPP; however, it shows a 6-fold increase in rate.