Rapid kinetic studies of PhyA from Selenomonas ruminantium, and a simplified means of production of a phosphate biosensor

Myo-inositol polyphosphates (IPs) are ubiquitous in nature and involved in various cellular events. Dephosphorylation of IPs by protein tyrosine phosphatase-like polyphosphatases (PTPLPs) occurs via a complex pathway, and the in vivo function of many of these enzymes remains unknown. In order to further our understanding of PTPLP catalyzed dephosphorylation of IPs; I present rapid kinetics studies of the representative PTPLP PhyA from Selenomonas ruminantium (PhyAsr). These studies revealed kinetic parameters of PhyAsr dimerization, and myo-inositol hexakisphosphate (IP6) binding to the homodimer. In addition to studying PhyAsr, I have developed a simplified methodology to produce a biosensor capable of detecting phosphate release in real-time. The phosphate biosensor is fluorescently labeled and utilizes Escherichia coli Phosphate-binding protein (PhoS). The results show that my proposed methodology yields a functional biosensor and is feasible for large-scale production. I envision this methodology to be versatile and useful for a large number of research applications where detection of free phosphate is required.

Authors

• Smith, Dustin D.
• University of Lethbridge. Faculty of Arts and Science

• Biochemistry
• Enzyme kinetics
• Phosphate sensor
• PTPLP
• myo-inositol
• Phosphatase
• Stopped-flow
• PhyA
• PhoS
• Phosphate release
• Phosphate binding protein
• Phytase

Identifier

hdl:10133/4763

OAI identifier oai:identifier

oai:opus.uleth.ca:10133/4763