Regina Puts

Abstract

The function of phosphoglycolate phosphatase (PGPase) in photosynthetic organisms such as green algae (Chlamydomonas reinhardti) is to recycle the 2-phosphoglycolate that is formed as a by-product of the Calvin cycle. The role of PGPase in nonphotosynthetic organisms is less understood, but one potential role that has been suggested is to remove the 2-phosphoglycolate that is formed as a result of DNA repair. This may be the biological role of PHO13 PGPase in Saccharomyces cerevisiae as well. PHO13 PGPase from S. cerevisiae is a member of the PGPase subfamily within the p-nitrophenylphosphatase (p-NPPase) family, which is within the Haloalkanoic Acid Dehalogenase (HAD) superfamily. pho13 has been subcloned into pET19b to create PHO13(His*Tag). PHO13(HT) was overexpressed and found to be soluble. It purified well by Ni2+-NTA affinity chromatography followed by size-exclusion chromatography. Its expression, solubility, and activity appeared comparable to the native PHO13, but PHO13(HT) was easier to purify due to the histidine tag. Both enzymes had comparable specific activities for both p-nitrophenylphosphate (p-NPP) and 2-phosphoglycolate (PG), pH optima around pH 8.0, optimal activity with ≥ 7 mM Mg2+, activity with Co2+ and Mn2+, negligible activity in the presence of Zn2+, and no activity for Ca2+. PHO13(HT) is now ready for x-ray crystal structure determination with our collaborator, Joseph Wedekind, at the University of Rochester.

Library of Congress Subject Headings

Phosphatases--Analysis; Saccharomyces cerevisiae--Biotechnology

Publication Date

5-1-2010

Document Type

Thesis

Advisor

Not listed

Comments

Note: imported from RIT’s Digital Media Library running on DSpace to RIT Scholar Works in December 2013. Physical copy available through RIT's The Wallace Library at: QP609.P5 P88 2010

Campus

RIT – Main Campus

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