dictyNews
Electronic Edition
Volume 46, number 27
September 25, 2020

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Abstracts
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Biochemical and biophysical analyses of hypoxia sensing prolyl 
hydroxylases from Dictyostelium discoideum and Toxoplasma gondii

Tongri Liu1, Martine I. Abboud1, Rasheduzzaman Chowdhury1, Anthony 
Tumber1, Adam P. Hardy1, Kerstin Lippl1, Christopher T. Lohans1, 
Elisabete Pires1, James Wickens1, Michael A. McDonough1, 
Christopher M. West2, and Christopher J. Schofield1

1Chemistry Research Laboratory, University of Oxford, 12 Mansfield 
Road, Oxford, OX1 3TA, United Kingdom; 
2Department of Biochemistry and Molecular Biology, Complex 
Carbohydrate Research Center, Center for Tropical and Emerging 
Global Diseases, 120 East Green Street - B122 Life Sciences Building, 
University of Georgia, Athens, GA 30602, United States of America.


J. Biol. Chem., in press

In animals, the response to chronic hypoxia is mediated by prolyl-
hydroxylases (PHDs) that regulate the levels of hypoxia inducible 
transcription factor a (HIFa). PHD homologues exist in other types of 
eukaryotes and prokaryotes where they act on non-HIF substrates. To 
gain insight into the factors underlying different PHD substrates and 
properties, we carried out biochemical and biophysical studies on PHD 
homologues from the slime mold, Dictyostelium discoideum, and the 
protozoan parasite, Toxoplasma gondii, both lacking HIF. The respective 
prolyl-hydroxylases (DdPhyA and TgPhyA) catalyze prolyl-hydroxylation 
of S-Phase Kinase Associated Protein 1 (Skp1), a reaction enabling 
adaptation to different dioxygen availability. Assays with full length Skp1 
substrates reveal substantial differences in the kinetic properties of 
DdPhyA and TgPhyA, both with respect to each other and compared with 
human PHD2; consistent with cellular studies TgPhyA is more active at 
low dioxygen concentrations than DdPhyA. TgSkp1 is a DdPhyA substrate 
and DdSkp1 is a TgPhyA substrate. No cross-reactivity was detected 
between DdPhyA/TgPhyA substrates and human PHD2. The human Skp1 
E147P variant is a DdPhyA and TgPhyA substrate, suggesting some 
retention of ancestral interactions. Crystallographic analysis of DdPhyA 
enables comparisons with homologues from humans, Trichoplax 
adhaerens, and prokaryotes, TgPhyA informing on differences in mobile 
elements involved in substrate binding and catalysis. In DdPhyA, two 
mobile loops that enclose substrates in the PHDs are conserved, but the 
C-terminal helix of the PHDs is strikingly absent. The combined results 
support the proposal that PHD homologues have evolved kinetic and 
structural features suited to their specific sensing roles.


submitted by: Chris West [[log in to unmask]]
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[End dictyNews, volume 46, number 27]