The regulation of oxygen homeostasis is crucial to our existence as aerobes. At the cellular level, oxygen sensing is implicated in a large number of biomedically relevant processes, including the immune response, pre-natal lung development, erythropoiesis, and angiogenesis. When oxygen levels become too low (hypoxia), the hypoxia inducible factor (HIF) induces the expression of over 70 genes, controlling cellular O2 regulation. The key players in the [O2] response are two enzymes known as HIF-hydroxylases, which regulate the activity level of HIF. The long-range goal of this proposal is to understand the oxygen- dependent reactivity of the HIF-hydroxylases. The HIF-hydroxylases belong to the Fe(ll), ?-ketoglutarate dependent oxygenase superfamily. The two known varieties of HIF-hydroxylases are HIF-prolyl hydroxylase (PHD) and HIF-asparaginyl hydroxylase (FIH), which hydroxylate discrete domains of the ? subunit of HIF (HIF?). PHD and FIH are the primary O? sensors in humans, inactivating HIF? under conditions of normal or elevated [O2]. Consequently, understanding the mechanism of these enzymes may provide avenues to control cellular responses to O2 levels. While much is known about ?KG oxygenases in general, the molecular details of O2 activation remain largely speculative, and the structural link between substrate and O2 binding is unclear. We will apply biophysical, mechanistic, and spectroscopic methods to understand the oxygen-dependent activity of HIF hydroxylases and a mechanistically related enzyme, AtsK. Kinetic isotope effects, mutagenesis, and single- turnover kinetics will be used to delineate the O2 activation mechanism. We will combine mechanistic and solvent-accessibility studies to probe the chemical and structural relationship between substrate and O2 binding. Non-productive reactions with O2 will be explored, as they may represent a physiological inactivation pathway. This research will identify structures unique to the HIF hydroxylases which regulate their oxygen-dependent reactivity, as well as better understanding of O2-activation in the broad class of ?KG oxygenases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM077413-04
Application #
7771713
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
2007-04-01
Project End
2012-02-29
Budget Start
2010-03-01
Budget End
2011-02-28
Support Year
4
Fiscal Year
2010
Total Cost
$201,312
Indirect Cost
Name
University of Massachusetts Amherst
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
153926712
City
Amherst
State
MA
Country
United States
Zip Code
01003
Hangasky, John A; Taabazuing, Cornelius Y; Martin, Cristina B et al. (2017) The facial triad in the ?-ketoglutarate dependent oxygenase FIH: A role for sterics in linking substrate binding to O2 activation. J Inorg Biochem 166:26-33
Taabazuing, Cornelius Y; Fermann, Justin; Garman, Scott et al. (2016) Substrate Promotes Productive Gas Binding in the ?-Ketoglutarate-Dependent Oxygenase FIH. Biochemistry 55:277-86
Pektas, Serap; Taabazuing, Cornelius Y; Knapp, Michael J (2015) Increased Turnover at Limiting O2 Concentrations by the Thr(387) ? Ala Variant of HIF-Prolyl Hydroxylase PHD2. Biochemistry 54:2851-7
Light, Kenneth M; Hangasky, John A; Knapp, Michael J et al. (2014) First- and second-sphere contributions to Fe(II) site activation by cosubstrate binding in non-heme Fe enzymes. Dalton Trans 43:1505-8
Hangasky, John A; Ivison, Geoffrey T; Knapp, Michael J (2014) Substrate positioning by Gln(239) stimulates turnover in factor inhibiting HIF, an ?KG-dependent hydroxylase. Biochemistry 53:5750-8
Taabazuing, Cornelius Y; Hangasky, John A; Knapp, Michael J (2014) Oxygen sensing strategies in mammals and bacteria. J Inorg Biochem 133:63-72
Hangasky, John A; Gandhi, Hasand; Valliere, Meaghan A et al. (2014) The rate-limiting step of O2 activation in the ?-ketoglutarate oxygenase factor inhibiting hypoxia inducible factor. Biochemistry 53:8077-84
Pektas, Serap; Knapp, Michael J (2013) Substrate preference of the HIF-prolyl hydroxylase-2 (PHD2) and substrate-induced conformational change. J Inorg Biochem 126:55-60
Light, Kenneth M; Hangasky, John A; Knapp, Michael J et al. (2013) Spectroscopic studies of the mononuclear non-heme Fe(II) enzyme FIH: second-sphere contributions to reactivity. J Am Chem Soc 135:9665-74
Hangasky, John A; Saban, Evren; Knapp, Michael J (2013) Inverse solvent isotope effects arising from substrate triggering in the factor inhibiting hypoxia inducible factor. Biochemistry 52:1594-602

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