Prolyl hydroxylase (PHD2) and `factor inhibiting HIF' (FIH) are the primary O2-sensors in human cells. PHD2 and FIH are Fe(II)- ?KG-dependent dioxygenases, catalyzing the oxidative decarboxylation of ?KG to produce succinate and CO2 to generate a ferryl oxidant, which hydroxylates specific amino acid residues on the hypoxia inducible factor (HIF) - PHD2 and FIH turn off HIF-controlled gene expression in response to increases in [O2]. The key to sensing O2 is to ensure that O2 activation only occurs after the substrate (HIF) binds to the enzyme. Contacts involved in substrate-triggered O2-activation are poorly understood for the HIF hydroxylases, as well as for the broader class of ?KG-dependent dioxygenases, yet are key to O2-sensing by HIF hydroxylases. The central hypothesis of this proposal is that changes in the second coordination sphere are induced by substrate binding, and these changes increase the rate of ligand exchange, and the O2-affinity at the cofactor. We will test this model using steady-state kinetic probes coupled with electronic spectroscopy and crystallography to correlate reactivity with Fe(II) geometry. These mechanistic studies will increase our understanding of O2 activation by the broader class of ?KG oxygenases, as well as providing specific insight into the reactivity of the HIF hydroxylases. We will extend this understanding into engineering of FIH to perform radical rebound chemistry leading to new C-X bond forming reactions.

Public Health Relevance

HIF hydroxylases are the primary O2-sensors in human cells, acting to control O2-homeostasis through the hypoxia-inducible factor (HIF). HIF controls over 100 genes involved in anaerobic metabolism, angiogenesis, and erythropoiesis, which are crucial for lung development, inflammation, and vascular remodeling. This research will provide molecular-level insight into functionally important parts of the HIF hydroxylases, and may provide a guide to design future therapeutics for ischemia, lung disease, or cancer.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM077413-09
Application #
9483303
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
2007-04-01
Project End
2019-05-31
Budget Start
2018-06-01
Budget End
2019-05-31
Support Year
9
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Massachusetts Amherst
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
153926712
City
Hadley
State
MA
Country
United States
Zip Code
Iyer, Shyam R; Chaplin, Vanessa D; Knapp, Michael J et al. (2018) O2 Activation by Nonheme FeII ?-Ketoglutarate-Dependent Enzyme Variants: Elucidating the Role of the Facial Triad Carboxylate in FIH. J Am Chem Soc 140:11777-11783
Chaplin, Vanessa D; Valliere, Meaghan A; Hangasky, John A et al. (2018) Investigations on the role of a solvent tunnel in the ?-ketoglutarate dependent oxygenase factor inhibiting HIF (FIH). J Inorg Biochem 178:63-69
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

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