# Outer sphere contributions within the thiol dioxygenase active site: a combined structure-function study

> **NIH NIH R15** · UNIVERSITY OF ALABAMA IN TUSCALOOSA · 2023 · $413,696

## Abstract

TITLE. Outer sphere contributions within the thiol dioxygenase active site: a combined structure-function
study
PROJECT SUMMARY. Thiol dioxygenases (TDOs) are a subset of non-heme mononuclear iron oxygenases
that catalyze the O2-dependent oxidation of thiol-bearing substrates to yield the corresponding sulfinic acid.
Cysteine dioxygenase (CDO) and cysteamine dioxygenase (ADO) are the only known mammalian TDOs. These
enzymes catalyze the oxidation of L-cysteine (CYS) and cysteamine (2-aminoethanthiol, CA) to produce cysteine
sulfinic acid (CSA) and hypotaurine (HT), respectively. Ultimately, CSA and HT feed into the mammalian
biosynthetic pathways for taurine (TAU). As the most abundant (sulfonic) amino acid derivative in the body,
TAU plays a variety of essential functions including osmoregulation, conjugation substrate in the synthesis of
bile salts, stabilization of skeletal muscle, maintenance of cardiac rhythm, and an essential neurotransmitter in
embryonic central nervous system development. Despite having structurally similar substrates, CDO and ADO
exhibit remarkable substrate-specificity; exhibiting no (or negligible) cross-reactivity. Significantly, changes in
the cellular expression and/or activity of mammalian TDOs (CDO and ADO) have been correlated with the onset
of various human diseases (cancer, neurodegenerative disorders, rheumatoid arthritis, as well as other metabolic
disorders). An emerging subset of TDOs are referred to as N-terminal cysteinyl dioxygenases (NCOs). These
enzymes are believed to function as physiologic O2-sensors by catalyzing the oxidation of protein N-terminal
CYS-residues to initiate protein degradation. A similar function has recently been reported for mammalian ADO
in controlling regulators of G protein signaling. Therefore ADO functions both as a small molecule TDO, specific
for cysteamine (CA), as well as a NCO by regulating the stability of specific proteins in an O2-dependent fashion.
A notable distinction within the NCO active site is the absence of a conserved sequence of spatially adjacent
amino acids (Ser-His-Tyr) forming a hydrogen bonding network. This structural motif is highly conserved among
structurally characterized TDOs and it is believed to regulate oxygen and thiol-substrate binding affinity at the
TDO Fe-site. The absence of this conserved sequence among NCOs suggests a novel mechanism for O2-sensing
in this class of enzymes. The central theme of the proposed activities is to investigate the role of outer-sphere
interactions within the TDO active site that influence substrate efficiency and recognition. The experiments
proposed involve a series of careful kinetic measurements to identify catalytically essential residues within the
enzymatic active site. In parallel, spectroscopic and computational methods will be employed to develop a
structural model for active site hydrogen-bonding interactions. Similar studies will be performed on members of
the NCO class of enzymes to provide a valuab...

## Key facts

- **NIH application ID:** 10579496
- **Project number:** 2R15GM117511-04
- **Recipient organization:** UNIVERSITY OF ALABAMA IN TUSCALOOSA
- **Principal Investigator:** Bradley S Pierce
- **Activity code:** R15 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $413,696
- **Award type:** 2
- **Project period:** 2015-09-15 → 2026-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10579496

## Citation

> US National Institutes of Health, RePORTER application 10579496, Outer sphere contributions within the thiol dioxygenase active site: a combined structure-function study (2R15GM117511-04). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/10579496. Licensed CC0.

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