# Switching Between One and Two Electron Mechanisms in the Nitroreductase Superfamily

> **NIH NIH R01** · JOHNS HOPKINS UNIVERSITY · 2022 · $316,418

## Abstract

PROJECT SUMMARY/ABSTRACT
 Bioinformatic tools can offer exquisite insight into the ever growing quantities of genomic
information. Gene sequences alone are usually sufficient to predict their membership in large
structural superfamilies as well as more specific subfamilies that may share related functions.
However, the scope of sequence data has so exceeded the realm of biochemical characterization that
many subfamilies lack any structural, chemical or biological description. Strategies for predicting
function within such groups are still in their infancy and will require a substantial investment in
biochemistry to develop the essential links between sequence, structure and function. The
nitroreductase superfamily offers an enticing platform to establish such a link to the redox chemistry
of the flavin mononucleotide associated with these enzymes. This endeavor will capitalize on the
extensive characterization of a few representative nitroreductases that lend their name to this
superfamily and the recent release of a sequence similarity network that introduces a sophisticated
order to more than 24,000 unique sequences.
 The iodotyrosine deiodinase group of this superfamily will now be investigated for its ability to
promote sequential single electron transfer and suppress hydride transfer as a counterpoint to the
ability of nitroreductases to act in the reverse by suppressing single electron transfer and promoting
hydride transfer. Experimental strategies will include isotope and viscosity effects on catalysis by
steady-state and rapid kinetics. Concurrently, substrate and flavin analogues will be used to measure
the relative efficiencies of proton and electron transfer during deiodination. Key residues involved in
these processes will then be identified by site-directed mutagenesis. A predictive understanding of
flavin's redox chemistry will be constructed from these results and prior knowledge derived from
nitroreductases. The principles developed by this comparison will be refined by two approaches.
One will use structural and mechanistic data to direct conversion of a native deiodinase into a
nitroreductase and vice versa. The other will examine the redox properties of superfamily members
that have not yet been tested but can be anticipated from the correlations developed by this project.

## Key facts

- **NIH application ID:** 10335771
- **Project number:** 5R01GM130937-04
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** STEVEN E ROKITA
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $316,418
- **Award type:** 5
- **Project period:** 2019-02-01 → 2024-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10335771

## Citation

> US National Institutes of Health, RePORTER application 10335771, Switching Between One and Two Electron Mechanisms in the Nitroreductase Superfamily (5R01GM130937-04). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10335771. Licensed CC0.

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