# Engineered flavin-dependent enzymes for probing redox environment and regulation

> **NIH NIH R00** · SCINTILLON INSTITUTE FOR PHOTOBIOLOGY · 2021 · $248,999

## Abstract

Abstract
Disturbances of the redox environment in various cellular compartments are linked to many pathologies,
including neurodegenerative diseases, cancer, cardiovascular disease and aging. Since the ratio of oxidized to
reduced nicotinamide adenine dinucleotides is a major contributor to the cellular redox environment,
engineering tools to perturb this ratio would enable the study the role of redox imbalances in driving these
pathologies. In this work we examine the fundamental mechanisms used by some microorganisms to control
their optimal redox environment, as well as the possible applications of these mechanisms in mammalian cells.
A number of microaerophilic bacteria and protozoa lack a conventional multi-complex respiratory chain, and
instead rely on enzymes which catalyze a H2O-forming NADH oxidase reaction to recycle NAD+ and to
eliminate toxic oxygen from the environment. Since the product of this reaction is benign water and not
hydrogen peroxide (H2O2), these enzymes represent attractive reagents to perturb metabolism in mammalian
cells. In this work we propose to engineer a bacterial H2O-forming NADH oxidase towards NADPH specificity.
This NADPH oxidase will then be expressed in different compartments of mammalian cells, and its effects on
cell viability and metabolism will be systematically evaluated. Since no tool has previously been reported to
safely increase the NADP+/NADPH ratio in cells in a compartment specific manner, our work will provide
fundamental insights into NADPH metabolism and its regulation, as well as into the sizes of NAD(P)H pools in
different cellular compartments. Our work also explores the mechanisms of how microaerophilic human
protozoan parasites like Giardia intestinalis, Trichomonas vaginalis and Entamoeba histolytica, which lack
conventional respiratory chains, control their redox environments in order to support energy metabolism. We
have identified in T.vaginalis a natural protein which represents a fusion between a flavodiiron core protein with
its redox partners: rubredoxin and rubredoxin oxidoreductase. This fusion protein catalyzes a four-electron
reduction of oxygen to water using reducing equivalents of NAD(P)H. Our studies of the structure and
mechanism of this fusion protein will provide insights into how these human parasites are able to maintain their
optimal redox environment. These mechanisms can be attractive targets for therapeutic intervention, allowing
us to combat diseases caused by human protozoan parasites.

## Key facts

- **NIH application ID:** 10112916
- **Project number:** 5R00GM121856-05
- **Recipient organization:** SCINTILLON INSTITUTE FOR PHOTOBIOLOGY
- **Principal Investigator:** Valentin Cracan
- **Activity code:** R00 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $248,999
- **Award type:** 5
- **Project period:** 2017-05-15 → 2022-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10112916

## Citation

> US National Institutes of Health, RePORTER application 10112916, Engineered flavin-dependent enzymes for probing redox environment and regulation (5R00GM121856-05). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10112916. Licensed CC0.

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