# Molecular mechanisms of alkane hydroxylase (AlkB) reactivity and selectivity

> **NIH NIH R01** · BARNARD COLLEGE · 2022 · $291,747

## Abstract

Project Summary
Reactions with atmospheric oxygen are required for many life-sustaining processes. The class-III diiron
proteins use oxygen to selectively oxidize lipids and to put OH groups into molecules in critical
biosynthetic pathways. Class-III diiron enzymes play essential roles in many aspects of lipid synthesis
and metabolism and are linked to human health problems including obesity, diabetes, attention-deficit
disorder, and neurodegeneration. They are also crucial in the natural bioremediation of oil. There is a
dearth of mechanistic information about this family of membrane enzymes, primarily because their
membrane-associated nature makes them very difficult to purify and study. Alkane monooxygenase
(AlkB) is a member of the class-III integral membrane diiron proteins along with fatty acid desaturases
and fatty acid hydroxylases. The amino acid sequence of AlkB indicates that it is not structurally similar
to other enzymes with similar functions. Determining its three-dimensional structure is a feat that has
eluded scientists for decades.
In an important step forward in preliminary work, PI Austin and co-Investigator Feng have solved the
first structure of AlkB with a bound substrate and shown that it serves as an excellent model system to
understand the catalytic mechanism of class-III diiron proteins. This breakthrough, together with the
establishment of a novel assay for rapid functional characterization and the development of a suite of
AlkB active AlkB homologs, paves the way to answering key questions about these important
metalloenzymes. The PIs will integrate structural, functional, biochemical, computational, and
spectroscopic studies to determine the three-dimensional structure of the diiron active site, identify
determinants of substrate specificity, learn how AlkB is activated by its partner protein, and probe how
the presence of a covalently bound electron-transfer partner, found only in a class of gram positive
bacteria, changes the reactivity of this enzyme family.
In so doing, they will expand the basic knowledge of strategies to break and make key chemical bonds,
which may lead to the development of new synthetic routes to make life-saving and life-extending
molecules. Their work will also provide critical insights to efforts to target this family of enzymes for
therapeutic purposes.

## Key facts

- **NIH application ID:** 10451683
- **Project number:** 5R01GM130989-03
- **Recipient organization:** BARNARD COLLEGE
- **Principal Investigator:** Rachel Narehood Austin
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $291,747
- **Award type:** 5
- **Project period:** 2020-09-15 → 2024-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10451683

## Citation

> US National Institutes of Health, RePORTER application 10451683, Molecular mechanisms of alkane hydroxylase (AlkB) reactivity and selectivity (5R01GM130989-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10451683. Licensed CC0.

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