# Learning the Steps to Metalloenzyme Choreography

> **NIH NIH R00** · EMORY UNIVERSITY · 2021 · $249,000

## Abstract

Abstract. Nature is the ultimate synthetic chemist, and metalloenzymes the catalysts of choice. To achieve their
unprecedented functional diversity, metalloenzymes modulate the structural and electronic properties of the
protein environment in which the reaction proceeds, thus enabling difficult transformations that are often
challenging for synthetic chemists. Although enormous progress has been made in the study of structural and
mechanistic enzymology, the dynamics of these reactions remain poorly understood. This proposal uses
innovative methods to characterize the concerted atomic and electronic variations that facilitate catalysis in two
medicinally-relevant classes of iron-containing metalloenzymes. Experiments will be rationally designed based
on known mechanistic behavior to!visualize otherwise transient catalytic intermediates both in solution and in
crystallo. The first specific aim focuses on the mode of substrate binding and ferryl-heme formation in the
immunosuppressive human enzyme indoleamine 2,3-dioxygenase. This project will be completed during the K99
funding period and will involve crystallographic characterization of both the reactant complex and an
enzymatically-generated ferryl species. Intermediates will be stabilized using substrate/cofactor mimics, site-
directed mutants, or freeze-trapping methods exploiting the pH dependence of the reaction. During the
independent phase, the second aim will use a similar approach applied to study the relatively uncharacterized
class of cobalamin-dependent radical S-adenosylmethionine methyltransferases, involved in the biosynthesis of
potent antibiotics. Although intermediate state models are desirable, any structure would provide unique insight
into the mechanism of this class as none have been published to-date. The third and final aim, to develop and
apply a laser pump/X-ray probe setup for the simultaneous collection of X-ray crystallographic and spectroscopic
real time data, will be pursued concurrently. Princeton University provides the ideal environment in which to
initiate pursuit of these goals, with excellent facilities and access to the world’s leading experts in bioinorganic
chemistry. These resources will be complemented by my co-mentor at the Pennsylvania State University. Having
received a formal education in physics, my short-term goals are to acquire wet lab skills necessary to generate,
characterize and troubleshoot my own samples. I will be trained in protein expression and purification as well as
UV-vis, EPR and sophisticated crystallographic characterization of metalloenzymes. During the mentored phase,
I will attend a formal course in heterologous expression and purification of proteins, as well as a number of other
workshops/conferences designed to increase my exposure to these techniques and learn the management skills
required to be a successful PI. The expertise I acquire in the K99 period will be necessary to study more complex
systems and develop laser pump/...

## Key facts

- **NIH application ID:** 10174959
- **Project number:** 5R00GM129460-04
- **Recipient organization:** EMORY UNIVERSITY
- **Principal Investigator:** Katherine Marie Davis
- **Activity code:** R00 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $249,000
- **Award type:** 5
- **Project period:** 2018-09-01 → 2023-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10174959

## Citation

> US National Institutes of Health, RePORTER application 10174959, Learning the Steps to Metalloenzyme Choreography (5R00GM129460-04). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10174959. Licensed CC0.

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