# Atomic-level characterization of self-regulatory mechanisms in large multidomain enzymes

> **NIH NIH R35** · IOWA STATE UNIVERSITY · 2024 · $364,287

## Abstract

PROJECT SUMMARY/ABSTRACT
Enzymes are remarkable nanomachines that play a myriad of essential functions in cellular metabolism.
Modulation of enzyme structure and flexibility by cofactor/substrate binding provides an important source of
regulation of enzyme function, yet our understanding of the fundamental mechanisms by which concerted protein
motion facilitate enzymatic activity is still largely incomplete. Indeed, while several studies have appeared in the
past two decades describing how conformational dynamics mediate the biological function of small proteins, our
understanding of how the coupling among multiple conformational equilibria determines the activity of large
multidomain systems continues to lag. The overall goal of this proposal is using and developing integrated
approaches combining NMR with complementary biophysical and biochemical tools to reveal how modulation of
local disorder upon cofactor/substrate binding affects concerted motions and regulates the activity of high
molecular weight enzymes that are essential for human and bacterial metabolism. This combination of tools
sensitive to protein motion brings a newly detailed picture of high molecular weight enzyme function. The
enzymes characterized in this proposal are Enzyme I (EI) of the bacterial phosphotransferase system (PTS),
and the AlkB family of nucleic acid demethylases – together these distinct classes of enzymes show how the
relationship between local disorder and concerted domain motions can be probed by this combination of tools
and demonstrate how essential these mechanisms are across diverse enzyme classes. In particular, the EI
enzymatic activity depends upon the synergistic action of four conformational equilibria that results in a series of
large intradomain, interdomain, and intersubunit structural rearrangements modulated by substrate binding.
Therefore, our efforts to uncover EI function at atomic level will reveal how modulation of local disorder mediates
long-range interdomain communication and, ultimately, regulates the activity of this essential bacterial enzyme.
The AlkB dioxygenases are flexible enzymes that are known to undergo modulation of their internal dynamics
upon substrate binding. Our studies will visualize conformational disorder in apo and holo AlkB enzymes with
unprecedented atomic-resolution details, and will reveal how residual disorder at the active site determines
substrate selectivity. In addition, we will investigate a number of complexes formed by AlkB proteins with their
inhibitors. We expect these results to indicate new strategies, based on selective perturbation of conformational
disorder, to develop AlkB inhibitors with subfamily selectivity. In summary, my research program will elucidate
the coupling between large scale conformational changes and function in two distinct classes of high molecular
weight multidomain enzymes, providing new insights for future therapies for obesity and cancer as well as novel
antibiotic targets....

## Key facts

- **NIH application ID:** 10837730
- **Project number:** 5R35GM133488-07
- **Recipient organization:** IOWA STATE UNIVERSITY
- **Principal Investigator:** Vincenzo Venditti
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $364,287
- **Award type:** 5
- **Project period:** 2019-09-01 → 2028-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10837730

## Citation

> US National Institutes of Health, RePORTER application 10837730, Atomic-level characterization of self-regulatory mechanisms in large multidomain enzymes (5R35GM133488-07). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10837730. Licensed CC0.

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