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

> **NIH NIH R35** · IOWA STATE UNIVERSITY · 2022 · $367,760

## 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 coupling protein
dynamics to enzymatic activity is still largely incomplete. Indeed, while our appreciation of how conformational
dynamics mediate biological function is predominantly based on structural studies on low-complexity, low-
molecular weight systems, enzymes are typically oligomeric, multidomain proteins whose biological function
depends on an intricate coupling among intradomain, interdomain, and intersubunit conformational equilibria.
Without a comprehensive, atomic-resolution understanding of conformational dynamics-mediated, self-
regulatory mechanisms in high-complexity, high-molecular weight enzymes, our ability to understand and
exploit ubiquitous phenomena in biology, such as allosterism and cooperativity, will continue to lag.
Here, we will use NMR combined with other biophysical and biochemical approaches to reveal how the
complex interplay between cofactor/substrate binding and conformational dynamics regulates the activity of
high molecular weight enzymes that are essential for human and bacterial metabolism. The systems of interest
in this proposal are Enzyme I (EI) of the bacterial phosphotransferase system (PTS), and the human RNA
demethylases FTO and Alkbh5. EI is a 128 kDa dimeric enzyme whose activity depends on the synergistic
action of four conformational equilibria that results in a series of large intradomain, interdomain, and
intersubunit structural rearrangements modulated by substrate binding. The PTS is a central regulator of
bacterial metabolism that controls multiple cellular functions, including virulence and biofilm formation, through
phosphorylation-dependent protein-protein interactions. Therefore, understanding EI activity at atomic level will
illuminate the fundamental mechanisms governing long-range interdomain communication in proteins, and may
suggest new therapeutic strategies to combat bacterial infections. The second part of the present proposal
focuses on enzymes that are capable of catalyzing oxidative demethylation of the N6-methyladenosine (m6A).
m6A is the most abundant modification in eukaryotic mRNA. Dynamic regulation of the m6A modification plays
an important role in gene expression, cellular response to external stimuli, oncogenesis, adipogenesis and in
development of other human diseases. We will investigate the mechanisms that regulate the function of the
human RNA demethylases FTO and Alkbh5 with atomic resolution. Our results will guide new strategies to
achieve selective inhibition of FTO and Alkbh5 to control gene expression and to contrast progression of
cancer. In summary, my research program will elucidate the coupling between large scale conformational
changes and...

## Key facts

- **NIH application ID:** 10408689
- **Project number:** 5R35GM133488-04
- **Recipient organization:** IOWA STATE UNIVERSITY
- **Principal Investigator:** Vincenzo Venditti
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $367,760
- **Award type:** 5
- **Project period:** 2019-09-01 → 2023-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10408689

## Citation

> US National Institutes of Health, RePORTER application 10408689, Atomic-level characterization of self-regulatory mechanisms in large multidomain enzymes (5R35GM133488-04). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10408689. Licensed CC0.

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