# Transformylase Enzymes Dihydrofolate Reductase

> **NIH NIH R01** · PENNSYLVANIA STATE UNIVERSITY, THE · 2022 · $329,025

## Abstract

Project Summary/Abstract
Combining isotopic labeling and gas cluster ion beam surface ionization mass spectrometry (GCIB-SIMS), we
found the purinosome metabolon consists of nine enzymes that channel phosphoribosyl pyrophosphate to
AMP and GMP. The metabolon greatly increases the pathway flux and unexpectedly favors partitioning of IMP
to AMP. Chemical imaging of the cell revealed “hot spots” of these metabolites. We presume that these are
located at the microtubule/mitochondrion interface based on temporal fluorescent imaging that found a
directional movement of the purinosome along the microtubules to the mitochondria. We uncovered two
mechanisms that govern purinosome assembly. One involves the participation of the chaperones HSP90/70
that act to fold two nascent enzymes prior to their incorporation into the purinosome. A second involves the
multiple phosphorylation that occurs on the majority of the pathway enzymes upon purinosome assembly. We
plan to exploit the unique isotopic labeling pattern that signals the presence of a functional purinosome to
survey human cell lines to ascertain the generality of this metabolon at endogenous levels. By extending our
chemical imaging capabilities, we will show that the cellular “hot spots” are loci of the pathway enzymes and
their respective metabolites. The determination of the actual number of enzyme copies within a purinosome will
be undertaken with a novel application of STORM. The actual organization of the metabolon will be probed by
a combination of intracellular photo- and enzyme- specific crosslinking. We will resort to APEX to validate the
identity of nearest neighbors within the purinosome and to discover other adjacent proteins that emanate from
nearby structures such as the mitochondrial membrane. Through a collaborative effort, cell lines that use a
functioning purinosome for purine synthesis will be examined in cells and in xenografts to ascertain the
metabolic requirements and the advantage of the metabolon for cellular proliferation. The proposed studies
have important ramifications for characterizing the importance of the purinosome to human cell viability.

## Key facts

- **NIH application ID:** 10353395
- **Project number:** 5R01GM024129-41
- **Recipient organization:** PENNSYLVANIA STATE UNIVERSITY, THE
- **Principal Investigator:** STEPHEN J BENKOVIC
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $329,025
- **Award type:** 5
- **Project period:** 1977-07-01 → 2025-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10353395

## Citation

> US National Institutes of Health, RePORTER application 10353395, Transformylase Enzymes Dihydrofolate Reductase (5R01GM024129-41). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10353395. Licensed CC0.

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