# Mechanisms of Iron-Sulfur Dependent Reactions

> **NIH NIH R35** · PENNSYLVANIA STATE UNIVERSITY, THE · 2024 · $370,099

## Abstract

PROJECT SUMMARY/ABSTRACT
The prevalence and significance of methylation reactions in biology is well established. Methyl groups are
appended to a wide array of biological molecules, including numerous small-molecule metabolites and natural
products, and various macromolecules, such as proteins, DNA, RNA, carbohydrates, and lipids. In the vast
majority of methylation reactions, S-adenosylmethionine (SAM) is the source of the appended methyl group. In
classical methyltransferase reactions, strong nucleophiles such as oxygen, nitrogen, and sulfur attack the sp3-
hybridized methyl group of SAM in a polar SN2 reaction, affording S-adenosylhomocysteine as a co-product.
Carbon atoms can also be methylated by this mechanism, but only if a suitably nucleophilic carbanion can be
generated. Relatively recently, it has come to light that SAM can be used to methylate inert carbon or
phosphinate phosphorous atoms via pathways involving radical intermediates. These noncanonical SAM-
dependent methylations are found in numerous biosynthetic pathways for antibiotic, antifungal, anticancer,
and herbicidal natural products, and are catalyzed exclusively by enzymes within the radical S-
adenosylmethionine superfamily. Radical SAM methylases currently consist of three classes (Class A, Class B,
and Class C) based on structural architecture, cofactor requirement, and mechanism of action. Class A
enzymes use a Cys dyad to catalyze methylation of sp2-hybridized carbon centers. Class B enzymes use
cobalamin cofactors to catalyze methylation of both sp2- and sp3-hybridized carbon centers. Class C enzymes
use two simultaneously bound molecules of SAM to methylate sp2-hybridized carbon centers. In all cases, the
appended methyl group derives from a second molecule of SAM. This work will continue our efforts to
understand how these radical SAM methylases work, with a particular focus on efforts to determine structures
of these enzymes with bound substrates, cofactors, and intermediates. Important systems include RNA
methylases that are involved in antibiotic resistance, as well as methylases that are involved in the biosynthesis
of important antibiotics, such as thiostrepton A, nosiheptide, and carbapenems, the antibiotics currently of last
resort.

## Key facts

- **NIH application ID:** 10806999
- **Project number:** 5R35GM122595-08
- **Recipient organization:** PENNSYLVANIA STATE UNIVERSITY, THE
- **Principal Investigator:** SQUIRE J. BOOKER
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $370,099
- **Award type:** 5
- **Project period:** 2017-04-01 → 2027-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10806999

## Citation

> US National Institutes of Health, RePORTER application 10806999, Mechanisms of Iron-Sulfur Dependent Reactions (5R35GM122595-08). Retrieved via AI Analytics 2026-06-04 from https://api.ai-analytics.org/grant/nih/10806999. Licensed CC0.

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