About

Squire J. Booker is an Emeritus Evan Pugh University Professor of Chemistry, Biochemistry, and Molecular Biology at Penn State University. His research focuses on elucidating the chemical mechanisms by which enzymes containing iron-sulfur clusters catalyze various chemical reactions. Most of his work deals with members of the Radical S-adenosylmethionine (SAM) Superfamily, a diverse group of enzymes that utilize radical chemistry to catalyze transformations involved in post-transcriptional and post-translational modifications, cofactor biosynthesis, secondary metabolite biosynthesis, and enzyme activation. These enzymes share a [4Fe-4S] cluster cofactor that facilitates the reductive cleavage of S-adenosylmethionine to generate the 5’-deoxyadenosyl radical, which is used to initiate radical chemistry on the substrate, leading to a wide array of transformations including methylations, sulfur insertions, decarboxylations, and complex rearrangements. Using biochemical, analytical, structural, and spectroscopic techniques, his lab characterizes these complex reaction mechanisms to provide insights relevant to human health and disease, and to understand how Nature solves difficult chemical problems.

Research topics

• Biochemistry
• Chemistry
• Biology
• Computational biology
• Stereochemistry
• Evolutionary biology
• Organic chemistry
• Combinatorial chemistry
• Physics

Selected publications

• Structural Basis for C ⁸ methylation of 23S ribosomal RNA by Cfr

  bioRxiv (Cold Spring Harbor Laboratory) · 2026-03-02

  articleOpen accessSenior authorCorresponding

  Abstract Cfr methylates C 8 of adenosine 2503 (A2503) in 23 S ribosomal RNA (rRNA) and will also methylate C 2 of A2503 after methylating C 8 . C 8 methylation confers resistance to more than five classes of clinically used antibiotics, highlighting it as a worrisome mechanism of antibiotic resistance. Here, we report the structure of Cfr, determined by cryogenic electron microscopy (Cryo-EM). Despite its small size (∼36 kDa), we exploit a transient protein–RNA crosslink that forms during catalysis, which requires Cys105 to resolve. Using a Cfr Cys105Ala variant and an 87-nucleotide strand of rRNA, we isolate the crosslinked species and determine its structure to 3.0 Å resolution. Notably, the 87-mer rRNA adopts an L-shaped conformation characteristic of tRNAs, rather than the conformation it assumes in the ribosome. One Sentence Summary Cryo-EM structure of Cfr, a radical S-adenosylmethionine methylase that confers antibiotic resistance

  PublisherDOI

• Structural and Spectroscopic Basis for Catalysis by a Class C Radical <i>S</i> -adenosylmethionine Methylase Involved in Nosiheptide/Nocathiacin Biosynthesis

  bioRxiv (Cold Spring Harbor Laboratory) · 2026-01-20

  articleOpen accessSenior authorCorresponding

  Abstract Nosiheptide (NOS) is a ribosomally synthesized and post-translationally modified peptide (RiPP) natural product that exhibits potent antibiotic activity against multiple bacterial pathogens. NOS features a core macrocyclic peptide containing thiazoles, dehydrated serine and threonine residues, and a 3-hydroxypyridine ring. In addition to the macrocycle, NOS possesses a side-ring system formed by a 3-methyl-2-indolic acid (MIA) bridge that connects to glutamyl and cysteinyl residues on the core peptide via ester and thioester linkages. This unique side-ring is installed by the class C radical S -adenosylmethionine (SAM) methylase NosN. Here, we report three X-ray crystal structures of the NosN homolog, NocN, at resolutions of 1.4 Å, 1.84 Å, and 1.78 Å under anaerobic conditions, representing the first structural characterization of a class C radical SAM methylase. The structures reveal clear electron density for two bound SAM molecules. Remarkably, the C5′ atom of SAM I , which coordinates to the [Fe 4 S 4 ] cluster, lies 3.5 Å from the methyl group of SAM II and is properly positioned for direct hydrogen atom abstraction. A structure containing a product mimic illustrates how NocN engages its substrate and identifies Tyr276 as a key catalytic residue. The structure further suggests that the sulfonium center of SAM II may undergo epimerization to facilitate radical attack. Finally, electron paramagnetic resonance spectroscopy identifies a paramagnetic species consistent with the addition of the SAM II -derived methylene radical to the MIA substrate.

  PublisherOA PDFDOI

• Structural basis for iterative methylation by a cobalamin-dependent radical <i>S</i> -adenosylmethionine enzyme in cystobactamids biosynthesis

  Proceedings of the National Academy of Sciences · 2026-01-21

  articleOpen accessSenior author

  Cystobactamids are nonribosomal peptide natural products that function as DNA gyrase inhibitors, exhibiting significant antibacterial activity. They are isolated from Cystobacter sp. Cbv34 and contain various alkoxy groups on para-aminobenzoic acid moieties, which are believed to play a crucial role in antibacterial functions. The alkoxy groups are generated by iterative methylations on a methoxy group by the cobalamin (Cbl)-dependent radical S -adenosylmethionine (SAM) enzyme CysS. CysS catalyzes up to three methylations to give ethoxy, isopropoxy, sec-butoxy, and tert-butoxy groups. For each methylation, CysS uses a ping–pong mechanism in which two molecules of SAM are consumed. One SAM is used to methylate cob(I)alamin, while another generates a 5′-deoxyadenosyl 5′-radical to initiate substrate methylation. However, little is known about how the enzyme promotes both Cbl methylation and iterative substrate methylation, which occur by polar S N 2 and radical processes, respectively. Here, we report three X-ray crystal structures of a homolog of CysS from Corallococcus sp. CA054B . Two were determined in the presence of methoxy- and ethoxy-containing substrates, showing how CysS accommodates substrates and products during iterative methylation. The third structure, determined in the absence of a substrate, exhibits structural changes that reorient the SAM’s conformation to allow for the methylation of cob(I)alamin.

  PublisherDOI

• Introducing the Tutorial Manuscript Type at the ACS Au Community Journals

  ACS Nanoscience Au · 2025-07-10

  editorialOpen access1st authorCorresponding
  PublisherDOI

• Iron–sulfur cluster with double duty

  Nature Catalysis · 2025-08-22 · 1 citations

  articleSenior author
  PublisherDOI

• Structural basis for catalysis by human lipoyl synthase

  Nature Communications · 2025-07-10 · 3 citations

  articleOpen accessSenior author

  Abstract Lipoic acid is an essential cofactor in five mitochondrial multiprotein complexes. In each complex, it is tethered in an amide linkage to the side chain of a conserved lysyl residue on a lipoyl carrier protein or lipoyl domain to afford the lipoyl cofactor. Lipoyl synthase catalyzes the last step in the biosynthesis of the lipoyl cofactor, the addition of two sulfur atoms to carbons 6 and 8 of an octanoyllysyl residue of the H protein, the lipoyl carrier protein of the glycine cleavage system. Lipoyl synthase, a member of the radical S-adenosylmethionine superfamily, contains two [Fe 4 S 4 ] clusters, one of which is sacrificed during catalysis to supply the appended sulfur atoms. Herein, we use X-ray crystallography to characterize several stages in lipoyl synthase catalysis and present a structure of an intermediate wherein the enzyme is cross-linked to the H protein substrate through a 6-mercaptooctanoyl ligand to a [Fe 3 S 4 ] cluster.

  PublisherDOI

• <i>ACS Bio &amp; Med Chem Au</i>: Introducing the 2024 Rising Stars in Biological, Medicinal, and Pharmaceutical Chemistry

  ACS Bio & Med Chem Au · 2025-04-08

  editorialOpen access1st authorCorresponding
  PublisherDOI

• In vitro maturation of fully active [FeFe]-hydrogenase in a defined system including the iron carrier NfuA

  Proceedings of the National Academy of Sciences · 2025-09-22 · 1 citations

  articleOpen access

  The [FeFe]-hydrogenase employs an active-site 6Fe H-cluster to catalyze the reversible reduction of protons to H 2 . A [4Fe-4S] subcluster of the H-cluster is synthesized by housekeeping iron-sulfur cluster assembly machinery, and then dedicated hydrogenase maturation enzymes, together with components of the glycine cleavage system, build and deliver a [2Fe] subcluster to generate the full H-cluster. Here, we report that the Escherichia coli iron-sulfur carrier protein NfuA supports in vitro maturation of fully active [FeFe]-hydrogenase, with H 2 production rates comparable to that of the in vivo - matured Chlamydomonas reinhardtii [FeFe]-hydrogenase ( Cr HydA). Inclusion of NfuA in the in vitro maturation process improves its efficacy by delivering the iron essential for formation of the [Fe II (cys)(CN)(CO) 2 ] – synthon at the dangler iron site of the HydG auxiliary cluster. NfuA serves an additional role in reconstituting and maintaining the catalytically essential iron-sulfur clusters on the maturase enzymes HydE, HydF, and HydG. Further inclusion of a high CO affinity myoglobin variant (Mb H64L ) sequesters free CO generated during the maturation process, minimizing formation of the CO-inhibited H ox -CO enzyme state, significantly increasing hydrogenase activity. The addition of NfuA and Mb H64L to the fully defined maturation system thus results in an in vitro [FeFe]-hydrogenase maturation system that generates highly active enzyme while providing insights into factors important to in vivo maturation.

  PublisherDOI

• Structural Basis for Iterative Methylation by a Cobalamin-dependent Radical S-Adenosylmethionine Enzyme in Cystobactamids Biosynthesis

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-11-17

  preprintOpen accessSenior authorCorresponding

  Abstract Cystobactamids are non-ribosomal peptide natural products that function as DNA gyrase inhibitors, exhibiting significant antibacterial activity. They are isolated from Cystobacter sp. Cbv34 and contain various alkoxy groups on para-aminobenzoic acid moieties, which are believed to play a crucial role in antibacterial functions. The alkoxy groups are generated by iterative methylations on a methoxy group by the cobalamin (Cbl)-dependent radical S-adenosylmethionine (SAM) enzyme CysS. CysS catalyzes up to three methylations to give ethoxy, isopropoxy, sec-butoxy, and tert-butoxy groups. For each methylation, CysS uses a ping-pong mechanism in which two molecules of SAM are consumed. One SAM is used to methylate cob(I)alamin, while another generates a 5′-deoxyadenosyl 5′-radical to initiate substrate methylation. However, little is known about how the enzyme promotes both Cbl methylation and iterative substrate methylation, which occur by polar S N 2 and radical processes, respectively. Here, we report three X-ray crystal structures of a homolog of CysS from Corallococcus sp. CA054B . Two were determined in the presence of methoxy- and ethoxy-containing substrates, showing how CysS accommodates substrates and products during iterative methylation. The third structure, determined in the absence of a substrate, exhibits structural changes that reorient the SAM’s conformation to allow for the methylation of cob(I)alamin.

  PublisherOA PDFDOI

• Radical Fluoromethylation Enabled by Cobalamin-Dependent Radical SAM Enzymes

  ACS Bio & Med Chem Au · 2025-05-06 · 6 citations

  articleOpen accessSenior authorCorresponding

  Fluorine is an important atom in many drugs because it can improve the efficacy and metabolic stability of many molecules. Strategies to incorporate monofluoromethyl groups in drugs have been limited and have received less attention than strategies for difluoromethylation or trifluoromethylation. Previously, we and others reported the enzymatic monofluoromethylation of several biologically relevant metabolites based on the transfer of a fluoromethyl group from analogs of S-adenosylmethionine (SAM) to various nucleophiles (carbon, oxygen, nitrogen, sulfur, and carbon) through a polar SN2 mechanism. However, this strategy is limited to molecules containing nucleophilic target atoms. Inspired by a subset of enzymes within the radical SAM superfamily that can methylate inert carbon atoms, we developed an enzymatic strategy to transfer fluoromethyl groups to unactivated carbon atoms. This strategy leverages the ability of halide methyltransferase to generate a transient fluoromethyl-containing SAM analog. Our studies show that S-adenosyl-L-(fluoromethyl)methionine can undergo reductive cleavage to a 5′-deoxyadenosyl 5′-radical, which initiates radical-dependent fluoromethylation through substrate hydrogen-atom abstraction. Adding fluoromethyl groups to unactivated C–H bonds using radical SAM enzymes is a powerful approach that can be used to derivatize molecules of interest where SN2-based fluoromethylation is precluded.

  PublisherDOI

Recent grants

• NIH Grant R21AI111419

  NIH · $398k · 2017

• Structure, Mechanism, and Regulation of Quinolinate Synthase, the First Committed Step in Bacterial NAD Biosynthesis

  NSF · $1.0M · 2012–2017

• NIH Grant R01GM103268

  NIH · $1.3M · 2017

• NIH Grant R01GM101957

  NIH · $1.0M · 2017

• PECASE: Mechanistic Studies on Cyclopropane Fatty Acid Synthase

  NSF · $557k · 2002–2008

Frequent coauthors

• Tyler L. Grove

  University of North Carolina at Chapel Hill

  49 shared

• Carsten Krebs

  Pennsylvania State University

  40 shared

• Douglas M. Warui

  Pennsylvania State University

  28 shared

• Alexey Silakov

  28 shared

• Nicholas D. Lanz

  United States Food and Drug Administration

  26 shared

• Bo Wang

  Pennsylvania State University

  24 shared

• Anthony J. Blaszczyk

  22 shared

• Hayley Knox

  Pennsylvania State University

  19 shared

Awards & honors

• Elected to the National Academy of Sciences (2019)
• Eberly Distinguished Chair in Science (2017)
• Elected to the American Academy of Arts and Sciences (2017)
• Lloyd N. Ferguson Distinguished Lecturer at Cal State, Los A…
• Penn State Faculty Scholar Medal (2016)