About

Amie Boal is a professor of Chemistry and of Biochemistry and Molecular Biology at Penn State University, located at 326 Benkovic, University Park, PA. Her research focuses on the structural differences between members of large metalloenzyme superfamilies that share common features but promote different reactions or use distinct cofactors. Her work involves understanding the mechanisms of various metalloenzymes, including heme oxygenase-like metalloenzymes, and exploring their roles in biological processes. Boal has contributed to the discovery, structure, and mechanism of enzymes such as tetraether lipid synthase and has provided structural insights into auxiliary cofactor usage by radical S-adenosylmethionine enzymes. She has also studied enzyme mechanisms related to hydroxylation, halogenation, and rare-earth element separation, among others. Her research has significant implications for biochemistry, inorganic chemistry, and biotechnological applications, including green technology and environmental sensing.

Research topics

• Chemistry
• Biochemistry
• Stereochemistry
• Biology
• Organic chemistry
• Crystallography
• Computational biology
• Inorganic chemistry
• Physics
• Internal medicine
• Photochemistry
• Combinatorial chemistry
• Cell biology

Selected publications

• Azetidine amino acid biosynthesis by non-haem iron-dependent enzymes

  Nature Chemistry · 2025-10-21 · 5 citations

  articleOpen access

  Abstract Azetidine, a four-membered aza-cycle, is a crucial structure in many bioactive compounds and drugs. However, their biosynthesis is frequently enigmatic. Here we report the mechanism of azetidine amino acid (polyoximic acid) biosynthesis in the polyoxin antifungal pathway. Genetic, enzymological and structural experiments revealed that PolF is a member of haem-oxygenase-like dimetal oxidase and/or oxygenase (HDO) superfamily, and this enzyme alone is sufficient for the transformation of l -isoleucine ( l -Ile) and l -valine to their azetidine derivatives via a 3,4-desaturated intermediate. Mechanistic studies of PolF suggested that a μ-peroxo-Fe(III) 2 intermediate is directly responsible for the unactivated C–H bond cleavage, and the post-H-abstraction reactions, including the C–N bond formation, probably proceed through radical mechanisms. We also found that PolE, a member of the DUF6421 family, is an Fe and pterin-dependent oxidase that catalyses the desaturation of l -Ile, assisting PolF by increasing the flux of l -Ile desaturation. The results provide important insights into azetidine biosynthesis and the catalytic mechanisms of HDO enzymes in general.

  PublisherOA PDFDOI

• Unusual O–H Activation-Initiated C–C Bond Cleavage Reaction by a Nonheme Fe Enzyme in Antifungal Nucleoside Biosynthesis

  Journal of the American Chemical Society · 2025-08-11 · 2 citations

  articleOpen accessCorresponding

  Fe(II)- and α-ketoglutarate (α-KG)-dependent enzymes catalyze diverse reactions, generally initiated by FeIV=O mediated cleavage of C–H bonds with bond dissociation energies (BDE) of up to ∼100 kcal/mol. Here, we report the discovery of a novel reaction initiated by a significantly more challenging O–H bond cleavage (>100 kcal/mol). This activity was identified in PolD, an enzyme that regulates the sugar size in antifungal nucleoside biosynthesis by catalyzing the transformation of a bicyclic eight-carbon sugar substrate, 5′-amino-6′-hydroxy-octosyl acid 2′-phosphate (AHOAP), into a monocyclic six-carbon product, aminohexuronic acid 2′-phosphate (AHAP). Our studies demonstrate that PolD catalyzes a two-step reaction, in which AHOAP is first oxidized to 5′-amino-6′-keto-octosyl acid 2′-phosphate (AKOAP) via typical C–H activation, followed by a unique C–C bond cleavage on AKOAP to AHAP initiated by O–H activation. X-ray crystal structures of PolD and its homologue, PasI, the latter solved in complex with AHOAP, succinate, and vanadyl, a structural mimic of the FeIV-oxo intermediate, reveal a substrate binding mode that is consistent with both C–H and O–H homolysis. A comparison of the three enzymes, PasI, PolD, and MalI, all of which exhibit distinct C–C bond cleavage activities, suggests that precise substrate positioning to bring the target OH group of AKOAP close to the FeIV-oxo intermediate is critical for hydrogen atom transfer from this functional group. These results indicate a novel reactivity of the FeIV═O intermediate in Fe/α-KG enzymes, thereby expanding the reaction scope of this enzyme superfamily. The results also reveal the molecular mechanism of the divergent biosynthesis of antifungal nucleosides.

  PublisherOA PDFDOI

• A Structurally Divergent Class Ia Ribonucleotide Reductase from a Tick-Borne Pathogen

  Biochemistry · 2025-08-28

  articleOpen accessSenior authorCorresponding

  Ribonucleotide reductases (RNRs) generate 2′-deoxynucleotides for DNA biosynthesis, a reaction essential to all life. Class I RNRs have two subunits, α and β. α binds and reduces the substrate, whereas β oxidizes one of the cysteines in α to a C3′–H-bond-cleaving thiyl radical to begin the reaction. The α-Cys oxidant in β is variously a tyrosyl radical (Y•) generated by a diiron or dimanganese cluster, a high-valent dimetal cluster [Mn(IV)/Fe(III) or Mn2(IV/III)], or a dihydroxylphenylalanine (DOPA) radical that operates without need of a transition metal. The metal (in)dependence of the Cys oxidant in β correlates loosely with sequence-similarity groupings. We show here that Francisella hispaniensis (Fh) β, which lies within an uncharacterized sequence cluster that contains orthologs from multiple human pathogens, harbors a Fe2(III/III)/Y• cofactor, as in class Ia RNRs from eukaryotes and Escherichia coli. Fh β has several unusual structural features that may reflect adaptation to the bacterium’s environment(s). In its apo form, an unwound helix everts a metal ligand toward solvent, and the radical-harboring Y points away from the diiron cluster. An additional aromatic residue (W194), conserved within the sequence cluster, is found close to the universally conserved W37, which is thought to mediate α-Cys oxidation in all class I enzymes. The Y• in resting β is remarkably resistant to reduction by hydroxyurea but becomes 8000 times more sensitive when β is engaged in turnover with α. These structural and functional distinctions could be counter measures against host redox defenses that would target the pathogen’s RNR and its cofactor.

  PublisherOA PDFDOI

• Heme Oxygenase–Like Metalloenzymes

  Annual Review of Biochemistry · 2025-03-27 · 13 citations

  reviewOpen accessSenior author

  Heme oxygenase (HO)-like metalloenzymes are an emerging protein superfamily diverse in reaction outcome and mechanism. Found primarily in bacterial biosynthetic pathways, members conserve a flexible protein scaffold shared with the heme catabolic enzyme, HO, and a set of metal-binding residues. Most HO-like metalloenzymes assemble a diiron cluster, although manganese-iron and mononuclear iron cofactors can also be accommodated. In the canonical HO-like diiron oxygenases/oxidases (HDOs), an Fe 2 (II/II) complex reacts with O 2 to form a peroxo-Fe 2 (III/III) intermediate ( P ), common to all HDOs studied to date. The HO-like scaffold confers both distinctive metal-binding properties, allowing for dynamic cofactor assembly and disassembly, and unusual reactivity to its associated metallocofactor. These features may prove to be important in HDO-mediated catalysis of the fragmentation and rearrangement reactions that remain unprecedented among other dinuclear iron enzymes. Much of the sequence space in the HO-like metalloenzyme superfamily remains unexplored, offering exciting opportunities for the discovery of new mechanisms and reactivities.

  PublisherDOI

• Cooperative ligand binding in a bacterial heme-based oxygen sensor

  Journal of Biological Chemistry · 2025-12-08

  articleOpen access

  <h2>Abstract</h2> Bacteria modulate essential phenotypes in response to external signals such as the availability of molecular oxygen (O₂). A class of direct O₂-sensing heme proteins, globin coupled sensors, have been implicated in O₂-dependent regulation of pathogenic phenotypes including biofilm formation, motility, and virulence. While cooperative O₂ binding is well known in both mammalian and prokaryotic hemoglobins, cooperative ligand binding previously has not been observed in bacterial sensor globins. This study explores the O₂-dependent allosteric communication between globin domains in the globin-coupled sensor protein from <i>Pectobacterium carotovorum</i> (<i>Pcc</i>GCS) through equilibrium O₂ binding measurements, X-ray crystallography, resonance Raman spectroscopy, and hydrogen-deuterium exchange mass spectrometry. Based on these experiments, we propose a model of allosteric regulation of O₂ binding that is directed by subtle changes in distal heme pocket protein conformation and transduced through dynamics of helices at the dimer interface of the <i>Pcc</i>GCS sensor globin. Together this work identifies cooperative ligand binding in a family of bacterial heme proteins, which could allow the bacteria to more robustly respond to small changes in O₂ levels. Furthermore, this work highlights the importance of heme pocket residues in transducing the O₂ binding event within the dimer and suggests a pathway for signal transduction in dimeric myoglobin-like sensor proteins.

  PublisherDOI

• eLife assessment: Nucleotide binding to the ATP-cone in anaerobic ribonucleotide reductases allosterically regulates activity by modulating substrate binding

  2024-07-05

  peer-reviewOpen access1st authorCorresponding
  PublisherDOI

• eLife Assessment: Activity modulation in anaerobic ribonucleotide reductases: nucleotide binding to the ATP-cone allosterically mediates substrate binding to the active site

  2024-04-05

  peer-reviewOpen access1st authorCorresponding

  A small, nucleotide-binding domain, the ATP-cone, is found at the N-terminus of most ribonucleotide reductase (RNR) catalytic subunits. By binding ATP or dATP it regulates the enzyme activity of all classes of RNR. Functional and structural work on aerobic RNRs has revealed a plethora of ways in which dATP inhibits activity by inducing oligomerization and preventing a productive radical transfer from one subunit to the active site in the other. Anaerobic RNRs, on the other hand, store a stable glycyl radical next to the active site and the basis for their dATP-dependent inhibition is completely unknown. We present biochemical, biophysical and structural information on the effects of ATP and dATP binding to the anaerobic RNR from Prevotella copri. The enzyme exists in a dimer-tetramer equilibrium biased towards dimers when two ATP molecules are bound to the ATP-cone and tetramers when two dATP molecules are bound. In the presence of ATP, P. copri NrdD is active and has a fully ordered glycyl radical domain (GRD) in one monomer of the dimer. Binding of dATP to the ATP-cone results in loss of activity and increased dynamics of the GRD, such that it can not be detected in the cryo-EM structures. The glycyl radical is formed even in the dATP-bound form, but the substrate does not bind. The structures implicate a complex network of interactions in activity regulation that involve the GRD more than 30 Å away from the dATP molecules, the allosteric substrate specificity site and a conserved but previously unseen flap over the active site. Taken together, the results suggest dATP inhibition in anaerobic RNRs acts by increasing the flexibility of the flap and GRD, thereby preventing both substrate binding and radical mobilisation.

  PublisherDOI

• Optimized Substrate Positioning Enables Switches in C–H Cleavage Site and Reaction Outcome in the Hydroxylation-Epoxidation Sequence Catalyzed by Hyoscyamine 6β-Hydroxylase

  ChemRxiv · 2024-02-21 · 2 citations

  preprintOpen access

  Hyoscyamine 6β-hydroxylase (H6H) is an Fe(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase that catalyzes the last two steps in the biosynthesis of scopolamine, a prolifically administered anti-nausea drug. After its namesake first reaction, H6H couples the newly installed C6-bonded oxygen to C7 to form the epoxide of scopolamine. Oxoiron(IV) (ferryl) intermediates initiate both reactions by cleaving C–H bonds, but it remains unclear how the enzyme switches target site and promotes (C6)O–C7 coupling in preference to C7 hydroxylation in the second step. In one possible epoxidation mechanism, the C6 oxygen would – analogously to mechanisms proposed for the Fe/2OG halogenases and, in the preceding paper, N-acetylnorloline synthase (LolO) – coordinate as alkoxide to the C7–H-cleaving ferryl intermediate to enable alkoxyl coupling to the ensuing C7 radical. Here we provide structural and kinetic evidence that H6H instead exploits the distinct spatial dependencies of competitive C–H-cleavage (C6 vs C7) and C–O-coupling (oxygen rebound vs cyclization) steps to promote the two-step sequence without substrate coordination or repositioning for the epoxidation step. Structural comparisons of ferryl-mimicking vanadyl complexes of wild-type H6H and a variant that preferentially hydroxylates C7 of 6-hydroxyhyoscyamine suggest that only a modest (~ 10°) shift in the Fe–O–H(C7) approach angle is sufficient to change the outcome. The observation that, in wild-type H6H, 2H2O solvent also increases the C7-hydroxylation:epoxidation ratio by ~ 8-fold implies that the latter outcome requires cleavage of the alcohol O-H bond, which, unlike in the LolO oxacyclization, is not accomplished in advance of C–H cleavage.

  PublisherOA PDFDOI

• eLife assessment: Novel sterol binding domains in bacteria

  2024-02-08

  peer-reviewOpen access1st authorCorresponding
  PublisherDOI

• Modulating metal-centered dimerization of a lanthanide chaperone protein for separation of light lanthanides

  Proceedings of the National Academy of Sciences · 2024-10-28 · 25 citations

  articleOpen accessCorresponding

  Elucidating details of biology’s selective uptake and trafficking of rare earth elements, particularly the lanthanides, has the potential to inspire sustainable biomolecular separations of these essential metals for myriad modern technologies. Here, we biochemically and structurally characterize Methylobacterium ( Methylorubrum ) extorquens LanD, a periplasmic protein from a bacterial gene cluster for lanthanide uptake. This protein provides only four ligands at its surface-exposed lanthanide-binding site, allowing for metal-centered protein dimerization that favors the largest lanthanide, La III . However, the monomer prefers Nd III and Sm III , which are disfavored lanthanides for cellular utilization. Structure-guided mutagenesis of a metal-ligand and an outer-sphere residue weakens metal binding to the LanD monomer and enhances dimerization for Pr III and Nd III by 100-fold. Selective dimerization enriches high-value Pr III and Nd III relative to low-value La III and Ce III in an all-aqueous process, achieving higher separation factors than lanmodulins and comparable or better separation factors than common industrial extractants. Finally, we show that LanD interacts with lanmodulin (LanM), a previously characterized periplasmic protein that shares LanD’s preference for Nd III and Sm III . Our results suggest that LanD’s unusual metal-binding site transfers less-desirable lanthanides to LanM to siphon them away from the pathway for cytosolic import. The properties of LanD show how relatively weak chelators can achieve high selectivity, and they form the basis for the design of protein dimers for separation of adjacent lanthanide pairs and other metal ions.

  PublisherDOI

Recent grants

• Structure determination of reaction intermediates in macromolecular complexes

  NIH · $729k · 2012–2016

• NIH Grant F32CA138027

  NIH · $55k · 2014

• Mapping the structural basis for mechanistic diversity in metalloenzyme superfamilies

  NIH · $4.4M · 2016–2026

• Structure determination of reaction intermediates in macromolecular complexes

  NIH · $110k · 2012–2014

Frequent coauthors

• J. Martin Bollinger

  Pennsylvania State University

  36 shared

• Carsten Krebs

  Pennsylvania State University

  35 shared

• Squire J. Booker

  Pennsylvania State University

  16 shared

• Alexey Silakov

  16 shared

• Christopher J. Pollock

  Cornell University

  16 shared

• Ryan J. Martinie

  Hamilton College

  15 shared

• Debangsu Sil

  Pennsylvania State University

  15 shared

• Irene Schaperdoth

  Dartmouth College

  15 shared

Labs

• Boal GroupPI

Awards & honors

• 2022 Early Career Award from the International Society for B…