About

Kylie Allen is an Associate Professor in the Department of Biochemistry at Virginia Tech. Her research is centered on methanogenic archaea, a diverse group of anaerobic microorganisms with complex energy metabolism dependent on one-carbon biochemistry to reduce CO2, formate, methyl compounds, and/or acetate to produce methane as a final end product. This process, known as methanogenesis, generates over a billion tons of methane each year, accounting for more than 90% of the methane produced on Earth. Her lab aims to uncover and characterize new enzymes, reactions, and biomolecules in methanogens, with the goal of understanding their unusual biochemistry and potential applications in methane bioconversion and energy production. Dr. Allen received her Ph.D. in Biochemistry from Washington State University in 2013 and her B.S. in Biology from Eastern Washington University in 2007. She has held positions at Virginia Tech since 2013, progressing from Postdoctoral Associate to Assistant Professor and then to Associate Professor in 2024.

Research topics

• Political Science
• Sociology
• Gender studies
• Social Science
• Epistemology
• Psychology
• Geography
• Medicine
• Social psychology
• Public relations
• Developmental psychology

Selected publications

• The HypA and HypB metallochaperones from Methanococcus maripaludis have unique metal-binding properties and a distinct nickel transfer mechanism

  Journal of Biological Chemistry · 2026-04-17

  articleOpen accessSenior author

  NiFe] hydrogenases are widespread microbial metalloenzymes that catalyze the reversible conversion of hydrogen (H2) to protons and electrons, playing key roles in energy metabolism.The biosynthesis of the NiFe(CN)2CO cofactor involves a suite of maturation proteins including the HypA and HypB nickel metallochaperones.Here, we define the metal-binding properties, nucleotide-dependent behavior, and functional interplay of HypA and HypB from the hydrogenotrophic methanogenic archaeon, Methanococcus maripaludis.Methanogens have multiple essential nickel-dependent enzymes, so they require efficient systems for nickel delivery that remain largely unexplored.Purified M. maripaludis HypA binds zinc or mononuclear iron at the C-terminal metal binding site, the latter of which has not been reported in other HypA proteins and may serve a unique regulatory role in methanogens.The G-protein metallochaperone HypB binds nickel at the G-domain, which stimulates GTPase activity.Size exclusion chromatography experiments reveal that HypA and HypB form complexes in the presence of nickel, and zinc-bound HypA is optimized for nickel transfer from HypB.The identity of the nucleotide bound to HypB (GDP or GTP) alters the oligomeric state of HypA-HypB complexes, supporting a GTPasemediated nickel delivery pathway.The HypA-HypB2 complex configuration is enriched and stable in the presence of GDP and nickel, indicating that this complex delivers nickel to the hydrogenase as opposed to HypA alone.Interestingly, affinity purification-mass spectrometry revealed that HypB interacts with several nickel-dependent proteins, suggesting that HypB may play a broader role in nickel homeostasis in M. maripaludis.Together, this work establishes a biochemical framework for HypAB-mediated nickel trafficking in methanogens.

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• Biochemical characterization of the self-sacrificing p-aminobenzoate synthase from Nitrosomonas europaea reveals key residues involved in selecting a Fe/Fe or Mn/Fe cofactor

  JBIC Journal of Biological Inorganic Chemistry · 2025-03-13 · 1 citations

  articleOpen accessSenior author

  A noncanonical route for p-aminobenzoate (pABA) biosynthesis in select bacteria utilizes a novel self-sacrificing heme oxygenase-like domain-containing oxidase/oxygenase (HDO) superfamily member. The recently characterized self-sacrificing pABA synthase from Chlamydia trachomatis ("CADD") requires manganese and likely employs a heterobimetallic Mn/Fe cofactor. A conserved active site tyrosine residue is cleaved from the protein backbone to serve as the substrate for pABA synthesis and a lysine residue is the amino group donor. Here, we investigated the orthologous pABA synthase from the ammonia-oxidizing bacterium, Nitrosomonas europaea, which we refer to as NePabS. Consistent with the previously studied C. trachomatis enzyme, purified NePabS produces pABA in vitro in a reaction that only requires a metal cofactor, molecular oxygen, and a reducing agent, but no other substrates. Interestingly, maximal activity was observed with the addition of only iron as opposed to manganese and iron; thus, NePabS utilizes the more traditional Fe/Fe cofactor employed by most characterized HDO superfamily members. The self-sacrificing residues were confirmed to be Tyr25 and Lys159, which are the corresponding self-sacrificing residues in the CADD reaction. Strikingly, we could switch the metal dependence (Fe/Fe to Mn/Fe) and significantly improve the activity (~ twofold) of NePabS by substituting two phenylalanine residues with tyrosine residues (F148Y/F177Y), thus rendering the enzyme more similar to CADD. These results demonstrate that these two aromatic residues play an essential role in dictating metal specificity and potentially the proposed radical translocation process that facilitates the tyrosine cleavage reaction for pABA synthesis.

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• Toward the Use of Methyl-Coenzyme M Reductase for Methane Bioconversion Applications

  Accounts of Chemical Research · 2024-08-27 · 14 citations

  articleOpen accessSenior authorCorresponding

  ConspectusAs the main component of natural gas and renewable biogas, methane is an abundant, affordable fuel. Thus, there is interest in converting these methane reserves into liquid fuels and commodity chemicals, which would contribute toward mitigating climate change, as well as provide potentially sustainable routes to chemical production. Unfortunately, specific activation of methane for conversion into other molecules is a difficult process due to the unreactive nature of methane C–H bonds. The use of methane activating enzymes, such as methyl-coenzyme M reductase (MCR), may offer a solution. MCR catalyzes the methane-forming step of methanogenesis in methanogenic archaea (methanogens), as well as the initial methane oxidation step during the anaerobic oxidation of methane (AOM) in anaerobic methanotrophic archaea (ANME). In this Account, we highlight our contributions toward understanding MCR catalysis and structure, focusing on features that may tune the catalytic activity. Additionally, we discuss some key considerations for biomanufacturing approaches to MCR-based production of useful compounds.MCR is a complex enzyme consisting of a dimer of heterotrimers with several post-translational modifications, as well as the nickel-hydrocorphin prosthetic group, known as coenzyme F430. Since MCR is difficult to study in vitro, little information is available regarding which MCRs have ideal catalytic properties. To investigate the role of the MCR active site electronic environment in promoting methane synthesis, we performed electric field calculations based on molecular dynamics simulations with a MCR from Methanosarcina acetivorans and an ANME-1 MCR. Interestingly, the ANME-1 MCR active site better optimizes the electric field with methane formation substrates, indicating that it may have enhanced catalytic efficiency. Our lab has also worked toward understanding the structures and functions of modified F430 coenzymes, some of which we have discovered in methanogens. We found that methanogens produce modified F430s under specific growth conditions, and we hypothesize that these modifications serve to fine-tune the activity of MCR.Due to the complexity of MCR, a methanogen host is likely the best near-term option for biomanufacturing platforms using methane as a C1 feedstock. M. acetivorans has well-established genetic tools and has already been used in pilot methane oxidation studies. To make methane oxidation energetically favorable, extracellular electron acceptors are employed. This electron transfer can be facilitated by carbon-based materials. Interestingly, our analyses of AOM enrichment cultures and pure methanogen cultures revealed the biogenic production of an amorphous carbon material with similar characteristics to activated carbon, thus highlighting the potential use of such materials as conductive elements to enhance extracellular electron transfer.In summary, the possibilities for sustainable MCR-based methane conversions are exciting, but there are still some challenges to tackle toward understanding and utilizing this complex enzyme in efficient methane oxidation biomanufacturing processes. Additionally, further work is necessary to optimize bioengineered MCR-containing host organisms to produce large quantities of desired chemicals.

  PublisherDOI

• Control of biofilm formation by an <i>Agrobacterium tumefaciens</i> pterin-binding periplasmic protein conserved among diverse <i>Proteobacteria</i>

  Proceedings of the National Academy of Sciences · 2024-06-13 · 4 citations

  articleOpen access

  Biofilm formation and surface attachment in multiple Alphaproteobacteria is driven by unipolar polysaccharide (UPP) adhesins. The pathogen Agrobacterium tumefaciens produces a UPP adhesin, which is regulated by the intracellular second messenger cyclic diguanylate monophosphate (c-di-GMP). Prior studies revealed that DcpA, a diguanylate cyclase-phosphodiesterase, is crucial in control of UPP production and surface attachment. DcpA is regulated by PruR, a protein with distant similarity to enzymatic domains known to coordinate the molybdopterin cofactor (MoCo). Pterins are bicyclic nitrogen-rich compounds, several of which are produced via a nonessential branch of the folate biosynthesis pathway, distinct from MoCo. The pterin-binding protein PruR controls DcpA activity, fostering c-di-GMP breakdown and dampening its synthesis. Pterins are excreted, and we report here that PruR associates with these metabolites in the periplasm, promoting interaction with the DcpA periplasmic domain. The pteridine reductase PruA, which reduces specific dihydro-pterin molecules to their tetrahydro forms, imparts control over DcpA activity through PruR. Tetrahydromonapterin preferentially associates with PruR relative to other related pterins, and the PruR-DcpA interaction is decreased in a pruA mutant. PruR and DcpA are encoded in an operon with wide conservation among diverse Proteobacteria including mammalian pathogens. Crystal structures reveal that PruR and several orthologs adopt a conserved fold, with a pterin-specific binding cleft that coordinates the bicyclic pterin ring. These findings define a pterin-responsive regulatory mechanism that controls biofilm formation and related c-di-GMP-dependent phenotypes in A. tumefaciens and potentially acts more widely in multiple proteobacterial lineages.

  PublisherOA PDFDOI

• Structural dynamics of the methyl-coenzyme M reductase active site are influenced by coenzyme F ₄₃₀ modifications

  bioRxiv (Cold Spring Harbor Laboratory) · 2024-01-08 · 1 citations

  preprintOpen accessCorresponding

  Abstract Methyl-coenzyme M reductase (MCR) is a central player in methane biogeochemistry, governing methanogenesis and the anaerobic oxidation of methane (AOM) in methanogens and anaerobic methanotrophs (ANME), respectively. The prosthetic group of MCR is coenzyme F 430 , a nickel-containing tetrapyrrole derivative. Additionally, a few modified versions of F 430 have been discovered, including the 17 2 -methylthio-F 430 (mt-F 430 ) that functions with ANME-1 MCR. This study employs molecular dynamics (MD) simulations to unravel the intricacies of the active-site dynamics of MCR from Methanosarcina acetivorans and ANME-1 when bound to the canonical F 430 compared to 17 2 -thioether coenzyme F 430 variants and substrates for methane formation. Overall, our simulations indicate that each MCR active site is optimized for a given version of F 430 and support the importance of the Gln to Val substitution in accommodating the 17 2 methylthio modification. Notably, modifications in the 17 2 position disrupt the canonical coordination among cofactors in M. acetivorans MCR, implicating structural perturbations, but evidence of active site reorganization to maintain substrate positions suggest that the modified F 430 s could be accommodated in a methanogenic MCR. We additionally report the first quantitative estimate of MCR intrinsic electric fields pivotal in driving methane formation. Our results suggest that the electric field aligned along the CH 3 -S-CoM thioether bond facilitates homolytic bond cleavage, coinciding with the proposed catalytic mechanism. Structural perturbations, however, weaken and misalign these electric fields, emphasizing the significance of the active site structure in maintaining their integrity. In conclusion, our results deepen the understanding of MCR active-site dynamics, the enzyme’s organizational role in intrinsic electric fields for catalysis, and the interplay between active site structure and electrostatics. This work not only advances our comprehension of MCR functionality but also provides a foundation for future investigations employing sophisticated models to capture the complex electronic properties of MCR active sites quantitatively.

  PublisherOA PDFDOI

• Structural Dynamics of the Methyl-Coenzyme M Reductase Active Site Are Influenced by Coenzyme F₄₃₀ Modifications

  Biochemistry · 2024-06-24 · 6 citations

  articleOpen accessCorresponding

  Methyl-coenzyme M reductase (MCR) is a central player in methane biogeochemistry, governing methanogenesis and the anaerobic oxidation of methane (AOM) in methanogens and anaerobic methanotrophs (ANME), respectively. The prosthetic group of MCR is coenzyme F430, a nickel-containing tetrahydrocorphin. Several modified versions of F430 have been discovered, including the 172-methylthio-F430 (mtF430) used by ANME-1 MCR. Here, we employ molecular dynamics (MD) simulations to investigate the active site dynamics of MCR from Methanosarcina acetivorans and ANME-1 when bound to the canonical F430 compared to 172-thioether coenzyme F430 variants and substrates (methyl-coenzyme M and coenzyme B) for methane formation. Our simulations highlight the importance of the Gln to Val substitution in accommodating the 172 methylthio modification in ANME-1 MCR. Modifications at the 172 position disrupt the canonical substrate positioning in M. acetivorans MCR. However, in some replicates, active site reorganization to maintain substrate positioning suggests that the modified F430 variants could be accommodated in a methanogenic MCR. We additionally report the first quantitative estimate of MCR intrinsic electric fields that are pivotal in driving methane formation. Our results suggest that the electric field aligned along the CH3-S-CoM thioether bond facilitates homolytic bond cleavage, coinciding with the proposed catalytic mechanism. Structural perturbations, however, weaken and misalign these electric fields, emphasizing the importance of the active site structure in maintaining their integrity. In conclusion, our results deepen the understanding of MCR active site dynamics, the enzyme’s organizational role in intrinsic electric fields for catalysis, and the interplay between active site structure and electrostatics.

  PublisherOA PDFDOI

• Biochemical and genetic studies define the functions of methylthiotransferases in methanogenic and methanotrophic archaea

  Frontiers in Microbiology · 2023-11-23 · 2 citations

  articleOpen accessSenior authorCorresponding

  Methylthiotransferases (MTTases) are radical S -adenosylmethionine (SAM) enzymes that catalyze the addition of a methylthio (-SCH 3 ) group to an unreactive carbon center. These enzymes are responsible for the production of 2-methylthioadenosine (ms 2 A) derivatives found at position A37 of select tRNAs in all domains of life. Additionally, some bacteria contain the RimO MTTase that catalyzes the methylthiolation of the S12 ribosomal protein. Although the functions of MTTases in bacteria and eukaryotes have been established via detailed genetic and biochemical studies, MTTases from the archaeal domain of life are understudied and the substrate specificity determinants of MTTases remain unclear. Here, we report the in vitro enzymatic activities of an MTTase (C4B56_06395) from a thermophilic Ca. Methanophagales anaerobic methanotroph (ANME) as well as the MTTase from a hyperthermophilic methanogen – MJ0867 from Methanocaldococcus jannaschii . Both enzymes catalyze the methylthiolation of N 6 -threonylcarbamoyladenosine (t 6 A) and N 6 - hydroxynorvalylcarbamoyladenosine (hn 6 A) residues to produce 2-methylthio- N 6 -threonylcarbamoyladenosine (ms 2 t 6 A) and 2-methylthio- N 6 - hydroxynorvalylcarbamoyladenosine (ms 2 hn 6 A), respectively. To further assess the function of archaeal MTTases, we analyzed select tRNA modifications in a model methanogen – Methanosarcina acetivorans – and generated a deletion of the MTTase-encoding gene (MA1153). We found that M. acetivorans produces ms 2 hn 6 A in exponential phase of growth, but does not produce ms 2 t 6 A in detectable amounts. Upon deletion of MA1153, the ms 2 A modification was absent, thus confirming the function of MtaB-family MTTases in generating ms 2 hn 6 A modified nucleosides in select tRNAs.

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• Control of Biofilm Formation by an <i>Agrobacterium tumefaciens</i> Pterin-Binding Periplasmic Protein Conserved Among Pathogenic Bacteria

  bioRxiv (Cold Spring Harbor Laboratory) · 2023-11-18

  preprintOpen access

  ABSTRACT Biofilm formation and surface attachment in multiple Alphaproteobacteria is driven by unipolar polysaccharide (UPP) adhesins. The pathogen Agrobacterium tumefaciens produces a UPP adhesin, which is regulated by the intracellular second messenger cyclic diguanylate monophosphate (cdGMP). Prior studies revealed that DcpA, a diguanylate cyclase-phosphodiesterase (DGC-PDE), is crucial in control of UPP production and surface attachment. DcpA is regulated by PruR, a protein with distant similarity to enzymatic domains known to coordinate the molybdopterin cofactor (MoCo). Pterins are bicyclic nitrogen-rich compounds, several of which are formed via a non-essential branch of the folate biosynthesis pathway, distinct from MoCo. The pterin-binding protein PruR controls DcpA activity, fostering cdGMP breakdown and dampening its synthesis. Pterins are excreted and we report here that PruR associates with these metabolites in the periplasm, promoting interaction with the DcpA periplasmic domain. The pteridine reductase PruA, which reduces specific dihydro-pterin molecules to their tetrahydro forms, imparts control over DcpA activity through PruR. Tetrahydromonapterin preferentially associates with PruR relative to other related pterins, and the PruR-DcpA interaction is decreased in a pruA mutant. PruR and DcpA are encoded in an operon that is conserved amongst multiple Proteobacteria including mammalian pathogens. Crystal structures reveal that PruR and several orthologs adopt a conserved fold, with a pterin-specific binding cleft that coordinates the bicyclic pterin ring. These findings define a new pterin-responsive regulatory mechanism that controls biofilm formation and related cdGMP-dependent phenotypes in A. tumefaciens and is found in multiple additional bacterial pathogens. SIGNIFICANCE Biofilms are bacterial communities attached to surfaces, physiologically distinct from free-living cells, and a common cause of persistent infections. Here we define the mechanism of a novel biofilm regulatory system based on excreted metabolites called pterins, that is conserved within a wide range of Gram-negative bacteria, including multiple pathogens of animals and plants. The molecular mechanism of pterin-dependent regulation is reported including structural determination of several members of a new family of pterin-binding proteins. Pterins are produced across all domains of life and mechanistic insights into this regulatory circuit could lead to new advances in antibiofilm treatments.

  PublisherOA PDFDOI

• Abstract 2544: Radical SAM aminomutases catalyzing beta-amino acid biosynthesis for salt tolerance in methanogenic archaea

  Journal of Biological Chemistry · 2023-01-01

  articleOpen access1st authorCorresponding
  PublisherDOI

• The <i>Chlamydia trachomatis p</i>‐aminobenzoate synthase <scp>CADD</scp> is a manganese‐dependent oxygenase that uses its own amino acid residues as substrates

  FEBS Letters · 2023-01-17 · 9 citations

  articleOpen accessSenior authorCorresponding

  CADD (chlamydia protein associating with death domains) is a p-aminobenzoate (pAB) synthase involved in a noncanonical route for tetrahydrofolate biosynthesis in Chlamydia trachomatis. Although previously implicated to employ a diiron cofactor, here, we show that pAB synthesis by CADD requires manganese and the physiological cofactor is most likely a heterodinuclear Mn/Fe cluster. Isotope-labeling experiments revealed that the two oxygen atoms in the carboxylic acid portion of pAB are derived from molecular oxygen. Further, mass spectrometry-based proteomic analyses of CADD-derived peptides demonstrated a glycine substitution at Tyr27, providing strong evidence that this residue is sacrificed for pAB synthesis. Additionally, Lys152 was deaminated and oxidized to aminoadipic acid, supporting its proposed role as a sacrificial amino group donor.

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Frequent coauthors

• Sally A. Lloyd

  Miami University

  68 shared

• Margaret Crosbie‐Burnett

  65 shared

• Leanne On November

  Centre for Social Justice

  64 shared

• Nathan Balter Blume

  Florida State University

  64 shared

• Edith Lewis

  64 shared

• Leanne Silvey

  Centre for Social Justice

  64 shared

• Maresa Murray

  64 shared

• Ann Lee

  64 shared