Kylie Allen

Program Focus

Research in my lab is centered on methanogenic archaea, a diverse group of anaerobic microorganisms with a 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, called methanogenesis, generates over a billion tons of methane each year, which accounts for >90% of the methane generated on Earth. Given that methane is a potent greenhouse gas as well as a powerful energy source, understanding methanogenic metabolism is an important endeavor. Methanogens contain a wealth of unusual biochemistry, much of which remains to be discovered or fully understood. My lab seeks to uncover and characterize new enzymes, reactions, and biomolecules in methanogens.

Current Projects

F430-3 function and biosynthesis
Coenzyme F430 is a unique nickel-containing tetrapyrrole that is required for the final step of methanogenesis catalyzed by methyl coenzyme M reductase (MCR). We recently discovered a modified version of coenzyme F430 denoted F430-3. The proposed structure of F430-3 contains a cyclized 3-mercaptopropionate moiety bound as a thioether. This is structurally similar to the only other described modified F430 that is found in archaea performing anaerobic oxidation of methane via reverse methanogenesis. We hypothesize that F430-3 tunes MCR to oxidize methane in methanogens. This is a highly desirable reaction for the possible generation of liquid fuels from methane using biocatalysis rather than inefficient chemical processes. Therefore, current research is focused on demonstrating the role/function of F430-3 as well as the genes/enzymes involved in its biosynthesis.

Compatible solute biosynthesis
In order to cope with salt stress in high salinity environments, organisms must accumulate ions or organic compounds inside the cell in order to keep water from leaving the cell via osmosis. The organic compounds used for this function are known as osmolytes or compatible solutes. The major compatible solutes in methanogens are N-acetyl-b-lysine and b-glutamate. In N-acetyl-b-lysine biosynthesis, b-lysine is converted to beta lysine by lysine-2,3-aminomutase (KAM), a member of the radical S-adenosyl-L-methionine (SAM) superfamily. Radical SAM enzymes catalyze diverse and complicated radical-mediated chemistry using a [4Fe-4S] cluster and SAM. b-glutamate is likely also generated via radical SAM dependent chemistry. We are currently working to characterize the KAM enzymes from methanogens and to determine the enzyme responsible for b-glutamate synthesis.

Methylated pterin biosynthesis
Methanopterin is a one-carbon group (C₁) carrier coenzyme present in methanogens that is structurally and functionally similar to folate, the more common C₁ transfer coenzyme.  One difference in the structure of methanopterin compared to folate is the presence of two methyl groups at the C-7 and C-9 positions of the pterin. We have identified a single radical SAM enzyme from Methanocaldococcus jannaschii that catalyzes the addition of both methyl groups to a pterin-containing precursor when expressed in E. coli. We are now working to purify and understand the mechanism utilized by this new radical SAM methylase.

We are also working on a collaborative project (Fuqua Lab, Indiana University Bloomington) to determine and characterize the enzymes involved in the biosynthesis of a newly discovered methylated monapterin involved in a pathway for biofilm regulation in bacteria. 

Dr. Allen's Pub Med Site

Gendron, A., Allen, K. (2022) Overview of Diverse Methyl/Alkyl-Coenzyme M Reductases and Considerations for Their Potential Heterologous Expression. Frontiers in Microbiology, https://doi.org/10.3389/fmicb.2022.867342

Tunckanat, T., Gendron, A., Sadler, Z., Neitz, A., Byquist, S., Lie, T., Allen, K. (2022) Lysine 2,3-aminomutase and a newly discovered glutamate 2,3-aminomutase produce b-amino acids involved in salt tolerance in methanogenic archaea. Biochemistry, https://doi.org/10.1021/acs.biochem.2c00014.

Allen, K., Wegener, G., Sublett Jr. D., Bodnar, R., Feng, X., Wendt, J., White, R. (2021) Biogenic formation of amorphous carbon by anaerobic methanotrophs and select methanogens. Science Advances, 7(44) DOI: 10.1126/sciadv.abg9739.

Macias-Orihuela, Y., Cast, T., Crawford, I., Brandecker, K., Thiaville, J., Murzin, A., de Crécy-Lagard, V., White, R., Allen, K. (2020) An unusual route for p-aminobenzoate biosynthesis in Chlamydia trachomatis involves a probable self-sacrificing diiron oxygenase. Journal of Bacteriology, 10.1128/JB.00319-20. (Spotlight Article)

Labine, M., DePledge, L., Ferier, N., Greenwich, J., Fuqua, C., Allen, K. (2020) Enzymatic and Mutational Analysis of the PruA Pteridine Reductase Involved in Pterin-Dependent Biofilm Control in Agrobacterium Tumefaciens. Journal of Bacteriology 202:e00098-20.  

White, R., Allen, K., and Wegener, G. (2019) Identification of a Redox Active Thioquinoxalinol Sulfate Compound Produced by an Anaerobic Methane-Oxidizing Microbial Consortium. ACS Omega, 4(27), 22613-22622.

Portugal, R., Shao, N., Whitman, W., Allen, K., and White, R. (2019) Identification and biosynthesis of 2-(1H-imidazol-5-yl)ethan-1-ol (histaminol) in methanogenic archaea. Microbiology, 165, 455-462.

Allen, K. and White, R. (2018) Identification of the Radical SAM Enzymes Involved in the Biosynthesis of Methanopterin and Coenzyme F₄₂₀ in Methanogens. Methods in Enzymology 606. In Press.

Allen, K. and White, R. (2016) Occurrence and biosynthesis of 3-mercaptopropionic acid in Methanocaldococcus jannaschii. FEMS Microbiology letters 363, fnw217.

Allen, K., Miller, D., Rauch, B., Perona, J., and White, R. (2015) Homocysteine is biosynthesized from aspartate semialdehyde and hydrogen sulfide in methanogenic archaea, Biochemistry 54, 3129-3132.

Feirer, N., Xu, J., Allen, K., Koestler, B., Waters, C., White, R., and Fuqua, C. (2015) A Pterin-Dependent Signaling Pathway Regulates a Dual Function Diguanylate Cyclase-Phosphodiesterase Protein Controlling Surface Attachment in Agrobacterium tumefaciens, mBio, 6(4).

Hu, K., Werner, W., Allen, K., Wang, S. (2015) Investigation of enzymatic C-P bond formation using multiple quantum HCP nuclear magnetic resonance spectroscopy, Magnetic Resonance in Chemistry 53, 267-272.

Allen, K., and White, R. (2014) Identification of structurally diverse methanofuran coenzymes in Methanococcales that are both N-formylated and N-acetylated, Biochemistry 53, 6199–6210.

Allen, K., and Wang, S., (2014) Spectroscopic characterization and mechanistic investigation of P-methyl transfer by a radical SAM enzyme from the marine bacterium Shewanella denitrificans OS217, Biochimica et Biophysica Acta. 1844, 2135-2144.

Allen, K., Wegner, G., and White, R. (2014) Discovery of multiple modified F430 coenzymes in methanogens and anaerobic methylotrophic archaea suggests new roles for F430 in Nature. Applied and Environmental Microbiology 80, 6403-6412. (Spotlight Article)

Allen, K., Xu, H., and White, R. (2014) Identification of a unique radical SAM methyltransferase likely involved in methanopterin biosynthesis in Methanocaldococcus jannaschii, Journal of Bacteriology 196, 3315-3323. (Selected by Faculty of 1000)

Allen, K., and Wang, S. (2014) Initial Characterization of Fom3 from Streptomyces wedmorensis: The Methyltransferase in Fosfomycin Biosynthesis, Archives of Biochemistry and Biophysics 543, 67-73.

Werner, W*.,Allen, K*., Hu, K., Helms, G., Chen, B., and Wang, S. (2011) In Vitro Phosphinate methylation by PhpK from Kitasatospora phosalacinea, Biochemistry 50, 8986-8988.

*Contributed equally