• Metalloenzymes are omnipresent and play critical roles in many essential processes for life to exist.
  

  H-cluster, an active center of the [Fe-Fe] hydrogenase

   Knowledge of their active-site structures and their mechanisms of catalysis is not only valuable from the perspective of fundamental science, but could also instruct the development of new applications and processes that have interesting biomedical, chemical and energy-related applications. Currently, our laboratory focuses on [Fe-Fe] hydrogenase, a class of enzymes that catalyzes reversible splitting of molecular hydrogen. Understanding the determining factors that govern functionality of this class of enzymes will open doors to industrial applications of these systems directly and inspire inorganic catalyst designs. Our laboratory is currently investigating a set of [Fe-Fe] hydrogenases from various Clostridia, which show aberrations in their behavior under an oxygenic environment. We perform protein film voltammetry, IR, and EPR spectroscopic experiments in conjunction with site-specific amino acid substitutions, to derive factors that can lead to understanding how oxygen damages the active center and how we can impose oxygen tolerance onto the oxygen-sensitive [Fe-Fe] hydrogenases.
  • FUNDING: DOE, NSF

 

• 

  An outline of the workflow for the proposed DEPR-based structural prediction methodology.

  Development of novel methods aiming to obtain determinants of structure-function relationships in proteins using EPR-based structural methods. For instance, measuring distances between two strategically placed spin-labels on proteins can provide unique information on structure and dynamics of proteins, especially in systems that are not readily accessible by conventional structural methods, such as intrinsically disordered proteins and amyloidogenic proteins. Currently, we research maximizing the information that could be obtained from a single spin-labeling experiment using a variety of advanced pulse EPR methods. We further apply our EPR-based methodology to a superfamily of iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenases involved in the biosynthesis of natural products.

  • FUNDING: NIH

The complete list of publications can be obtained at Google Scholars:

https://scholar.google.com/citations?user=334TBfIAAAAJ&hl=en

Selected publications:

2023:

 

Evidence of Atypical Structural Flexibility of the Active Site Surrounding of an [FeFe] Hydrogenase from Clostridium beijerinkii
Corrigan PS , Majer SH, Silakov A
J. Am. Chem. Soc. 2023, 145, 20, 11033–11044
https://doi.org/10.1021/jacs.2c13458

 

Evidence for Porphyrin-Mediated Electron Transfer in the Radical SAM Enzyme HutW
M Brimberry, P Corrigan, A Silakov*, WN Lanzilotta*
Biochemistry 62 (6), 1191-1196
https://doi.org/10.1021/acs.biochem.2c00474

2022:

Use of Noncanonical Tyrosine Analogues to Probe Control of Radical Intermediates during Endoperoxide Installation by Verruculogen Synthase (FtmOx1)
Chi-Yun Lin*, Angel L. Muñoz Hernández, Tatiana N. Laremore, Alexey Silakov*, Carsten Krebs*, Amie K. Boal*, and J. Martin Bollinger Jr.*
ACS Catal. 2022, 12, 12, 6968–6979
https://doi.org/10.1021/acscatal.2c01037

Using peptide substrate analogs to characterize a radical intermediate in NosN catalysis
Wang B, Silakov A, Booker SJ
Methods in enzymology 666, 469-487
https://doi.org/10.1016/bs.mie.2022.02.008

2020:

 

Investigation of the Unusual Ability of the [FeFe] Hydrogenase from Clostridium beijerinckii to Access an O2-Protected State.
Patrick S. Corrigan, Jonathan L. Tirsch, and Alexey Silakov*
J. Am. Chem. Soc. 2020, 142, 28, 12409–12419.
https://doi.org/10.1021/jacs.0c04964

 

Impact of Atomizer Age and Flavor on in vitro Toxicity of Aerosols from a Third-Generation Electronic Cigarette against Human Oral Cells
Jose F Urena, Lauren Ebersol, Alexey Silakov, Ryan J. Elias, and Joshua D. Lambert
Chem. Res. Toxicol. 2020
https://doi.org/10.1021/acs.chemrestox.0c00028

Narrow-Spectrum Antibiotic Targeting of the Radical SAM Enzyme MqnE in Menaquinone Biosynthesis
Ayala G. Carl, Lawrence D. Harris, Mu Feng, Lars U. Nordstrøm, Gary J. Gerfen, Gary B. Evans, Alexey Silakov*, Steven C. Almo*, and Tyler L. Grove*
Biochemistry 2020, 59, 27, 2562–2575
https://doi.org/10.1021/acs.biochem.0c00070

 

2019:

An Unexpected Species Determined by X-ray Crystallography that May Represent an Intermediate in the Reaction Catalyzed by Quinolinate Synthase
Olga A. Esakova, Alexey Silakov*, Tyler L. Grove, Douglas M. Warui, Neela H. Yennawar*, and Squire J. Booker*
J. Am. Chem. Soc. 2019, 141, 36, 14142–14151
https://doi.org/10.1021/jacs.9b02513

Analysis of RNA methylation by phylogenetically diverse Cfr radical SAM enzymes reveals an iron-binding accessory domain in a clostridial enzyme
James D. Gumkowski, Ryan J. Martinie, Patrick S. Corrigan, Juan Pan, Matthew R. Bauerle, Mohamed Almarei, Squire J. Booker, Alexey Silakov, Carsten Krebs* and Amie K. Boal*
Biochemistry. 2019 Jul 23; 58(29): 3169–3184.
https://dx.doi.org/10.1021%2Facs.biochem.9b00197

2018:

Metal-free class Ie ribonucleotide reductase from pathogens initiates catalysis with a tyrosine-derived dihydroxyphenylalanine radical
Elizabeth J. Blaesi, Gavin M. Palowitch, Kai Hu, Amelia J. Kim, Hannah R. Rose, Rahul Alapati, Marshall G. Lougee, Hee Jong Kim, Alexander T. Taguchi, Kong Ooi Tan, Tatiana N. Laremore, Robert G. Griffin, View ORCID ProfileCarsten Krebs, Megan L. Matthews, Alexey Silakov, J. Martin Bollinger Jr., Benjamin D. Allen, and Amie K. Boal
PNAS October 2, 2018 115 (40) 10022-10027; 2018
https://doi.org/10.1073/pnas.1811993115

 

Two Distinct Mechanisms for C–C Desaturation by Iron(II)- and 2-(Oxo)glutarate-Dependent Oxygenases: Importance of α-Heteroatom Assistance
Noah P. Dunham, Wei-chen Chang, Andrew J. Mitchell, Ryan J. Martinie, Bo Zhang, Jonathan A. Bergman, Lauren J. Rajakovich, Bo Wang, Alexey Silakov*, Carsten Krebs*, Amie K. Boal*, and J. Martin Bollinger Jr.*
J. Am. Chem. Soc. 2018, 140, 23, 7116–7126
https://doi.org/10.1021/jacs.8b01933

2017:

Vanadyl as a Stable Structural Mimic of Reactive Ferryl Intermediates in Mononuclear Non-heme-iron Enzymes
Ryan J. Martinie,Christopher J. Pollock, Megan L. Matthews, J. Martin Bollinger, Jr.* Carsten Krebs* and Alexey Silakov*
Inorg Chem. 2017 Nov 6; 56(21): 13382–13389.
https://dx.doi.org/10.1021%2Facs.inorgchem.7b02113

 

Evidence for a Di-μ-oxo Diamond Core in the Mn(IV)/Fe(IV) Activation Intermediate of Ribonucleotide Reductase from Chlamydia trachomatis
Ryan J. Martinie, Elizabeth J. Blaesi, Carsten Krebs, J. Martin Bollinger, Jr., Alexey Silakov*, and Christopher J. Pollock*
J. Am. Chem. Soc. 2017, 139, 5, 1950–1957
https://doi.org/10.1021/jacs.6b11563