Keywords

Biochemistry, Bioinorganic chemistry             

Contact Information

Office: 256
Lab: Rooms 208, 254
Chemistry and Biochemistry Building
P.O. Box 173400
Bozeman, MT 59717

Email: eshepard@montana.edu

Education & Professional Preparation

  • Rocky Mountain College, B.S. Chemistry, 1999
  • Montana State University, Graduate Teaching and Research Assistant, NSF Integrative Graduate Education and Research Traineeship Fellow, 2000 - 2005
  • Montana State University, Biochemistry, Ph.D., 2006
  • Montana State University, Postdoctoral Fellow, 2006 – 2011
  • Montana State University, Senior Research Scientist, 2011-2016
  • Montana State University, Assistant Research Professor, current

Selected Publications (42 total)

Shepard E.M., Byer A.S., Betz J.N. Peters J.W., Broderick J.B. (2016) A redox active [2Fe-2S] cluster on the hydrogenase maturase HydF, Biochemistry, 55, 3514-3527.  doi: 10.1021/acs.biochem.6b00528

Shepard E.M., Dooley D.M. (2015) Inhibition and oxygen activation in copper amine oxidases, Acc. Chem. Res.48, 1218-1226.  doi: 10.1021/ar500460z

Broderick J.B., Duffus B.R., Duschene K.S., Shepard E.M. (2014) Radical S-adenosylmethionine enzymes, Chem. Rev. 114, 4229-4317.  doi: 10.1021/cr4004709

Shepard E.M., Duffus B.R., George S.J., McGlynn S.E., Challand M.R., Swanson K.D., Roach P.L., Cramer S.P., Peters J.W., Broderick J.B. (2010) [FeFe]-hydrogenase maturation: HydG-catalyzed synthesis of carbon monoxide, J. Amer. Chem. Soc.132, 9247-9249.  doi: 10.1021/ja1012273

Shepard E.M., McGlynn S.E., Bueling A.L., Grady-Smith C.S., George S.J., Winslow M.A., Cramer S.P., Peters J.W., Broderick J.B. (2010) Synthesis of the 2Fe subcluster of the [FeFe]-hydrogenase H cluster on the HydF scaffold, Proc. Natl. Acad. Sci. USA107, 10448-10453.  doi: 10.1073/pnas.1001937107

Synthetic Biomimetic Design of Radical S-Adenosylmethionine Maquettes from Experiments and Theory

Research Summary:  A collaborative team at Montana State University comprised of Dr. Eric Shepard (PI), Dr. Joan Broderick (co-PI), Dr. Valérie Copié (co-PI), and Dr. Robert Szilagyi (co-PI) will examine the minimal protein structural requirements for generating and controlling organic radical chemistry in biology.  The largest known superfamily of enzymes in nature is the radical S-adenosylmethionine (SAM) superfamily; these metalloenzymes harbor an iron-sulfur cluster that is responsible for the coordination and cleavage of the essential cofactor SAM.  The cleavage of SAM generates a radical intermediate species, known as the 5’-deoxyadenosyl radical, which is responsible for propagating an incredibly diverse array of chemical transformations essential to life that include DNA repair, RNA modification, protein activation, vitamin biosynthesis, and cofactor formation.  At the core of radical SAM catalytic processes is a redox active [4Fe-4S] cluster that harbors a site differentiated Fe site that promotes the bidentate coordination of SAM.  Using a retrosynthetic approach, we will employ parallel synthetic, spectroscopic, and computational investigations to develop and characterize iron-sulfur cluster coordinated, short oligopeptide maquette complexes that are functional in SAM chemistry.  Beyond the misleading simplicity of the radical formation reaction by inner-sphere electron-transfer from the iron sulfur cluster to the sulfonium group of SAM, essential aspects of 5’-deoxyadenosyl radical formation require studies toward understanding the origin of selectivity in SAM-based sulfonium bond cleavage, the control that the protein environment imparts, and the reversibility of the SAM cleavage event that speaks to the role of SAM as either cofactor or cosubstrate.  We will address these chemical, structural, and mechanistic issues through structure-function relationships and the insights gained from [4Fe-4S] maquette studies will contribute to our understanding of how and why nature uses this platform as its preferred method for generating and propagating radical reactions.  

 

 

 

 

 

 

 

Left.  A radical SAM maquette. A maquette is a minimal synthetic oligopeptide designed to act as a framework, or model system, of the full functional protein.  The illustration highlights the bidentate coordination of SAM to the unique Fe of a [4Fe-4S] cluster, as well as the sulfonium functionality of SAM.  Right.  A local Bozeman sculptor, Ott Jones, is seen here using a clay bison maquette to direct design of his wildlife sculptures. Photo credit (copyright, 2016, BigSky Journal: http://bigskyjournal.com/Article/artist-of-the-west-a-story-in-clay).

Role of HydF in Hydrogenase Maturation

Research Summary:  A collaborative team at Montana State University comprised of Dr. Joan Broderick (PI) and Dr. Eric Shepard (co-PI) will examine the mechanism of active site metal cluster assembly in [FeFe]-hydrogenase.  The potential for harnessing biological hydrogen production as an energy solution, especially through metabolic engineering, cannot be fully realized without a complete fundamental understanding of how the complex metal clusters at the active sites of hydrogenase enzymes function and are assembled.  Physical biochemical approaches will be used to elucidate the reactions catalyzed by the three specific hydrogenase maturase enzymes common to all organisms that express [FeFe]-hydrogenase.

The active site metal cluster of [FeFe]-hydrogenase, referred to as the H-cluster, is a highly unusual modified iron-sulfur cluster; this H-cluster exists as a [4Fe-4S] cubane bridged to a 2Fe subcluster that contains three carbon monoxide, two cyanide, and a bridging dithiomethylamine group as ligands.  Three specific gene products, denoted HydE, HydF, and HydG, are now known to function in the assembly and maturation of functional [FeFe]-hydrogenase (HydA) via their specific role in the biosynthesis of the uniquely decorated 2Fe subcluster of the H-cluster.  Two of these enzymes (HydE and HydG) belong to the radical S-adenosylmethionine (SAM) superfamily.  The third protein, HydF, is a GTPase that functions as a scaffold/carrier during H-cluster assembly. 

While substantial work has delineated aspects of H-cluster maturation, much remains unknown about the specific roles of HydE, HydF, and HydG.  Our work has provided critical insights into the chemical function and mechanism of the radical SAM maturases HydE and HydG, while working towards the goal of determining how the products of these enzymes assemble on the scaffold protein HydF to achieve H-cluster synthesis.  The goals of this project are to develop a molecular-level understanding of the reactions catalyzed by HydE, HydF, and HydG; current evidence provides support for the stepwise nature of H-cluster biosynthesis and the novel chemical transformations that accompany this process.  This project will not only enhance the ability to promote biohydrogen energy solutions through genetic engineering and biomimicry, but stands to also help define valuable paradigms for complex metal cluster assembly in biology.