Uniformed Services University of the Health Sciences
Department of Biochemistry and Molecular Biology
4301 Jones Bridge Road, C1094
Bethesda, Maryland 20814-4799
Fax: (301) 295-3512
Lab: (301) 295-3274
Archaea (formerly called archaebacteria) are organisms that are genetically distinct from Eubacteria and Eukaryotes. These organisms are recognized as the third domain of life, and are frequently found living in extreme environments such as hot springs, under-sea thermal vents, or highly alkaline and/or concentrated saline conditions. Among the Archaea, the largest group is the methanogens. Methanogens are capable of using one-carbon (C-1) substrates for their sole source of both carbon and energy, and synthesize all of their cellular components starting from the most rudimentary of organic substrates (CO2 for example).
Research in our laboratory is focused on understanding the enzymatic mechanisms involved in biosynthesis and cleavage of two-carbon acetyl units in Archaea. Acetyl-CoA synthesis from one-carbon precursors (and also cleavage of acetyl-CoA to yield C-1 products) is catalyzed by a multi-enzyme complex, known as ACDS (the acetyl-CoA decarbonylase/synthase complex). Both the carbon-sulfur and the carbon-carbon bond of acetyl-CoA are formed by the ACDS complex in a highly unusual biochemical mechanism involving metal-based carbonyl group insertion, and/or methyl group migration. Biochemical techniques, including spectroscopy, chromatography, electrochemistry, and kinetic and thermodynamic measurements, are being used to explore the enzymology of this fascinating multi-step reaction. Immunological and electron microscopic techniques are being employed to elucidate the structures of individual protein subcomponents, such as the 220 kDa Ni-Fe/S protein subcomponent to yield ultimately the 3-dimensional, quaternary structure of the entire ACDS complex.
In a second project we are using molecular biological techniques to investigate important members of a new class of zinc-based alkyltransferase enzymes. Studies are underway to elucidate the biochemistry of two methyltransferase isoenzymes (MT2-A and MT2-M) that catalyze specific methyl group transfer reactions in separate C-1 metabolic pathways. The two isozymes are also divergently expressed and highly regulated by C-1 substrate availability. Thus, the molecular mechanisms underlying the control of MT2 are of great interest for the potential to discover new regulatory mechanisms in the methanogenic Archaea. Molecular genetic and biochemical experiments are now in progress to identify DNA-binding regulatory proteins involved in control of MT2 expression in different C-1 pathways.
"Tight coupling of partial reactions in the acetyl-CoA decarbonylase/synthase (ACDS) multienzyme complex from Methanosarcina thermophila: Acetyl C-C bond fragmentation at the A cluster promoted by protein conformational changes." Gencic S., Duin E.C., Grahame D.A. (2010) J. Biol. Chem. Mar 4. [Epub ahead of print]
"Two separate one-electron steps in the reductive activation of the A cluster in subunit beta of the ACDS complex in Methanosarcina thermophila." Gencic, S. and Grahame, D.A. (2008) Biochemistry 47, 5544-5555.
"A single operon-encoded form of the acetyl-CoA decarbonylase/synthase multienzyme complex responsible for synthesis and cleavage of acetyl CoA in Methanosarcina thermophila." Grahame, D.A., Gencic, S., and DeMoll (2005) Arch. Microbiol. 184, 32-40.
"Chemically distinct Ni sites in the A-cluster in subunit beta of the acetyl-CoA decarbonylase/synthase complex from Methanosarcina thermophila: Ni L-edge and X-ray magnetic circular dichroism analyses." Funk, T., Gu, W., Friedrich, S., Wang, H., Gencic, S., Grahame, D.A., and Cramer, S.P. (2004) J. Am. Chem. Soc. 126, 88-95.
"The A-cluster in subunit beta of the acetyl-CoA decarbonylase/synthase complex from Methanosarcina thermophila: Ni and Fe K-edge XANES and EXAFS analyses." Gu, W., Gencic, S., Cramer, S.P., and Grahame, D.A. (2003) J. Am. Chem. Soc. 125, 15343-15351.