Contact Information

Biochemistry and Molecular Biology (BIO)


Uniformed Services University of the Health Sciences
Department of Biochemistry and Molecular Biology
4301 Jones Bridge Road, C1094
Bethesda, Maryland 20814-4799
B3022
Phone: 301-295-3555
Fax: (301) 295-3512
Lab: (301) 295-3274
Email: david.grahame@usuhs.edu

PubMed listing

David Grahame, Professor, Department of Biochemistry and Molecular Biology

David Grahame

Professor

Research interests

In our laboratory we study redox-active metalloenzymes and their functions in microbial metabolism in anaerobic bacteria and archaea. Archaea are genetically distinct from eukaryotes and bacteria, and represent a unique domain of living organisms. Anaerobic bacteria and archaea impact human life in many ways; they play critical roles in global nitrogen and carbon cycles, they are useful to detoxify environmental and municipal wastes, and as part of the human gut microbiome they aid in digestion and the development of the immune system. Anaerobic microorganisms also can be pathogenic, and various species of Clostridia cause diseases such as tetanus, gangrene, acute ulcerative gingivitis, botulism and antibiotic-associated inflammatory diarrhea (C. difficile). Thus, research to reveal how specialized metalloenzymes function in anaerobic metabolism is useful to exploit the metabolic potential of these organisms for industrial, agricultural, and biomedical purposes.

Anaerobic methane producers, the methanogens, are the largest group of archaea, and we have been investigating the mechanism by which methanogens carry out large scale synthesis/cleavage of two-carbon acetyl units from one-carbon precursors/products. The reaction is catalyzed by a large 2,000 kDa multienzyme complex known as the ACDS complex (acetyl-CoA decarbonylase/synthase) that can make up as much as 25% of the soluble protein in the cell. Both the carbon-carbon and the carbon-sulfur bond of acetyl-CoA are formed/broken by the ACDS complex in a highly unusual biochemical mechanism involving metal-based carbonyl group insertion, and/or methyl group migration. Different catalytic roles of the five ACDS subunits include: CO dehydrogenase (αε), acetyl-CoA synthase (β) and B12 corrinoid methyltransferase (γδ) as shown below.

Scheme 1. ACDS complex partial reactions in the overall synthesis and cleavage of acetyl CoA. (H4SPt stands for tetrahydrosarcinapterin, a methanogen folate analog.)

Scheme 1. ACDS complex partial reactions in the overall synthesis and cleavage of acetyl CoA. (H4SPt stands for tetrahydrosarcinapterin, a methanogen folate analog.)

We use molecular biological and biochemical techniques to explore the various reactions in this important multi-step process.

Recently, we carried out a side-by-side comparison of the enzymatic properties of the isolated archaeal subunit with the bacterial acetyl-CoA synthase, a subunit that is part of a smaller, two-subunit protein known as CO dehydrogenase/acetyl-CoA synthase (CODH/ACS). Our results revealed unexpected protein conformational control over the organometallic chemistry that takes place at the A cluster, a Ni- and Fe- containing active site metal center. Comparison of different ACS constructs showed acetyl C-C bond fragmentation was promoted by interdomain interactions involving the bacterial ACS N-terminal domain (a region of the protein not found in archaea). Our analyses of the role of a nearby phenylalanine residue showed remarkable effects on catalysis. We see that Nature provided the reactive Ni center with a repositionable aromatic shield, whose action is linked to the protein conformational state so as to selectively avoid CO substrate inhibition and to capitalize on the most productive sequence of steps in the reaction, maximizing the efficiency of acetyl-CoA synthesis. The enzyme thereby acquired the means to handle special requirements of an atypical organometallic mechanism, not found in organisms outside of anaerobic bacteria and archaea. A scheme that illustrates the individual steps and the connection with open/closed conformational states is shown below.

Scheme 2. Different modes of CO binding to acetyl-CoA synthase and the role of a conserved phenylalanine in the coordination environment of Ni.

Scheme 2. Different modes of CO binding to acetyl-CoA synthase and the role of a conserved phenylalanine in the coordination environment of Ni.

Our overall goal in studies on the mechanisms and functions of metalloenzymes is to provide a fundamental understanding of the unusual metabolic and physiological adaptations of important anaerobic microorganisms.

Selected publications

Gencic, S., Kelly, K., Ghebreamlak, S., Duin, E. C., and Grahame, D. A. (2013) Different modes of carbon monoxide binding to acetyl-CoA synthase and the role of a conserved phenylalanine in the coordination environment of nickel. Biochemistry 52, 1705-1716.

Grahame, D. A. (2011) Methods for analysis of acetyl-CoA synthase: Applications to bacterial and archaeal systems. Methods Enzymol. 494, 189-217.

Gencic S., Duin E.C., Grahame, D. A. (2010) 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." J. Biol. Chem. 285, 15450-15463.

Gencic, S. and Grahame, D. A. (2008) Two separate one-electron steps in the reductive activation of the A cluster in subunit beta of the ACDS complex in Methanosarcina thermophila. Biochemistry 47, 5544-5555.

Grahame, D. A., Gencic, S., and DeMoll (2005) A single operon-encoded form of the acetyl-CoA decarbonylase/synthase multienzyme complex responsible for synthesis and cleavage of acetyl CoA in Methanosarcina thermophila. Arch. Microbiol. 184, 32-40.