Professor of Biochemistry & Chemistry
Ph.D., Marquette University, 1975
r.armstrong@vanderbilt.edu

Structural And Mechanistic Enzymology

Research efforts in Professor Armstrong's laboratory are embodied in three projects directed at elucidating the mechanisms of action of enzymes involved in the metabolism of foreign or xenobiotic molecules. These catalysts, known as detoxication enzymes, are essential components of any organism's ability to resist chemical insult.

The first project is a study of glutathione transferases, a family of enzymes involved in the metabolism of electrophilic molecules such as expoxides, alkyl halides and a,b-unsaturated carbonyl compounds. From studies of the physical organic chemistry occurring in the active site, aspects of the kinetic, chemical, and stereochemical mechanisms of these enzymes have been elucidated. In addition, high-resolution three-dimensional structures of several glutathione transferases have been solved and are being used as a guide in the construction of chimeric or hybrid enzymes with altered catalytic properties. The functional properties of the mutant enzymes provide insight into the specific role of various amino acid residues in the region of the active site. The site-general and site-specific incorporation of unnatural amino acids into this enzyme is being investigated as a tool to refine our understanding of the mechanism of catalysis.

Many detoxication enzymes are membrane-bound and pose unique problems for mechanistic analysis. In a second project two membrane-bound detoxication enzymes, enzymes epoxide hydrolase and UDP-glucuronosyltransferase are being investigated. Efficient expression systems are being developed for these enzymes to facilitate structural and mechanistic studies. The discovery that epoxide hydrolase proceeds via a covalent ester intermediate has aided in the identification of active site residues that participate in catalysis and has helped define the evolutionary relationship of this protein with other hydrolase enzymes.

Microorganisms also have detoxication enzymes which allow them to use many organic compounds as energy sources or to resist the toxic effect of antibiotics. This later phenomenon contributes to the erosion of the efficacy of clinically useful antibiotics and represents a serious human health problem. The objectives of the third research project are to elucidate the catalytic mechanisms and structures of enzymes involved in the resistance of microorganisms to the antibiotic fosfomycin. These objectives include, (i) the construction of high-level expression systems for fosfomycin resistance proteins; (ii) elucidation of the catalytic mechanisms of the enzymes, by spectroscopic, steady state and pre-steady state kinetic techniques; and (iii) determination of the three-dimensional structures by X-ray crystallography. The information will provide a rational basis for the design of new drugs to counter antibiotic resistance.

Selected Publications

Brown DW, Schaab MR, Birmingham WR, Armstrong RN. Evolution of the Antibiotic Resistance Protein, FosA, Is Linked to a Catalytically Promiscuous Progenitor. Biochemistry. 2009, 48 (9): 1847-1849.

Busenlehner LS, Branden G, Namslauer I, Brzezinski P, Armstrong RN. Structural elements involved in proton translocation by cytochrome c oxidase as revealed by backbone amide hydrogen-deuterium exchange of the E286H mutant. Biochemistry. 2008, 47 (1): 73 -83.

Kinsley N, Sayed Y, Mosebi S, Armstrong RN, Dirr HW. Characterization of the binding of 8-anilinonaphthalene sulfonate to rat class Mu GST M1-1. Biophysical Chemistry. 2008, 137
(2-3): 100-104.

Thompson LC, Ladner JE, Codreanu SG, Harp J, Gilliland GL, Armstrong RN. 2-hydroxychromene-2-carboxylic acid isomerase: A kappa class glutathione transferase from Pseudomonas putida. Biochemistry. 2007, 46 (23): 6710-6722.

Fillgrove KL, Pakhomova S, Schaab MR, Newcomer M, Armstrong RN. Structure and mechanism of the genomically encoded fosfomycin resistance protein, FosX, from Listeria monocytogenes. Biochemistry. 2007, 46 (27): 8110-8120.

Rigsby RE, Brown DW, Dawson E, Lybrand TP, Armstrong RN. A model for glutathione binding and activation in the fosfomycin resistance protein, FosA. Archives of Biochemistry and Biophysics. 2007, 464 (2): 277-283.

Busenlehner LS, Alander J, Jegerscohld C, Holm PJ, Bhakat P, Hebert H, Morgenstern R, Armstrong RN. Location of substrate binding sites within the integral membrane protein microsomal glutathione transferase-1. Biochemistry. 2007, 46 (10): 2812-2822.

Busenlehner LS, Salomonsson L, Brzezinski P, Armstrong RN. Mapping protein dynamics in catalytic intermediates of the redox-driven proton pump cytochrome coxidase. Proceedings of the National Academy of Sciences of the United States of America. 2006, 103 (42): 15398-15403.

Thompson LC, Walters J, Burke J, Parsons JF, Armstrong RN, Dirr HW. Double mutation at the subunit interface of glutathione transferase rGSTM1-1 results in a stable, folded monomer. Biochemistry. 2006, 45 (7): 2267-2273.

Rigsby RE, Fillgrove KL, Beihoffer LA, Armstrong RN. Fosfomycin resistance proteins: A nexus of glutathione transferases and epoxide hydrolases in a metalloenzyme superfamily. Biochemistry. 2005, 401: 367-379.

Armstrong RN. Mechanistic diversity and the evolution of enzyme function. FASEB Journal. 2005, 19 (5): A1371-A1371 Part 2 Suppl. S.

Walsby CJ, Telser J, Rigsby RE, Armstrong RN, Hoffman BM. Enzyme control of small-molecule coordination in FosA as revealed by P-31 pulsed ENDOR and ESE-EPR. Journal of the American Chemical Society. 2005, 127 (23): 8310-8319.

Codreanu SG, Thompson LC, Hachey DL, Dirr HW, Armstrong RN. Influence of the dimer interface on glutathione transferase structure and dynamics revealed by amide H/D exchange mass spectrometry. Biochemistry. 2005, 44 (31): 10605-10612.

Busenlehner, L. S.; Armstrong, R. N. Insights Into Enzyme Structure And Dynamics Elucidated By Amide H/D Exchange Mass Spectrometry. Archives of Biochemistry and Biophysics. 2005, 433 (1): 34-46.

Ladner, J. E.; Parsons, J.F.; Rife, C. L.; Armstrong, R. N. Parallel Evolutionary Pathways For Glutathione Transferases: Structure And Mechanism of The Mitochondrial Class Kappa Enzyme rGSTK1-1. Biochemistry. 2004, 43: (2), 352-361.

Pakhomova, S.; Rife, C. L.; Armstrong, R. N.; et al. Structure of Fosfomycin Resistance Protein Fosa from Transposon Tn2921. Protein Science. 2004, 13: (5), 1260-1265.

Svensson, R.; Alander, J.; Armstrong, R. N.; et al. Kinetic. Characterization of Thiolate Anion Formation and Chemical Catalysis of Activated Microsomal Glutathione Transferase 1. Biochemistry. 2004, 43: (27), 8869-8877.

Busenlehner, L. S.; Codreanu, S. G.; Holm, P. J.; Armstrong, R. N. Stress Sensor Triggers: Conformational Response of the Integral Membrane Protein Microsomal Glutathione Transferase 1. Biochemistry. 2004, 43: (35), 11145-11152.

Armstrong, R. N.; Marnett, L. J. Cracking Open Access. Chemical & Engineering News. 2004, 82: (43), 7.

Rigsby, R. E.; Rife, C. L.; Fillgrove, K. L.; Armstrong, R. N. Phosphonoformate: A Minimal Transition State Analogue Inhibitor of The Fosfomycin Resistance Protein, FosA. Biochemistry. 2004, 43 (43): 13666-13673.

Armstrong, R. N. Mechanistic and Genomic Imprints of The Evolution of Antibiotic Resistancein Pathogenic Microorganisms. Biochemistry. 2003, 42: (28), 55.

Stourman, N. V.; Rose, J. H.; Vuilleumier, S.; Armstrong, R. N. Catalytic Mechanism Of Dichloromethane Dehalogenase from Methylophilus sp Strain DM11. Biochemistry. 2003, 42: (37), 11048-11056..

Rife, C. L.; Parsons, J. F.; Xiao, G. Y.; Armstrong, R. N. Conserved Structural Elements in Glutathione Transferase Homologues Encoded in The Genome of Escherichia Coli. Proteins-Structure Function and Genetics. 2003, 53: (4), 777-782.

Fillgrove, K. L.; Pakhomova, S.; Newcomer, M. E.; Armstrong, R. N. Mechanistic Diversity of Fosfomycin Resistance in Pathogenic Microorganisms. Journal of the American Chemical Society. 2003, 125: (51).

Smoukov, Stoyan; Telser, Joshua; Bernat, Bryan A.; Rife, Chris L.; Armstrong, Richard N.; Hoffman, Brian M. EPR Evidence for the Interaction Between Substrates and the Mn(II) Active Site of the Bacterial Antibiotic Resistance Enzyme, FosA: A Better Way to Examine Mn(II). Journal of the American Chemical Society. 2002, 124: 2318-2326.

Specialties

• VICB
• Mechanistic enzymology
• Biochemistry

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