Organisms living in aerobic environments require mechanisms which prevent or limit damage to cellular components by reactive oxygen species; these species arise from the incomplete reduction of oxygen during respiration or from exposure to external agents such as light, radiation, redox-cycling drugs or stimulated host phagocytes. A variety of enzymatic and nonenzymatic systems have evolved within living organisms to counter such damage. In bacteria such as Salmonella typhimurium and Escherichia coli, expression of a set of antioxidant enzymes is controlled in a coordinate fashion by an oxidation- reduction-sensitive regulatory protein, OxyR. Using a high-expression OxyR mutant of S. typhimurium, a novel enzymatic activity responsible for the NAD(P)H-linked reduction of toxic organic hydroperoxides was discovered. This alkyl hydroperoxide reductase (AhpR), which is the focus of studies in my laboratory, was subsequently shown to be separable into two protein components, designated AhpF and AhpC. AhpF is an FAD-containing protein related to another well- characterized flavoprotein, thioredoxin reductase, and catalyzes the transfer of electrons from NAD(P)H to AhpC. The smaller AhpC protein is without a chromaphoric cofactor and serves directly as the peroxide-reducing component (homologues of AhpC are widespread in biological systems and have been designated "peroxiredoxins"). Our studies of the catalytic mechanism of AhpR indicate that both component proteins operate through cycling of protein- derived cystine disulfides between oxidized (disulfide) and reduced (dithiol) states. Anaerobic titrations of each protein with reductants have confirmed the presence and essentiality of three redox centers in AhpF (one FAD and two disulfide centers) and one redox-active disulfide center per monomer in AhpC (Poole, 1996; Poole, Godzik, et al, 2000). We have also shown that an unusual oxidized cysteine derivative, cysteine-sulfenic acid (Cys-SOH), is generated transiently through direct oxidation of Cys46, one of the two cysteine residues of AhpC, by the hydroperoxide substrate (Ellis & Poole, 1997a & b). Related oxidized cysteine derivatives have been identified in the two other known heme- and metal-independent peroxide reductases (NADH peroxidase and glutathione peroxidases) and may play a role in signal transduction by the OxyR protein itself.
Studies of the structural and chemical bases for the enzymatic functions of AhpF and AhpC involve a wide range of biochemical techniques. Site-directed and PCR-based mutagenesis experiments have been designed to address specific structure-function questions. Characterization of native and mutant proteins involve enzymological techniques, such as spectral titrations and rapid reaction kinetic measurements, protein chemistry methodology to define cysteine content and redox status, and structural work, which includes protein crystallization and analytical ultracentrifugation.
Poole, L.B., and Ellis, H.R. (1996) Flavin-dependent alkyl hydroperoxide reductase from Salmonella typhimurium. 1. Purification and enzymatic activities of overexpressed AhpF and AhpC proteins. Biochemistry 35, 56-64. PDF of article
Poole, L.B. (1996) Flavin-dependent alkyl hydroperoxide reductase from Salmonella typhimurium. 2. Cystine disulfides involved in catalysis of peroxide reduction. Biochemistry 35, 65-75. PDF of article
Poole, L.B., Chae, H.Z., Flores, B.M., Reed, S.L., Rhee, S.G., and Torian, B.E. (1997) Peroxidase activity of a TSA-like antioxidant protein from a pathogenic amoeba. Free Radical Biol. Med. 23, 955-959. PDF of article
Li Calzi, M., and Poole, L.B. (1997) Requirement for the two AhpF cystine disulfide centers in catalysis of peroxide reduction by alkyl hydroperoxide reductase. Biochemistry 36, 13357-13364. PDF of article
Ellis, H. R., and Poole, L.B. (1997) Roles for the two cysteine residues of AhpC in catalysis of peroxide reduction by alkyl hydroperoxide reductase from Salmonella typhimurium.Biochemistry 36, 13349-13356. PDF of article
Ellis, H. R., and Poole, L.B. (1997) Novel application of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole to identify cysteine sulfenic acid in the AhpC component of alkyl hydroperoxide reductase. Biochemistry 36, 15013-15018. PDF of article
Poole, L.B. (1997) The Salmonella typhimurium alkyl hydroperoxide reductase enzyme system, in Flavins and Flavoproteins 1996 (K.J. Stevenson, V. Massey and C.H. Williams, Jr., eds) University of Calgary Press, Calgary, Alberta, Canada, 751-760.
Higuchi, M., Yamamoto, Y., Poole, L.B., Shimada, M., Sato, Y., Takahashi, N., and Kamio, Y. (1999) Functions of two types of NADH oxidases in energy metabolism and oxidative stress of Streptococcus mutans. J. Bacteriol. 181, 5940-5947. PDF of article
Poole, L.B. (1999) Flavin-linked redox components required for AhpC reduction in alkyl hydroperoxide reductase systems. In Flavins and Flavoproteins 1999, (S. Ghisla, P. Kroneck, P. Macheroux and H. Sund, eds.) Agency for Scientific Publications, Berlin, Germany, 195-202.
Reynolds, C.M., and Poole, L.B. (1999) Functional characterization of the N-terminus of AhpF by chimeric construction with TrR. In Flavins and Flavoproteins 1999, (S. Ghisla, P. Kroneck, P. Macheroux and H. Sund, eds.) Agency for Scientific Publications, Berlin, Germany, 681-684.
Higuchi, M., Yamamoto, Y., Poole, L.B., Shimada, M., Sato, Y., Takahashi, N., and Kamio, Y. (1999) Functional and regulatory studies of two distinct NADH oxidases from Streptococcus mutans. In Flavins and Flavoproteins 1999, (S. Ghisla, P. Kroneck, P. Macheroux and H. Sund, eds.) Agency for Scientific Publications, Berlin, Germany, 691-694.
Poole, L.B., Higuchi, M., Shimada, M., Li Calzi, M., and Kamio, Y. (2000) Streptococcus mutans H2O2-forming NADH oxidase is an alkyl hydroperoxide reductase protein. Free Radical Biol. Med. 28, 108-120. PDF of article
Poole, L.B., Godzik, A., Nayeem, A. and Schmitt, J.S. (2000) AhpF can be dissected into two functional units: tandem repeats of two thioredoxin-like folds in the N-terminus mediate electron transfer from the thioredoxin reductase-like C-terminus to AhpC. Biochemistry 39, 6602-6615. PDF of article, PDF of supporting info
Reynolds, C.M., and Poole, L.B. (2000) Attachment of the N-terminal domain of Salmonella typhimurium AhpF to Escherichia coli thioredoxin reductase confers AhpC reductase activity but does not affect thioredoxin reductase activity. Biochemistry 39, 8859-8869. PDF of article, PDF of supporting info
Yamamoto, Y., Higuchi, M., Poole, L.B., and Kamio, Y. (2000) Identification of a new gene responsible for the oxygen tolerance in aerobic life of Streptococcus mutans. Biosci. Biotechnol. Biochem. 64, 1106-1109.
Yamamoto, Y., Higuchi, M., Poole, L.B., and Kamio, Y. (2000) Role of the dpr product in oxygen tolerance in Streptococcus mutans. J. Bacteriol. 182, 3740-3747. PDF of article
Poole, L.B., Reynolds, C.M., Wood, Z.A., Karplus, P.A., Ellis, H.R. and Li Calzi, M. (2000) AhpF and other NADH:peroxiredoxin oxidoreductases, homologues of low Mr thioredoxin reductase. Eur. J. Biochem. 267, 6126-6133. PDF of article
Baker, L.M.S., Raudonikiene, A., Hoffman, P.S., and Poole, L.B. (2001) An essential thioredoxin-dependent peroxiredoxin system from Helicobacter pylori: genetic and kinetic characterization. J. Bacteriol. 183, 1961-1973. PDF of article
Reynolds, C.M., and Poole, L.B. (2001) Activity of one of two engineered heterodimers of AhpF, the NADH:peroxiredoxin oxidoreductase from Salmonella typhimurium, reveals intrasubunit electron transfer between domains. Biochemistry 40, 3912-3919. PDF of article
Wood, Z.A., Poole, L.B., and Karplus, P.A. (2001) Structure of intact AhpF reveals a mirrored thioredoxin-like active site and implies large domain rotations during catalysis. Biochemistry 40, 3900-3911. PDF of article
Reynolds, C.M., Meyer, J., and Poole, L.B. (2002) An NADH-dependent bacterial thioredoxin reductase-like protein, in conjunction with a glutaredoxin homologue, form a unique peroxiredoxin (AhpC) reducing system in Clostridium pasteurianum. Biochemistry 41, 1990-2001. PDF of article, PDF of supporting material
Conway, M.E., Yennawar, N., Wallin, R., Poole, L.B., Hutson, S.M. (2002) Identification of a peroxide-sensitive redox switch at the CXXC motif in the human mitochondrial branched chain aminotransferase. Biochemistry 41, 9070-9078. PDF of article.
Poole, L.B., and Ellis, H.R. (2002) Methods to identify cysteine sulfenic acid in AhpC alkyl hydroperoxide reductase. Meth. Enzymol. 348, 122-136.
Yamamoto, Y., Poole, L.B., Hantgan, R.R., and Kamio, Y. (2002) An iron-binding protein, Dpr, from Streptococcus mutans prevents iron-dependent hydroxyl radical formation in vitro. J. Bacteriol. 184, 2931-2939. PDF of article.
Conway, M.E., Yennawar, N., Wallin, R., Poole, L. B., and Hutson, S.M. (2003) Human mitochondrial branched chain aminotransferase: Structural basis for substrate specificity and role of redox active cysteines. Biochim. Biophys. Acta 1647: 61-65. PDF of article.
Schröder, E., Jönsson, T., and Poole, L. (2003) Hydroxyapatite chromatography: altering the phosphate-dependent elution profile of protein as a function of pH. Anal. Biochem. 313: 176-178. PDF of article.
Baker, L.M.S., and Poole, L.B. (2003) Catalytic mechanism of thiol peroxidase from Escherichia coli: sulfenic acid formation and overoxidation of essential Cys61. J. Biol. Chem. 278: 9203-9211. PDF of article.
Conway, M.E., Poole, L.B., and Hutson, S.M. (2004) Roles for cysteine residues in the regulatory CXXC motif of human mitochondrial branched chain aminotransferase enzyme. Biochemistry 43: 7356-64. PDF of article.
Choi, M.-H., Sayed, D., Poole, L.B., Hirata, K., Henderman, S., Torian, B. and Reed, S.L.: An unusual surface peroxiredoxin protects invasive Entamoeba histolytica from oxidant attack. Mol. Biochem. Parasitol. 143: 80-89 (2005). PDF of article.
Roberts, B.R., Wood, Z.A., Jönsson, T.J., Poole, L.B., and Karplus, P.A.: Oxidized and synchrotron cleaved structures of the disulfide redox center in the N-terminal domain of Salmonella typhimurium AhpF. Prot. Sci. 14: 2414-2420 (2005). PDF of article.
|Born in PA, raised in Maryland|
|B.A. in Biology and Chemistry from Wake Forest University in Winston-Salem, NC, 1980, Magna Cum Laude, with Honors in Biology|
|NSF Undergraduate Research Fellowship, 1979, protein folding and chemical modification research with Dr. Donald Wetlaufer in the Dept. of Chemistry at the University of Delaware|
|U.S. Peace Corps Volunteer, 1980-1982, Bingkor, Sabah, East Malaysia (on the island of Borneo), Form 4 and 5 Science and Math Teacher|
|Ph.D. in Biochemistry in 1988 from Bowman Gray School of Medicine of Wake Forest University, entitled "The Streptococcal Flavoprotein NADH Peroxidase: Purification, Analysis of Structural and Redox Properties, and Identification of the Active-Site Cysteinyl Derivative," with Dr. Al Claiborne|
|Postdoctoral Studies, 1988-1991, with Dr. John Gerlt, Dept. of Chemistry and Biochemistry at the University of Maryland in College Park, NIH Fellowship on staphylococcal nuclease structure and function (a Lawton Chiles Biotechnology Fellowship Award, NRSA GM13211)|
|Assistant Professor of Biochemistry, Dept. of Biochemistry, Wake Forest University School of Medicine, 1992-1997|
Associate Professor of Biochemistry, Dept. of Biochemistry, Wake Forest University School of Medicine, 1997-2006
Professor of Biochemistry and Director of the Center for Structural Biology, Dept. of Biochemistry, Wake Forest University School of Medicine, 2006-present
NIH RO1 GM50389, Mechanistic studies of alkyl hydroperoxide reductase
NIH R21 CA112145, Profiling of redox-sensitive signaling proteins
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