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case western reserve university

BIOCHEMISTRY
 
 

Dr. Marianne Pusztai-Carey

Assistant Professor

Pub Med:

Dr. Marianne Pusztai-Carey

1) Studying function and structure relationship regarding the transcarboxylase enzyme complex 2) Mode of action studies of the insecticidal proteins produced by Bacillus thuringiensis

1) The enzyme transcarboxylase (TC) is a nano-machine of 1,200 kDa that carries out the transfer of a carboxylate group from methylmalonyl-CoA to pyruvate. The reaction utilizes three different kinds of subunits. TC is a paradigm for a complex multisubunit enzyme since the subunits can be assembled and disassembled. The individual subunits are being characterized by a number of biophysical techniques with future emphasis being on the use of FRET (fluorescence resonance energy transfer) to determine the dynamic process of CO2- transfer between subunits. 2) Bacillus thuringiensis (Bt) is a natural aerobic Gram positive spore forming soil bacterium which produces multiple forms of insect specific delta endotoxin proteins. Bt is a key agent for biological insect control and an environmentally friendly alternative to chemical insecticides. Bt proteins target the midguts of insects, their mode of action is not fully understood. My involvement in the Bt research includes: A/ Outbreak of West Nile virus infection warrants to speed up the research into the details of the mechanism of mosquito control by Bt. Results with Bt israelensis, the major mosquitocidal agent point to the direction of a detergent-like action of this protein on the insect midgut membranes -in collaboration with scientists at University of Mississippi B/ The agricultural and forestry industries are gradually introducing transgenic plants expressing Bt. The constant exposure of the insect pests to the same insecticide leads to insect resistance. Understanding the mode of action of Bt on the insects will help to avoid or overcome this resistance. Prime target of the studies is the isolation and characterization of insect midgut receptors: in collaboration with scientists at Auburn University, Alabama and CSIRO, Canberra, Australia C/ Bt transgenic plants have brought about strong public resistance towards Bt. Part of this project is to study the effect of Bt on beneficial insects in collaboration with the Biology and Ecophysiology Laboratory at Besancon Cedex, France

Selected References

  • Pius S. Padayatti, Marion S. Helfand, Monica A. Totir, Marianne P. Carey, Andrea M. Hujer, Paul R. Carey, Robert A. Bonomo and Focco van den Akker: Tazobactam forms a stoichiometric trans-enamine intermediate in the E166A variant of SHV-1 beta-lactamase: 1.63 Å crystal structure, Biochemistry, 2003. in print

  • Marion S. Helfand, Monica A. Totir, Marianne P. Carey, Andrea M. Hujer, Robert A. Bonomo, and Paul R. Carey. Following the Reactions of Mechanism-Based Inhibitors with b-Lactamase by Raman Crystallography. Biochemistry; 2003; 42(46) 13386 - 13392

  • Zheng X., Rivera-Hainaj, R. E., Zheng, Y., Pusztai-Carey, M., Hall, P.R., Yee, V.C. and Carey, P.R.: Substrate binding induces a cooperative conformational change in the 12S subunit of transcarboxylase: Raman crystallographic evidence. Biochemistry, 41, 10741-10746, 2002

  • Rivera-Hainaj, R. E., Pusztai-Carey, M., Reddy, D.V., Choowongkomon, K., Sonnichsen, F. D. and Carey, P. R. Characterization of the carboxylate Delivery Module of Transcarboxylase: Following the Spontaneous Decarboxylation of the 1.3S-CO2- Subunit by NMR and FTIR Spectroscopies Biochemistry 41, 2191-2197, 2002

  • Agrawal, A., Simpson, M.J., Black, S., Carey, M.P. and Samols, D.: A C-reactive protein mutant that does not bind to phosphocholine and pneumococcal C-polysaccharide, J Immunol. 169, 3217-22. 2002

  • Monnerat, R., Masson, L., Brousseau, R., Pusztai-Carey, M., Bordat, D., Frutos, R: Differential Activity and Activation of Bacillus thuringiensis Insecticidal Proteins in Diamondback Moth, Plutella xylostella. Current Microbiology 39:159-162, 1999

  • Hilbeck, A., Moar, W. J., Pusztai-Carey, M., Filippini, A. and Bigler, F. : Toxicity of Bacillus thuringiensis CryIAb toxin to the Predator Chrysoperla carnea (Neuptera: Chrysopidae). Environ. Entomol. 27, 1255-1263, 1998

  • Masson, L., Erlandson, M., Pusztai-Carey, M., Brousseau, R., Juarez-Perez, V. and Frutos, R.: A holistic approach for determining the entomopathogenic potential of Bacillus thuringiensis Appl Environ Microbiol 64(12): 4782-8, 1998

  • Butko, P., Huang, F., Pusztai-Carey, M., Surewicz, W. K.: Interaction of the delta endotoxin CytA from Bacillus thuringiensis var.israelensis with lipid membranes. Biochemistry 36(42),12862-8 1997

  • Liu, Y.-B., Tabashnik, B. E. and Pusztai-Carey, M.: Field-Evolved Resistance to Bacillus thuringiensis Toxin CryIC in Diamondback Moth (Lepidoptera: Plutellidae). Journal of Economic Entomology, 89, 129-135, 1996

  • Pusztai-Carey, M., Carey, P., Lessard, T. and Yaguchi, M.: Identification, quantitation and purification of insecticidal proteins from Bacillus thuringiensis. US Patent No.5,523,211, 1996

  • Butko, P., Huang, F., Pusztai-Carey, M. and Surewicz, W. K. Surewicz. Membrane permeabilization induced by cytolotic d-endotoxin CytA from Bacillus thuringiensis var. israelensis. Biochemistry, 35, 11355-11360, 1996

  • Moar, W. J., Pusztai-Carey, M., Van Faassen, H., Bosch, D., Frutos, R., Luo, and Adang, M. J. Development of Bacillus thuringiensis CryIC resistance by Spodoptera exigua, (Hübner) (Lepidoptera: Noctuidae). Appl. Environ. Microbiol. 61(6), 2086-2092, 1995

  • Pusztai-Carey, M., Carey, P., Lessard, T. and Yaguchi, M.: Isolation, quantitation and purification of insecticidal proteins from endotoxins of Bacillus thuringiensis. US Patent No.5,356,788.1995

  • Pusztai-Carey, M. A novel method for quantitation and isolation of individual toxins from multi-gene Bacillus thuringiensis strains. In Bacillus thuringiensis Biotechnology and Environmental Benefits, 1, 191-199, 1995 Ed. T-Y Feng Hua Shiang Yuan Publishing Co., Taipei, Taiwan.

  • Tonge, P. J., Anderson, V. E., Fausto, R., Kim, M., Pusztai-Carey, M. and Carey, P. R. Localised electron polarization in a substrate analog binding to the active site of enoyl-CoA hydratase: Raman Spectroscopic and conformational analyses of rotamers of hexadienoyl thiolesters. Biospectroscopy. 1(6) 387-394, 1995

  • Moar, W. J., Pusztai-Carey, M. and Mack, T. P. Toxicity of purified proteins and the HD-1 Strain from Bacillus thuringiensis against lesser cornstalk borer (Lepidoptera: Pyralidae). Biol. Microbial Control, 88, 606-609, 1995

  • Grochulski, P., Masson, L., Borisova, S., Pusztai-Carey, M., Schwartz, J.L., Brousseau, R. and Cygler. M. Bacillus thuringiensis CryIA(a) Insecticidal Toxin: Crystal Structure and Channel Formation. J. Mol. Biol. 254, 447- 464,1995

  • Butko, P., Cournoyer, M., Pusztai-Carey, M. and Surewicz, W. K. Membrane interactions and surface hydrophobicity of Bacillus thuringiensis delta-endotoxin cryIC. FEBS Letters 340, 89-92, 1994

  • Borisova S., Grochulski P., van Faassen, H., Pusztai-Carey, M., Masson L. and Cygler M.: Crystallization and preliminary X-ray diffraction studies of the Lepidopteran-specific insecticidal crystal protein CryIA(a). J. Mol. Biol. 243, 530-532, 1994