76100 Rehovot, Israel
telephone: +972 (8) 934-3828
Members of the group:
Standing (left to right): Shoshana Tel-Or, Yael Hacham
Sitting (left to right): Aaron Palmon, Oren Bogin, Yigal Burstein, Moshe Peretz
Inna Levin, Ph.D email@example.com Moshe Peretz, Ph.D. firstname.lastname@example.org Shoshana Tel-Or email@example.com Yael Hacham firstname.lastname@example.org Gal Meiri (Ph.D. student) email@example.com Edi Goihberg (Ph.D. student) firstname.lastname@example.org Collaboration: Virginia Buchner, Ph.D. email@example.com Visiting scientists: Herzl Ben-Hur, M.D. Aaron Palmon, D.M.D., Ph.D. firstname.lastname@example.org
Structure-thermostability relationship studies in thermophilic enzymesThe goals of this ongoing research program are (1) to identify, characterize, clone, sequence, and overexpress adh genes from a variety of hyperthermophilic, thermophilic, mesophilic, and psychrophilic microorganisms; (2) to determine the three dimensional structures of these ADHs by X-ray crystallography (in collaboration with Dr. Felix Frolow); (3) to analyze and compare the detailed three dimensional structures of the enzymes and to predict structural features conferring thermal stability in the thermophilic enzyme; (4) to test the validity of the predictions by altering the mesophilic enzymes to include the relevant features of the thermophilic enzyme using site-directed mutagenesis. To this end, we are comparing the molecular properties of five highly homologous ADHs: one from the extreme thermophile Thermoanaerobacter brockii (TBADH) and the others from the pseudo-thermophiles and mesophiles Clostridium beijerinckii (CBADH), Entamoeba histolytica (EHADH) and Entamoeba invadens (EIADH). All four highly homologous ADHs (60%-76% sequence identity and 72%-86% sequence similarity) are tetrameric, medium-chain, NADP-linked, zinc-containing enzymes with similarly broad substrate ranges. A fifth highly homologous ADH (62% identity) from Mycoplasma pneumoniae (MPADH), is being characterized. TBADH was purified from the extreme thermophilic bacterium T. brockii, which expresses enzymic activity at temperatures up to 85oC. TBADH was characterized as a class A enzyme (transfers the pro-R hydrogen from the pyridine 4 position of the reduced coenzyme), and its complete amino acid sequence was determined.
Crystalline alcohol dehydrogenase from the extreme
thermophile Thermoanaerobacter brockii
(photo by Dr. Yakov Korkhin)
The structural genes encoding TBADH and it mesophilic counterparts, CBADH, EHADH, and EIADH were cloned, sequenced, and overexpressed in Escherichia coli. Characterization of the active-site metal ion and the associated ligand amino acids, using atomic absorption spectroscopy analysis and site-directed mutagenesis, verified the presence of a catalytic zinc and the absence of a structural metal ion in TBADH, as well as the involvement of Cys-37, His-59, and Asp-150 of TBADH in zinc coordination. To study the role of certain structural peptide segments (peptide stretch, alpha-helix, beta-strand, peptide loop, etc.), regions or even whole protein domains, in conferring thermal stability, we engineered a series of 12 CB adh and TB adh chimeric genes and expressed them in E. coli. The results suggested that major structural elements of thermal stability might reside among the 9 discrepant amino acid residues between the N-terminal 50-amino acid residues of TBADH and CBADH.
Ribbon type representation of the tetramer of T. brockii alcohol dehydrogenase
Our results indicated that TBADH and its mesophilic counterparts, CBADH, EHADH, EIADH, and MPADH, would provide an excellent and reliable model system for studying the molecular basis of enzyme thermostability. To this end, the respective crystal structures of holo- and apo-CBADH were determined at 2.05 Angstrom; and 2.15 Angstrom; resolution, and that of holo-TBADH at 2.5 Angstrom; resolution (in collaboration with Dr. Felix Frolow). The 3-D structures of these proteins were the first to be determined for a prokaryotic ADH, as well as for an NADP(H)-dependent ADH. CBADH and TBADH have very similar three-dimensional structures. The monomers are composed of two domains: a cofactor-binding domain and a catalytic domain.
Super-position of the backbone structures of the monomeric subunits of TBADH (in green)
and CBADH (in red).
The tetramers are composed of two dimers, each structurally homologous to the dimer of alcohol dehydrogenases of vertebrates. The dimers form tetramers by means of contacts between surfaces opposite the interdomain cleft, thus leaving it accessible from the surface of the tetramer. The tetramer encloses a large internal cavity with a positive surface potential. A molecule of NADP(H) binds in the interdomain cleft to the cofactor-binding domain of each monomer. The specificity of the two bacterial alcohol dehydrogenases toward NADP(H) is determined by residues Gly-198, Ser-199, Arg-200 and Tyr-218, with the latter three forming hydrogen bonds with the 2´-phosphate of the cofactor. Upon NADP(H) binding to CBADH, Tyr-218 undergoes a rotation of approximately 120' that facilitates stacking interactions with the adenine moiety and hydrogen bonding with one of the phosphate oxygens. In apo-CBADH the catalytic zinc is tetracoordinated by side chains of residues Cys-37, His-59, Asp-150 and Glu-60; in holo-CBADH, Glu-60 is retracted from zinc in three of the four monomers, whereas in holo-TBADH, Glu-60 does not participate in Zn coordination.
A view of the interface (residues 268-278) between subunits A (green) and B (yellow) of TBADH and CBADH. Residues 273-276 of both subunits are colored red and the cavities between them are in blue. Note the smaller cavity at the interface of TBADH relative to larger one in CBADH. Amino acid residues are indicated by their number and the one letter code. The figure was generated using the program INSIGHT (MSI). Cavities were calculated by the program SURFNET 1.4.
The thermal stability of CBADH was enhanced after strategic substitution of amino acid residues with prolines from the homologous thermophilic TBADH. We used site-directed mutagenesis to replace eight complementary residue positions in CBADH, one residue at a time, with proline, yielding eight enzymatically active single-proline mutants and one double-proline mutant. The proline residues that appear to be crucial for the increased thermal stability of CBADH are located at a b-turn and a terminating external loop in the polypeptide chain. The findings of this study imply that at least two of the eight extra prolines in TBADH contribute to its thermal stability.
Intrasubunit salt-bridge within the Rossmann Fold of TBADH. Glu224 (red) located in alpha-helix D forms a salt-bridge (3.94 Angstrom) with Lys254 (blue) located in alpha-helix E. The position of the neighboring residue Glu280 of the same subunit is also shown. The coloring for alpha-helices is light blue, for beta-strands is light green and loops are in yellow. Amino acid residues are indicated by their number and the one letter code. The figure was generated using the program INSIGHT (MSI).
The thermal stability of CBADH was also enhanced by improving the intersubunit hydrophobic interactions and by salt-bridge formation. We used site-directed mutagenesis to replace strategic hydrophobic residues in non-conserved regions at the interface between two subunits with the corresponding amino acids from TBADH. We also introduced a short ion-pair network at the interface between two other subunits of CBADH. Of three hydrophobic mutants, only a double mutant showed substantially increased thermal stability. Thermostability was also augmented in the mutant of CBADH with the added ion-pair-network. The results imply that the amino acid substitutions in CBADH mutants with enhanced thermal stability reinforce the quaternary structure of the molecule by strengthening existing intersubunit hydrophobic interactions and by forming new salt bridges.
Intersubunit ion-pair network in TBADH. Lys257 (blue), Asp237 (red) of subunit A, Arg304 (blue) of subunit D and Glu165 of subunit A, form a four-membered ion-pair network. Subunit A is colored purple and subunit D is yellow. Amino acid residues are indicated by their number and the one letter code. The figure was generated using the program INSIGHT (MSI).
Click here for list of all publications since 2000 (including DOIs) [Submit additions and corrections]
L.J.W. Shimon, E. Goihberg, M. Peretz, Y. Burstein and F. Frolow, "Structure of alcohol dehydrogenase from Entamoeba histolytica", Acta Cryst. D 62, 541-547 (2006). [Read online]
E. Goihberg, O. Dym, S. Tel-Or, I. Levin, M. Peretz, and Y. Burstein, "A single proline substitution is critical for the thermostabilization of Clostridium beijerinckii alcohol dehydrogenase", Proteins: Structure, Function, and Bioinformatics 66, 196-204 (2007). [Read online]
S. Albeck, Y. Burstein, O. Dym, Y. Jacobovitch, N. Levi, R. Meged, Y. Michael, Y. Peleg, J. Prilusky, G. Schreiber, I. Silman, T. Unger, and J. L. Sussman. Three-dimensional structure determination of proteins related to human health in their functional context at The Israel Structural Proteomics Center (ISPC), Acta Crystallographica D 61, 1364-1372 (2005). [Read online]
O. Kleifeld, L. Rulisek, O. Bogin, A. Frenkel, Z. Havlas, Y. Burstein, and I.Sagi. Higher metal-ligand coordination in the catalytic site of cobalt-substituted Thermoanaerobacter brockii alcohol dehydrogenase lowers the barrier for enzyme catalysis. Biochemistry 43, 7151-7161 (2004). [Read online]
Levin I, Meiri G, Peretz M, Burstein Y, Frolow F. The ternary complex of Pseudomonas aeruginosa alcohol dehydrogenase with NADH and ethylene glycol. Protein Science 13 (6), 1547-1556 (2004). [Read online]
O. Bogin, I. Levin, Y. Hacham , S. Tel-Or, M. Peretz, F. Frolow and Y. Burstein. Structural basis for the enhanced thermal stability of alcohol dehydrogenase mutants from the mesophilic bacterium Clostridium beijerinckii: Contribution of salt bridging. Protein Sci. 11, (11) 2561-74 (2002). [Read online]
L. J. W. Shimon, M. Peretz, E. Goihberg, Y. Burstein, and F. Frolow, Thermophilic alcohol dehydrogenase from the mesophile Entamoeba histolytica: crystallization and preliminary X-ray characterization, Acta Crystallographica D 58, 546-548 (2002). [Read online]
A. Palmon, S. Tel-Or, E. Shai, B. Rager-Zisman and Y. Burstein. Development of a highly sensitive quantitative competitive PCR assay for the Detection of Murine Cytomegalovirus DNA. J. Virol. Methods, 86(2), 107-14 (2000). [Read online]
O. Kleifeld, A. Frenkel, O. Bogin, M. Eisenstein, V. Brumfeld, Y. Burstein and I. Sagi. Inhibition studies of alcohol dehydrogenase from Thermoanaerobacter brockii suggest a possible structure for a catalytic transition state. Biochemistry 39, 7702-7711 (2000).[Read online]
Y. Korkhin, A. J. Kalb(Gilboa), M. Peretz, O. Bogin, Y. Burstein and F. Frolow. Oligomeric integrity - the structural key to thermal stability in bacterial alcohol dehydrogenases. Protein Sci. 8, 1241-1249 (1999).[Read online]
Bogin O, Peretz M, Burstein Y, Probing structural elements of thermal stability in bacterial oligomeric alcohol dehydrogenases. I. Construction and characterization of chimeras consisting of secondary ADHs from Thermoanaerobacter brockii and Clostridium beijerinckii, Letters in Peptide Science 5 (5-6), 399-408 (1998).
Bogin O, Peretz M, Hacham Y, Korkhin Y, Frolow F, Kalb AJ, Burstein Y, Enhanced thermal stability of Clostridium beijerinckii alcohol dehydrogenase after strategic substitution of amino acid residues with prolines from the homologous thermophilic Thermoanaerobacter brockii alcohol dehydrogenase, Protein Science 7 (5), 1156-1163 (1998) [Read online]
Y. Korkhin, A. J. Kalb(Gilboa), M. Peretz, O. Bogin, Y. Burstein and F. Frolow. NADP-dependent bacterial alcohol dehydrogenases: crystal structure, cofactor binding and cofactor specificity of the ADHs of Clostridium beijerinckii and Thermoanaerobacter brockii. J. Mol. Biol. 278, 965-979 (1998). [Read online]
N. Trainin, M. Pecht, Y. Burstein and B. Rager-Zisman. Thymic hormones, viral infections and psychoneuroimmunology. In Psyconeuroimmunology, Stress, and Infection, (H. Friedman, T.W. Klein and A.L. Friedman, eds.) CRC Press, Inc. Boca Raton, Chap. 11, pp.215-229 (1996).
L.N. Levi, N. Ben Aroya, S. Tel-Or, A. Palmon, Y. Burstein, and Y. Koch. Expression of the gene for the receptor of gonadotropin-releasing hormone in rat mammary gland. FEBS Letters 379, 186-190 (1996).[read online]
R. Ophir, M. Pecht, Y. Keisari, G. Rashid, S. Lourie, S. Ben-Efraim, N. Trainin and Y. Burstein. Thymic humoral factor-gamma-2 (THF-gamma-2) immunotherapy reduces the metastatic load and restores immunocompetence in 3LL tumor-bearing mice receiving anticancer chemotherapy. Immunopharmac. Immunotoxicol. 18, 209-236 (1996).
Y. Khorkin, F. Frolow, O. Bogin, M. Peretz, A. J. Kalb (Gilboa) and Y. Burstein. Crystalline alcohol dehydrogenase from the mesophilic bacterium Clostridium beijerinckii and the thermophilic bacterium Thermoanaerobium brockii: preparation, characterization and molecular symmetry. Acta Crystallographica D52, 882-886 (1996).[Read online]
B. Rager-Zisman, A. Palmon, S. Blagerman, S. Tel-Or, D. Bennarouch, M. Pecht, N. Trainin and Y. Burstein. Novel therapeutic strategies of cytomegalovirus infection. Natural Immunity 14, 250-261 (1995).
D. Sternberg, J. Honigwachs-Sha'anani, N. Brosh, Z. Malik, Y. Burstein, D. Zipori. Restrictin-P Stromal Activin-A, kills its target-cells via an apoptotic mechanism Growth Factors 12, 277-287 (1995).
N. Brosh, D. Sternberg, J. Honigwachs-Sha'anani, B-C. Lee, Y. Shav-Tal, E. Tzehoval, L.M. Shulman, J. Toledo, Y. Hacham, P. Carmi, W. Jiang, J. Sasse, F. Horn, Y. Burstein and D. Zipori. The plasmacytoma growth inhibitor Restrictin-P is an antagonist of interleukin-6 and interleukin-11: identification as a stroma-derived Activin A. J. Biol. Chem. 270, 29594-29600 (1995).[Read online]