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2000 | 47 | 3 | 807-814
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Preliminary crystallographic studies of Y25F mutant of periplasmic Escherichia coli L-asparaginase.

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Periplasmic Escherichia coli L-asparaginase II with Y25F mutation in the active-site cavity has been obtained by recombinant techniques. The protein was crystallized in a new hexagonal form (P6522). Single crystals of this polymorph, suitable for X-ray diffraction, were obtained by vapor diffusion using 2-methyl-2,4-pentanediol as precipitant (pH 4.8). The crystals are characterized by a = 81.0, c = 341.1 Å and diffract to 2.45 Å resolution. The asymmetric unit contains two protein molecules arranged into an AB dimer. The physiologically relevant ABA'B' homotetramer is generated by the action of the crystallographic 2-fold axis along [1, -1, 0]. Kinetic studies show that the loss of the phenolic hydroxyl group at position 25 brought about by the replacement of Y with F strongly impairs kcat without significantly affecting Km.
Physical description
  • Department of Macromolecular Physics, Faculty of Physics, A. Mickiewicz University, Poznań, Poland
  • Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznań, Poland
  • Institut für Physiologische Chemie, Philipps Universität, Marburg, Germany
  • Alberts, S.R., Bretscher, M., Wiltsie, J.C., O'Neill, B.P., Mokri, B. & Witzig, T.E. (1999) Thrombosis related to the use of L-asparaginase in adults with acute lymphoblastic leukemia: A need to consider coagulation monitoring and clotting factor replacement. Leuk. Lymphoma 32, 489-496.
  • Aung, H.P., Bocola, M., Schleper, S. & Röhm, K.H. (2000) Dynamics of a mobile loop at the active site of E. coli asparaginase II. Eur. J. Biochem. (submitted).
  • Bagert, U. & Röhm, K.H. (1989) On the role of histidine and tyrosine residues in E. coli asparaginase. Chemical modification and 1H-nuclear magnetic resonance studies. Biochem. Biophys. Acta 999, 36-41.
  • Bonthron, D.T. & Jaskólski, M. (1997) Why a benign mutation kills enzyme activity. Structure-based analysis of the A176V mutant of Sacchromyces cerevisiae L-asparaginase. Acta Biochim. Polon. 44, 491-504.
  • Broome, J.D. (1961) Evidence that the L-asparaginase activity of guinea pig serum is responsible for its antilymphoma effects. Nature 191, 1114-1115.
  • Chakrabarti, R. (1997) L-Asparaginase perspectives on the mechanisms of action and resistance. Int. J. Pediatr. Hematol. Oncol. 4, 597-611.
  • Chibnall, A.C. (1930). Protein Metabolism in the Plant. Yale University Press, New Haven.
  • Clementi, A. (1922) La desamidation enzymatique de l'asparagine chez les différentes espéces animales et la signification physiologique de sa présence dans l'organisme. Arch. Intern. Physiol. 19, 369.
  • Crowther, R.A. & Blow, D.M. (1967) A method for positioning a known molecule in an unknown crystal structure. Acta Cryst. 23, 544-549.
  • Derst, C., Henseling, J. & Röhm, K.H. (1992) Probing the role of threonine and serine residues of E. coli asparaginase II by site-specific mutagenesis. Protein Eng. 5, 785-789.
  • Derst, C., Wehner, A., Specht, V. & Röhm, K.H. (1994) States and functions of tyrosine residues in Escherichia coli asparaginase II. Eur. J. Biochem. 224, 533-540.
  • Dodson, G. & Wlodawer, A. (1998) Catalytic triads and their relatives. Trends Biochem. Sci. 23, 347-352.
  • Epp, O., Steigemann, W., Formanek, H. & Huber, R. (1971) Crystallographic evidence for the tetrameric subunit structure of L-asparaginase from Escherichia coli. Eur. J. Biochem. 20, 432-437.
  • Ettinger, L.J., Ettinger, A.G., Avramis, V.I. & Gaynon, P.S. (1997) Acute lymphoblastic leukaemia: A guide to asparaginase and pegaspargase. Biodrugs 7, 30-39.
  • Harms, E., Wehner, A., Jennings, M.P., Pugh, K.J., Beacham, I.R. & Röhm, K.H. (1991) Construction of expression systems for Escherichia coli asparaginase II and two-step purification of the recombinant enzyme. Protein Exp. Purif. 2, 144-150.
  • Hill, J.M., Roberts, J., Loeb, E., Khan, A., MacLellan, A. & Hill, R.W. (1967) L-Asparaginase therapy for leukemia and other malignant neoplasms. Remission in human leukemia. J. Am. Med. Assoc. 202, 116-122.
  • Itai, A., Yonei, M., Mitsui, Y. & Itaka, Y.J. (1976) Crystallographic study on the orthorhombic crystal of L-asparaginase from Escherichia coli HAP. J. Mol. Biol. 105, 321-325.
  • Jakob, C.G., Lewinski, K., Lacount, M.W., Roberts, J. & Lebioda, M. (1997) Ion binding induces closed conformation in Pseudomonas 7A glutaminase-asparaginase (pga): Crystal structure of the PGA-SO42--NH4+ complex at 1.7 Å resolution. Biochemistry 36, 923-931.
  • Jancarik, J. & Kim, S.-H. (1991) Sparse matrix sampling: A screening method for crystallization of proteins. J. Appl. Cryst. 24, 409-411.
  • Kidd, J.G. (1953) Regression of transplanted lymphomas induced in vivo by means of normal guinea pig serum. J. Exp. Med. 98, 565-605.
  • Kozak, M. & Jaskólski, M. (2000) Crystallization and preliminary crystallographic studies of a new crystal form of Escherichia coli L-asparaginase II (Ser58Ala mutant). Acta Cryst. D56, 509-511.
  • Lubkowski, J., Palm, G.J., Gilliland, G.L., Derst, C., Röhm, K.H. & Wlodawer, A. (1996) Crystal structure and amino acid sequence of Wolinella succinogenes L-asparaginase. Eur. J. Biochem. 241, 201-207.
  • Lubkowski, J., Wlodawer, A., Ammon, H.L., Copeland, T.D. & Swain, A.L. (1994a) Structural characterization of Pseudomonas 7A glutaminase-asparaginase. Biochemistry 33, 10257- 10265.
  • Lubkowski, J., Wlodawer, A., Housset, D., Weber, I.T., Ammon, H.L., Murphy, K.C. & Swain, A.L. (1994b) Refined crystal structure of Acinebacter glutaminaficans glutaminase-asparaginase. Acta Cryst. D50, 826-832.
  • Matthews, B.W. (1968) Solvent contents of protein crystals. J. Mol. Biol. 33, 491-497.
  • McPherson, A. (1982) The Preparation and Analysis of Protein Crystals. John Wiley, New York.
  • Miller, M., Rao, J.K.M., Wlodawer, A. & Gribskov, M. (1993) A left-handed crossover involved in amidohydrolase catalysis. Crystal structure of Erwinia chrysanthemi L-asparaginase with bound L-aspartate. FEBS Lett. 328, 275-279.
  • Navaza, J. (1994) AMoRe: An automated package for molecular replacement. Acta Cryst. D50, 157-163.
  • Ortlund, E., Lacount, M.W., Lewinski, K. & Lebioda, L. (2000) Reactions of Pseudomonas 7A glutaminase-asparaginase with diazo analogues of glutamine and asparagine result in unexpected covalent inhibitions and suggests an unusual catalytic triad Thr-Tyr-Glu. Biochemistry 39, 1199-1204.
  • Otwinowski, Z. & Minor, W. (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307-327.
  • Palm, G., Lubkowski, J., Derst, C., Schleper, S., Röhm, K.H. & Wlodawer, A. (1996) A covalently bound catalytic intermediate in Escherichia coli asparaginase: Crystal structure of a Thr-89-Val mutant. FEBS Lett. 390, 211-216.
  • Polikarpov, I., de Oliveira, R.T. & Abrahăo-Neto, J. (1999) Preparation and preliminary X-ray diffraction studies of a new crystal form of L-asparaginase from Escherichia coli. Acta Cryst. D55, 1616-1617.
  • Rao, J.K.M., Gribskov, M., Lubkowski, J., Miller, M., Swain, A.L. & Wlodawer, A. (1996) A comparison of the crystal structures of bacterial L-asparaginases. Techniques in Protein Chemistry VII (Marshak, R.M., ed.) Academic Press, San Diego.
  • Röhm, K.H. & Van Etten, R.L. (1986) The 18O isotope effect in 12C nuclear magnetic resonance spectroscopy: Mechanistic studies on asparaginase from Escherichia coli. Arch. Biochem. Biophys. 244, 128-136.
  • Swain, A.L., Jaskólski, M., Housset, D., Rao, J.K.M. & Wlodawer, A. (1993) Crystal structure of Escherichia coli L-asparaginase, an enzyme used in cancer therapy. Proc. Natl. Acad. Sci. U.S.A. 90, 1474-1478.
  • Teng, T.Y. (1990) Mounting of crystals for macromolecular crystalography in free-standing thin film. J. Appl. Cryst. 23, 387-391.
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