1616 Polikarpov et al.  L-Asparaginase Acta Cryst. (1999). D55, 1616±1617 crystallization papers Acta Crystallographica Section D Biological Crystallography ISSN 0907-4449 Preparation and preliminary X-ray diffraction studies of a new crystal form of L-asparaginase from Escherichia coli I. Polikarpov, a * R. T. de Oliveira a and J. Abraha Äo-Neto b a Laborato Ârio Nacional de Luz Sõ Âncrotron, Caixa Postal 6192, CEP 13083-970, Campinas SP, Brazil, and b Department of Biochemical and Pharmaceutical Technology, Pharmaceutical Sciences School, University of Sa Äo Paulo, PO Box 66083, CEP 05315-970, Sa Äo Paulo, SP, Brazil Correspondence e-mail: igor@lnls.br # 1999 International Union of Crystallography Printed in Denmark ± all rights reserved l-Asparaginase is an enzyme which hydrolyzes asparagine to produce aspartic acid and ammonia. It is an effective chemotherapeutic drug, especially in the treatment of acute lymphoblastic leukaemia in children. The enzyme from Escherichia coli was crystallized in a new crystal form with space group C2, unit-cell parameters a = 76.3 (0), b = 134.6 (2), c = 64.8 (7) A Ê ,  = 110.5 (1)  and a dimer in the asymmetric unit. Synchrotron-radiation diffraction data have been collected to 1.95 A Ê resolution. Received 13 May 1999 Accepted 22 July 1999 1. Introduction l-Asparaginase, the enzyme which hydrolyzes asparagine to produce aspartic acid and ammonia, is isolated from various bacterial sources and is an effective and widely used chemotherapeutic drug, especially in the treatment of acute lymphoblastic leukaemia in children (Chakrabarti & Schuster, 1997; Hill et al., 1967). The enzyme works by depleting the tumour of its source of asparagine from the circulation, as tumours, especially of lymphoid origin, are incapable of intracellular asparagine synthesis and are l-asparaginase dependent. Despite its effectiveness, however, the use of l-asparaginase as a chemotherapeutic agent is somewhat limited because of its toxicity, side effects and the spontaneous acquisition of resistance to this enzyme by tumour cells (Alberts et al. , 1999; Ettinger et al., 1997; Sadoff et al., 1997; Tozuka et al., 1997). Rational modi®cations of l-asparaginase as a drug, directed toward improvements in stability and lowering of side effects, depend to a large extent on the availability of a well re®ned three-dimensional structure of the enzyme and a deeper understanding of its conformational ¯exibility. Crystallographic structures of bacterial l-asparaginases from Erwinia chrysanthemi (Miller et al., 1993), Pseudomonas 7a (Lubkowski et al., 1994; Jakob et al., 1997), Wolinella succinogenes (Lubkowski et al., 1996) and Escherichia coli (Swain et al., 1993; Palm et al., 1996) have been described. The active E. coli l-asparaginase is a homotetramer of 142 kDa, with 326 amino- acid residues per monomer. The native E. coli enzyme has previously been crystallized in the monoclinic space group P2 1 and the structure has been re®ned to 2.3 A Ê resolution (Swain et al., 1993), whereas the T98V mutant of the enzyme produced crystals in the orthorhombic space group P2 1 2 1 2 1 and diffracted to 2.2 A Ê resolution (Palm et al., 1996). Both crystal forms contain one tetramer of l-asparaginase per asymmetric unit cell. Here, we report a new crystal form of the native E. coli enzyme in the monoclinic space group C2 with one dimer per asymmetric unit; the crystals diffract to 1.95 A Ê resolution at the synchrotron source. 2. Methods, results and discussion l-asparaginase was purchased in a lyophilized form from Merck, Sharp and Dohme and was used in crystallization trials without further puri®cation. Preliminary screening of the crystallization conditions was performed using a sparse-matrix screen at 291 K (Crystal Screens I and II, Hampton Research Corp.) and re®nement of the crystallization conditions was then carried out. The crystals were grown at room temperature using the hanging-drop vapour-diffusion technique by mixing equal volumes (2 ml+2 ml) of a protein solution concentrated to 20 mg ml 1 and a reservoir solution containing 30% 2-methyl-2,4- pentanediol, 4% PEG 3350, 0.1 M 2-(N-morpholino)ethanesulfonic acid buffer pH 6. Crystals of dimensions 0.5  0.3  0.2 mm appeared after one week and were brought to the dedicated protein crystal- lography beamline (Polikarpov, Oliva et al., 1997; Polikarpov, Perles et al. , 1997) at the Laborato  rio Nacional de Luz Sõ Âncrotron (Campinas, SP, Brazil) for data collection. X-ray diffraction data were collected at room temperature using a MAR345 image plate. The synchrotron-radiation wavelength was set to 1.38 A Ê to optimize the X-ray ¯ux and minimize absorption errors. The ®rst image was subjected to the autoindexing routine of DENZO (Otwinowski, 1993), from which the best re®ned solution was a C-centred ortho- rhombic cell. Following an optimum strategy of