QUARTERLY Preliminary crystallographic studies of Y25F mutant of periplasmic Escherichia coli L-asparaginase �

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 (P6 5 22). Single crystals of this polymorph, suitable for X-ray dif-fraction, were obtained by vapor diffusion using 2-methyl-2,4-pentanediol as precipi-tant (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 k cat without significantly affecting K m. L-Asparaginases (EC 3.5.1.1) hydrolyze L-asparagine to L-aspartate, with the release of ammonia (Fig. 1). L-Asparaginase activity was first discovered in the blood plasma of guinea pig by Clementi (1922). Later, enzymes with analogous activities were isolated from bacteria and plants. The studies on mice performed by Kidd (1953) showed antitumor activity of the guinea pig serum against certain lympho-mas. Broome (1961) correlated this effect with Clementi's observations and ascribed it to asparaginase activity. Since then the

antitumor activity of asparaginases has been the subject of numerous studies.
In Escherichia coli two forms of this enzyme, termed type I (cytosolic) and type II (periplasmic) have been found.They are characterized by, respectively, high and low K M for asparagine.Only some L-asparaginases, those with high affinity towards the substrate (K M about 10 -5 M), show antitumor activity.Type I and type II isoenzymes have been found in both eukaryotic and prokaryotic organisms but their evolutionary relation is obscure (Bonthron & Jaskólski, 1997).Type II enzymes isolated from E. coli (EcAII) and Erwinia chrysanthemi (ErA) are successful drugs in the treatment of acute lymphoblastic leukemia, leukemic lymphosarcoma and lymphosarcoma (Hill et al., 1967;Chakrabarti, 1997).Their therapeutic effect is related to depleting the circulating pools of asparagine and, in consequence, to decreasing the source of asparagine for the tumor cells, which (in contrast to normal cells) are incapable of intracellular asparagine synthesis and therefore are asparagine-dependent.However, the clinical advantages of asparaginases are limited because of toxic side effects and instances of spontaneous resistance of the tumor cells (Alberts et al., 1999;Ettinger et al., 1997).
The crystal structures of several type II bacterial asparaginases are known.They include the enzyme from E. coli -EcAII with bound aspartate (Swain et al., 1993; Protein Data Bank, PDB, entry 3ECA), and its active-site T89V mutant with covalently bound product (Palm et al., 1996; PDB entry 4ECA) as well as enzymes from E. chrysanthemi -ErA (Miller et al., 1993), Wolinella succinogenes -WsA (Lubkowski et al., 1996; PDB entry 1WSA), Acinetobacter glutaminasificans -AGA (Lubkowski et al., 1994b; PDB entry 1AGX) and Pseudomonas 7A -PGA (Lubkowski et al., 1994a; PDB entry 3PGA; Jakob et al., 1997; PDB entry 4PGA).Type II asparaginases are active as homotetramers with nearly ideal 222 symmetry.The identical subunits (in EcAII 326 amino acids each) composing a homotetramer are denoted A, B, C, D (Fig. 2).The active site is created by subunits A and C (or B and D).Therefore, the asparaginase tetramer is more accurately described as a dimer of dimers.The molecules of the reaction product, L-aspartate, found in the crystal structures of the enzymes, define the location of the active site (Fig. 3) and the key surrounding residues: T12, Y25, S58, T89, D90, and K162 (in EcAII sequence).The role of these residues has been confirmed by mutagenesis and kinetic studies (Röhm & Van Etten, 1986; Bagert & Röhm, 1989;Derst et al., 1992;Derst et al., 1994).One of the proposed mechanisms of action suggests similarity to the reaction catalyzed by serine proteases (Rao et al., 1996).The role of the S-H-D catalytic triad of serine proteases can be played in L-aspara- ginases by a similar triad, T89-K162-D90.This triad is conserved in the sequences (Bonthron & Jaskólski, 1997) and in all known three-dimensional structures of the bacterial enzymes (Dodson & Wlodawer, 1998).Alternatively, T12 has been implicated as the primary nucleophile (Palm et al., 1996;Ortlund et al., 2000).However, for its activation a hydrogen abstracting group is required.The mutated residue, Y25, is involved in the native enzyme in binding of the reaction product in the active site through its side-chain OH group.The presence of a hydrogen bond between the hydroxyl groups of Y25 and T12 suggest that Y25 could be involved in the activation of T12 (Palm et al., 1996).Crystallographic studies of covalent complexes between Pseudomonas 7A glutaminase-asparaginase and diazo analogs of natural substrates also suggest that residue Y35 (equivalent to Y25 in EcAII) can be directly involved in the catalysis  Y25 in EcAII catalysis may be due to the fact, that this residue is part of a mobile loop that closes upon the active site after substrate binding.This structural element is disordered in nearly all asparaginase structures but is well-defined in PGA and especially in the covalent intermediate of mutant T89V (Palm et al., 1996).Recent stopped-flow studies with EcAII indicate that the rate of loop closure decreases by three orders of magnitude when Y25 is replaced with other residues (Aung et al., 2000).
The crystal structure of the Y25F mutant is of interest because it could be helpful in explaining the mechanism of substrate recognition and docking and the role of this residue in catalysis.

MATERIALS AND METHODS
Protein expression and purification.The Y25F mutant of EcAII was constructed and purified as described previously for the native form (Harms et al., 1991).For mutagenesis of the ansB gene subcloned in M13mp19, the oligonucleotide 5¢-AAA TCT AAC TTC ACA GTG GGT-3¢ was employed to replace Y25 with F (mutagenic changes underlined).
Crystallization.A new polymorphic form of E. coli L-asparaginase II has been obtained for the Y25F mutant using new crystallization conditions.The crystallization experiments were conducted at 20°C using the vapor diffusion method and the hanging or sitting drop technique (McPherson, 1982).The protein fraction was desalted and then concentrated using Centricon-10 concentrators.The protein concentration, determined by UV absorption at 280 nm, was 10-15 mg/ml in 10 mM sodium citrate buffer, pH 4.8.Initial crystallization conditions (precipitant and pH) were established by the sparse-matrix method (Jancarik & Kim, 1991) using Crystal Screen II (Hampton Research, California, U.S.A.).Protein samples, 5 ml, were mixed on siliconized cover slips (for hanging drop experiments) or on polypropylene bridges (for sitting drop experiments) with equal amounts of reservoir solutions.The droplets were equilibrated against 1 ml reservoir solution in 24-well cell culture plates.The best crystals were obtained when the reservoir contained 46-48% 2-methyl-2,4-pentanediol (MPD), 100 mM sodium citrate buffer, pH 4.8, and 10-20 mM CaCl 2 .Prismatic hexagonal crystals (Fig. 4) appeared after about 2 days and reached maximum dimensions of 0.6 ´0.3 0.3 mm within one week.The crystals for X-ray diffraction experiments were mounted in thin-walled quartz capillary with a small amount of mother liquor.
Diffraction experiments.X-ray diffraction data were collected at room temperature using synchrotron radiation at l = 0.98 Å  (EMBL, Hamburg Outstation c/o DESY, beamline X11) and a 345-mm MarResearch image plate scanner.The crystal-to-detector distance was 372 mm and the oscillation range 1.5°.74,462 reflections (with I/s(I) > 0.0) were collected to 2.45 Å resolution (Fig. 5).They were merged to give a unique data set of 23,517 reflections characterized by R int = 0.097 and < I/s(I) > = 7.9 (Table 1).Indexing and integration of the images was done in Denzo and scaling of the intensity data in Scalepack of the "HKL" program package (Otwinowski & Minor, 1997).
Structure solution.The crystal structure was solved by molecular replacement using the AMoRe program package (Navaza, 1994).X-ray diffraction data between 15-3.5 Å resolution were used and the AB dimer of native EcAII (Swain et al., 1993) served as molecular probe.The rotation function was calculated using a Patterson radius of 45 Å.The best rotation peak had correlation coefficient of 0.112 and was 1.62 times higher than the next peak.In the next step, a translation search was performed for these peaks using the Crowther & Blow (1967) translation function.In the P6 5 22 space group, it produced for the first rotation peak a solution with very high correlation coefficient (0.716) and low R-factor (0.314).This best molecular replacement solution after rigid-body optimization is characterized by a correlation coefficient 0.769 and R-factor 0.288.In contrast, no translation solution could be found in the enantiomorphic P6 1 22 space group.

Kinetic features of the Y25F mutant
The loss of the phenolic hydroxyl group at position 25 brought about by the replacement of Y with F strongly impairs k cat without significantly affecting K m (Derst et al., 1994).With L-Asn as the substrate, k cat decreases by a factor of 2000 while with AHA the decrease is only 200.Other Y25 mutants (Y25A, Y25G) have comparable kinetic properties indicating that it is indeed the OH group which is required.Recent stopped-flow studies with the double mutant W66Y/Y25W show that Y25 is crucially involved in the closure of the active-site loop (Aung et al., 2000) which brings in position another essential residue, i.e.T12.This function of Y25 alone could account for the observed loss of activity in Y25F.The evidence for a role of Y25 as a general base (Ortlund et al., 2000) is still rather circumstantial, and further experiments are needed to substantiate or refute such an assumption.

Crystallography
The crystal was very stable in the X-ray beam allowing collection of a complete data set from a single specimen.Our attempts to collect diffraction data at low temperature were unsuccessful.Flash freezing (Teng, 1990) increased Vol.47 E. coli L-asparaginase II mutant 811 The edge of the detector corresponds to 2.45 Å resolution.
the mosaicity of the crystals and spot size, which in combination with the huge c parameter (341.

Table 1 . Summary of data collection
1 Å) limited the accessible d-spacings.

Table 2 . Crystallographic data for different polymorphic forms of E. coli L-asparaginase II
As indicated by the successful performance of molecular replacement, the asymmetric unit of the crystal can be conveniently described as containing the AB dimer.