CRYSTAL STRUCTURE OF S58A MUTANT OF E. COLI L-ASPARAGINASE II

M. Kozak¹, M.Jaskólski^1,2

¹Deptartment of Crystallography, A.Mickiewicz University, Poznan, Poland,
²Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland
maciej@krystal.amu.edu.pl

Keywords: amidohydrolases, leukemia, asparaginase, cryobiocrystallography, mutagenesis.

L-Asparaginases catalyse the hydrolysis of L-asparagine to L-aspartate, with release of ammonia. Enzymes isolated from Escherichia coli (EcaII) and Erwinia chrysanthemi (ErA) have been used in the treatment of acute lymphoblastic leukemia, leukemic lymphosarcoma and lymphosarcoma. These enzymes are found in the periplasm and are classified as type II. Bacteria also produce type I asparaginases, found in cytosol, which, having low substrate affinity, are not suitable as antileukemic drugs. The crystal structures of several type II bacterial asparaginases are known. They include the enzyme from Escherichia coli with bound aspartate, and its T89V mutant with covalently bound product as well as enzymes from Erwinia chrysanthemi, Wolinella succinogenes, Acinetobacter glutaminasificans and Pseudomonas 7A. Asparaginases are biologically active as 222-symmetric homotetramers. In the case of EcAII, the ABCD tetramer can be considered as a dimer of intimate dimers (AC and BD) within which the four active sites are formed.

The S58A mutant of EcAII was prepared in the Institute of Physiological Chemistry, Philipps University, Marburg, by Prof. K.H. Röhm. The mutated residue, S58, is involved in binding the reaction product in the active site through its side-chain OH group. The S58A mutant is therefore of interest because of its potential to have different substrate/product affinity or improved differential affinity with respect to glutamine, which is a minor but highly undesired substrate of this class of enzymes.

Single crystals of the mutant were grown by the hanging drop vapor-diffusion method and in agarose gel. The X-ray diffraction data were collected at the Brookhaven synchrotron at 120K (A) and using CuKa radiation at room temperature (B) to a maximal resolution of 2.6 A and 2.33 A, respectively. The crystal form is orthorhombic, space group P2₁2₁2 with unit cell dimensions: a = 226.9, b = 128.3, c = 61.9 A

The structure was solved by molecular replacement using the "active" AC dimer from the EcAII (PDB code: eca3) as the probe. Patterson search followed by rigid body refinement gave an unambiguous solution - one complete tetramer and one AC dimer in the asymmetric unit. The second complete tetramer is generated by the crystallographic dyad along [001]. The model was refined using X-PLOR and partly rebulit using "O". In the structure, we found the reaction product, aspartic acid, in all six independent active sites. The mutation reduced the number of hydrogen bonds between the enzyme and the bound product. This could be responsible for the increased differential affinity towards the substrate. At the current stage of refinement the model is characterized by R=0.191, R-free 0.253 and rmsd (bonds) = 0.014 A.