Crystallization and Preliminary X-ray Diffraction Study of a Mutant of L-Asparaginase from Wolinella succinogenes
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CTURE OF MACROMOLECULAR COMPOUNDS
Crystallization and Preliminary X-ray Diffraction Study of a Mutant of L-Asparaginase from Wolinella succinogenes V. I. Timofeeva,b, N. V. Bulushovac, N. E. Zhukhlistovaa, and I. P. Kuranovaa,b,* a
Shubnikov Institute of Crystallography of Federal Scientific Research Centre “Crystallography and Photonics,” Russian Academy of Sciences, Moscow, 119333 Russia b National Research Centre “Kurchatov Institute,” Moscow, 123098 Russia cState Research Institute of Genetics and Selection of Industrial Microorganisms of the National Research Center “Kurchatov Institute,” Moscow, 117545 Russia *e-mail: [email protected] Received March 4, 2019; revised March 4, 2019; accepted March 19, 2019
Abstract—The double mutant of Wolinella succinogenes L-asparaginase (Was72) with the V23Q and K24T substitutions in the C-terminal region of the N-terminal loop enclosing the active site was crystallized in the apo form and in complexes with L-aspartic and L-glutamic acids. This mutant exhibits glutaminase activity eight times lower compared to the wild-type enzyme. Crystals of the apo enzyme were grown in two modifications (sp. gr. P21 and sp. gr. P22121). Crystals grown in the presence of both aspartic and glutamic acids belong to the same space group (P21) but have different unit cell parameters. The X-ray diffraction data sets were collected from all types of crystals at 1.65–2.00 Å resolution. All X-ray diffraction data sets are suitable for the determination of the three-dimensional structure of the enzyme. DOI: 10.1134/S1063774519060221
INTRODUCTION Enzymes of the asparaginase family are commonly found in microorganisms. They belong to threonine amidohydrolases (EC 3.5.1.1) and catalyze the hydrolysis of asparagine to aspartic acid and ammonia. A remarkable property of L-asparaginases is their pronounced antitumor activity, due to which these enzymes are used in medicine as effective anticancer agents for the treatment of acute lymphoblastic leukemia, lymphosarcomas, and reticulosarcomas [1–3]. The antitumor activity of L-asparaginases is attributed to their ability to inhibit the metabolism of asparagine essential for tumor cell growth [2]. However, the antitumor activity of asparaginases is accompanied by adverse effects because of their toxicity. The latter is partially associated with glutaminase activity of the enzymes. L-Glutamine plays an important role in blood nitrogen transport and, consequently, a longterm decrease in its concentration during therapy with asparaginases causes serious disorders in the body. Hence, only a few asparaginases, for instance, the enzymes from Erwinia chrysanthemi and Wolinella succinogenes [4–6], exhibiting low glutaminase activity, can be used as drugs. The discovery and engineering of new asparaginases as effective therapeutic agents are challenging problems. Different representatives of this class of enzymes have been extensively studied [4–10]. Of particular interest is to reveal the
factors that have an effect on the ratio of asparaginase to glutaminase
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