A novel l-asparaginase from Aquabacterium sp. A7-Y with self-cleavage activation

Abstract

We have identified a novel l-asparaginase, abASNase3, from Aquabacterium sp. A7-Y. abASNase3 is composed of 306 amino acids and exhibits 34 % sequence homology to human asparaginase (hASNase3). Further analysis revealed that abASNase3 belongs to the N-terminal nucleophile (Ntn) family of hydrolases. Previous reports about the Ntn hydrolase family and the results of our study suggest that abASNase3 must form two subunits by self-cleavage between Gly189 and Thr190 to attain catalytic activity. The two subunits remained tightly associated to build a single functional unit. The optimum pH for abASNase3 was found to be 8.0 in Tris–HCl buffer and the enzyme was found to be stable over a broad pH range from pH 6.0 to 12.0. The optimum temperature for abASNase3 was found to be approximately 40 °C, and the enzyme was stable below 65 °C. abASNase3 showed high substrate specificity toward l-asparagine and had no or only slight activity toward d-asparagine, l-glutamine and d-glutamine. abASNase3 was significantly activated by Mg²⁺ and was substantially inhibited by Ni²⁺, Cu²⁺, Mn²⁺ and Co²⁺. The Michaelis–Menten constant and turnover number of abASNase3 for l-asparagine were estimated to be 3.37 × 10⁻² M and 8.72 × 10⁻³ s⁻¹, respectively. Our results indicate that abASNase3 is a novel l-asparaginase in the Ntn family of hydrolases.