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

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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 Mg(2+) and was substantially inhibited by Ni(2+), Cu(2+), Mn(2+) and Co(2+). 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.