Structural and functional analyses of an L-asparaginase from Geobacillus thermopakistaniensis

Genome sequence of Geobacillus thermopakistaniensis contains an open reading frame annotated as a type II L-asparaginase (ASNase(Gt)). Critical structural analysis disclosed that ASNase(Gt) might be a type I L-asparaginase. In order to determine whether it is a type I or type II L-asparaginase, we have performed the structural-functional characterization of the recombinant protein as well as analyzed the localization of ASNase(Gt) in G. thermopakistaniensis. ASNase(Gt) exhibited optimal activity at 52 degrees C and pH 9.5. There was a > 3-fold increase in activity in the presence of beta-mercaptoethanol. Apparent V-max and K-m values were 2735 U/mg and 0.35 mM, respectively. ASNase(Gt) displayed high thermostability with >80 % residual activity even after 6 h of incubation at 55 degrees C. Recombinant ASNase(Gt) existed in oligomeric form. Addition of beta-mercaptoethanol lowered the degree of oligomerization and displayed that tetrameric form was the most active, with a specific activity of 4300 U/mg. Under physiological conditions, ASNase(Gt) displayed >50 % of the optimal activity. Localization studies in G. thermopakistaniensis revealed that ASNase(Gt) is a cytosolic protein. Structural and functional characterization, and localization in G. thermopakistaniensis displayed that ASNase(Gt) is not a type II but a type I L-asparaginase.