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

Abstract

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 °C and pH 9.5. There was a > 3-fold increase in activity in the presence of β-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 °C. Recombinant ASNase_Gt existed in oligomeric form. Addition of β-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.

Keywords: G. thermopakistaniensis; L-Asparaginase; Oligomerization; Structural analysis; Thermostability.