Structural elements of thermostability in the maltogenic amylase of Geobacillus thermoleovorans

Maltogenic amylase of Geobacillus thermoleovorans (Gt-MamyIII), which has the highest thermostability among bacterial maltogenic amylases, has been used as a model enzyme to understand the role of networked salt bridges in thermoadaptation. The role of intra-chain cross-domain salt bridge networks in the thermostabilization of maltogenic amylase of G. thermoleovorans was confirmed by site-directed mutagenesis and CD analysis. The amino acid pairs in seven salt bridges have been mutated singly and pair-wise, and their free energy of thermal inactivation has been calculated. Among seven, single and double mutations in the amino acids corresponding to four salt bridges (lys306.glu336, arg403.asp65, arg497.glu523 and lys524.glu523) decrease T1/2 and Tm of Gt-MamyIII significantly. Moreover, glu523 forms networked salt bridges with arg497 and lys524. OE1 of glu523 forms electrostatic interactions with NH1 of arg497, NH2 of arg497 and NZ of lys524 at a distance of 2.33, 2.02 and 0.33Å, respectively. The mutations in three buried amino acids led to a decline in T1/2 and Tm. The buried as well as networked cross-domain salt bridges thus appear to play a significant role in the thermostabilization of Gt-MamyIII. The salt bridges lys306.glu336 and arg403.asp65, which are isolated and partially accessible, play a minor role in its thermostabilization.