Aromatic residues in N-terminal domain of archaeal trehalase affect the folding and activity of catalytic domain.

ABSTRACT

Considering the structure of the bacterial GH15 family glucoamylase (GA), Thermoplasma trehalase Tvn1315 may be composed of a ß-sandwich domain (BD) and a catalytic domain (CD). Tvn1315 BD weakly binds to insoluble ß-glucans, such as cellulose, and helps fold CD. To determine how aromatic residues contribute to proper folding and enzyme activity, we performed alanine scanning for 32 aromatic residues in the BD. The study did not identify a single residue involved in glucan binding. However, several aromatic residues were found to be involved in BD or CD folding and in modulating the activity of the full-length enzyme. Among those aromatic residue mutations, the W43A mutation led to reduced solubility of the BD and full-length protein and resulted in a full-length enzyme with significantly lower activity. The activity of W43F and W43Y was significantly higher than that of W43A. In addition, Ala substitutions of Tyr83, Tyr113, and Tyr17 led to a reduction in trehalase activity, but Phe substitutions of these residues could be tolerated, as these mutants maintained activities similar to WT activity. Thus, these aromatic residues in BD may interact with CD and modulate enzyme activity. KEY POINTS • Aromatic residues in the BD are involved in BD and CD folding. • Aromatic residues in the BD near the CD active site modulate enzyme activity. • BD interacts with CD and closely modulates enzyme activity.