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

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.

Evaluation of the roles of hydrophobic residues in the N-terminal region of archaeal trehalase in its folding.

Thermoplasma trehalase Tvn1315 is predicted to be composed of a ß-sandwich domain (BD) and a catalytic domain (CD) based on the structure of the bacterial GH15 family glucoamylase (GA). Tvn1315 as well as Tvn1315 (Δ5), in which the 5 N-terminal amino acids are deleted, could be expressed in Escherichia coli as active enzymes, but deletion of 10 residues (Δ10) led to inclusion body formation. To further investigate the role of the N-terminal region of BD, we constructed five mutants of Δ5, in which each of the 5th to 10th residues of the N-terminus of Tvn1315 was mutated to Ala. Every mutant protein could be recovered in soluble form, but only a small fraction of the Y9A mutant was recovered in the soluble fraction. The Y9A mutant recovered in soluble form had similar specific activity to the other proteins. Subsequent mutation analysis at the 9th position of Tvn1315 in Δ5 revealed that aromatic as well as bulky hydrophobic residues could function properly, but residues with hydroxy groups impaired the solubility. Similar results were obtained with mutants based on untruncated Tvn1315. When the predicted BD, Δ5BD, Δ10BD, and BD mutants were expressed, the Δ10BD protein formed inclusion bodies, and the BD mutants behaved similarly to the Δ5 and full-length enzyme mutants. These results suggest that the hydrophobic region is involved in the solubilization of BD during the folding process. Taken together, these results indicate that the solubility of CD depends on BD folding. KEY POINTS: • N-terminal hydrophobic region of the BD is involved in the protein folding. • The N-terminal hydrophobic region of the BD is also involved in the BD folding. • BD is able to weakly interact with the insoluble ß-glucan.

Functional dissection of the N-terminal sequence of Clostridium sp. G0005 glucoamylase: identification of components critical for folding the catalytic domain and for constructing the active site structure.

Clostridium sp. G0005 glucoamylase (CGA) is composed of a ß-sandwich domain (BD), a linker, and a catalytic domain (CD). In the present study, CGA was expressed in Escherichia coli as inclusion bodies when the N-terminal region (39 amino acid residues) of the BD was truncated. To further elucidate the role of the N-terminal region of the BD, we constructed N-terminally truncated proteins (Δ19, Δ24, Δ29, and Δ34) and assessed their solubility and activity. Although all evaluated proteins were soluble, their hydrolytic activities toward maltotriose as a substrate varied: Δ19 and Δ24 were almost as active as CGA, but the activity of Δ29 was substantially lower, and Δ34 exhibited little hydrolytic activity. Subsequent truncation analysis of the N-terminal region sequence between residues 25 and 28 revealed that truncation of less than 26 residues did not affect CGA activity, whereas truncation of 26 or more residues resulted in a substantial loss of activity. Based on further site-directed mutagenesis and N-terminal sequence analysis, we concluded that the 26XaaXaaTrp28 sequence of CGA is important in exhibiting CGA activity. These results suggest that the N-terminal region of the BD in bacterial GAs may function not only in folding the protein into the correct structure but also in constructing a competent active site for catalyzing the hydrolytic reaction.