Department of Chemistry and Life Science, Kogakuin University, 2,665-1 Nakano-Cho, Hachioji, Tokyo, 192-0015, Japan.
Appl Microbiol Biotechnol. 2024 Aug 15;108(1):441. doi: 10.1007/s00253-024-13205-3.
Considering the structure of the bacterial GH15 family glucoamylase (GA), Thermoplasma trehalase Tvn1315 may be composed of a β-sandwich domain (BD) and a catalytic domain (CD). Tvn1315 BD weakly binds to insoluble β-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.
考虑到细菌 GH15 家族葡糖淀粉酶（GA）的结构，嗜热栖热菌海藻糖酶 Tvn1315 可能由 β-三明治结构域（BD）和催化结构域（CD）组成。Tvn1315 BD 可弱结合不溶性β-葡聚糖，如纤维素，并有助于折叠 CD。为了确定芳香族残基如何有助于正确折叠和酶活性，我们对 BD 中的 32 个芳香族残基进行了丙氨酸扫描。该研究未鉴定出一个与葡聚糖结合有关的单个残基。然而，发现几个芳香族残基参与 BD 或 CD 折叠，并调节全长酶的活性。在这些芳香族残基突变中，W43A 突变导致 BD 和全长蛋白的溶解度降低，并且全长酶的活性显著降低。W43F 和 W43Y 的活性明显高于 W43A。此外，Ala 取代 Tyr83、Tyr113 和 Tyr17 导致海藻糖酶活性降低，但这些残基的 Phe 取代可以耐受，因为这些突变体保持与 WT 活性相似的活性。因此，BD 中的这些芳香族残基可能与 CD 相互作用并调节酶活性。关键点：• BD 中的芳香族残基参与 BD 和 CD 折叠。• CD 活性位点附近的 BD 中的芳香族残基调节酶活性。• BD 与 CD 相互作用并紧密调节酶活性。
