Laboratory for Evolution and Development, Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao, 266003, China.
Department of Marine Biology, Ocean University of China, Qingdao, 266003, China.
Mar Biotechnol (NY). 2019 Aug;21(4):448-462. doi: 10.1007/s10126-019-09891-0. Epub 2019 May 3.
Klotho, a putative aging suppressor, shares sequence similarity with members of the glycosidase family 1. It has been identified in several vertebrate species, but only mouse Klotho has so far been proven to exhibit β-glucuronidase activity. Thus, the argument that Klotho from animals other than mouse has glycosidase activity remains open. Moreover, little information is available regarding the structure-activity relationship of Klotho. Here, we demonstrate the presence of a single klotho gene in the amphioxus Branchiostoma japonicum, Bjklotho, which possesses two tandem domains named BjKL1 and BjKL2, and each of them has two glutamic acid residues that have been shown to be involved in the catalytic activity of family 1 glycosidase. Enzymatic activity assays of the recombinant proteins BjKL1 and BjKL2 revealed that only BjKL2 displayed β-glucosidase activity, but BjKL1 did not. Structural analysis showed that there existed nine consecutive but not conserved residues in the β6α6 loop, which affects the conformational form in the entrance to the catalytic pocket of BjKL1 and BjKL2, thereby leading to a subtle difference in the enzyme-substrate binding and interaction. Furthermore, the substitution of the nine residues 354QNRVDPNDT362 in BjKL1 by the residues 884EDNVVVGAA892 in BjKL2 resulted in significant increase in β-glucosidase activity in the BjKL1 mutant. Our results indicate that BjKL2 possesses β-glucosidase, the first data as such in invertebrates. We also identify, for the first time, the residues 884EDNVVVGAA892 in BjKL2 a sequence critical and indispensable for glucosidase.
Klotho 是一种假定的衰老抑制剂，与糖苷酶家族 1 的成员具有序列相似性。它已在几种脊椎动物物种中被鉴定出来，但迄今为止，只有小鼠 Klotho 被证明具有β-葡萄糖醛酸酶活性。因此，动物（而非小鼠）Klotho 具有糖苷酶活性的说法仍有待证实。此外，关于 Klotho 的结构-活性关系的信息也很少。在这里，我们在文昌鱼（Branchiostoma japonicum）中鉴定出单个 klotho 基因，命名为 Bjklotho，它具有两个串联结构域，分别命名为 BjKL1 和 BjKL2，每个结构域都有两个谷氨酸残基，这些残基被证明参与了家族 1 糖苷酶的催化活性。对重组蛋白 BjKL1 和 BjKL2 的酶活性测定表明，只有 BjKL2 显示出β-葡萄糖苷酶活性，而 BjKL1 则没有。结构分析表明，在β6α6 环中存在 9 个连续但不保守的残基，这影响了 BjKL1 和 BjKL2 催化口袋入口的构象形式，从而导致酶-底物结合和相互作用存在细微差异。此外，用 BjKL2 中的残基 884EDNVVVGAA892 取代 BjKL1 中的 354QNRVDPNDT362 九个残基，导致 BjKL1 突变体的β-葡萄糖苷酶活性显著增加。我们的结果表明，BjKL2 具有β-葡萄糖苷酶活性，这是首次在无脊椎动物中发现。我们还首次确定了 BjKL2 中残基 884EDNVVVGAA892 对糖苷酶活性是关键且不可或缺的。
