Abstract
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.
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The authors acknowledge the substantive input from all members of the Evolution & Development laboratory.
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This work was supported by the grants of the Natural Science Foundation of China (31601862; U1401211).
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Ma, Z., Qu, B., Zhong, S. et al. Subtle Difference Generates Big Dissimilarity: Comparison of Enzymatic Activity in KL1 and KL2 Domains of Lancelet Klotho. Mar Biotechnol 21, 448–462 (2019). https://doi.org/10.1007/s10126-019-09891-0
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DOI: https://doi.org/10.1007/s10126-019-09891-0