Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China.
FEBS J. 2011 Sep;278(17):3032-40. doi: 10.1111/j.1742-4658.2011.08223.x. Epub 2011 Jul 26.
β-Propeller phytases (BPPs) with tandemly repeated domains are abundant in nature. Previous studies have shown that the intact domain is responsible for phytate hydrolysis, but the function of the other domain is relatively unknown. In this study, a new dual-domain BPP (PhyH) from Bacillus sp. HJB17 was identified to contain an incomplete N-terminal BPP domain (PhyH-DI, residues 41-318) and a typical BPP domain (PhyH-DII, residues 319-644) at the C-terminus. Purified recombinant PhyH and PhyH-DII required Ca(2+) for phytase activity, showed activity at low temperatures (0-35 °C) and pH 6.0-8.0, and remained active (at 37 °C) after incubation at 60 °C and pH 6.0-12.0. Compared with PhyH-DII, PhyH is catalytically more active against phytate (catalytic constant 27.72 versus 4.17 s(-1)), which indicates the importance of PhyH-DI in phytate degradation. PhyH-DI was found to hydrolyze phytate intermediate D-Ins(1,4,5,6) P(4), and to act synergistically (a 1.2-2.5-fold increase in phosphate release) with PhyH-DII, other BPPs (PhyP and 168PhyA) and a histidine acid phosphatase. Furthermore, fusion of PhyH-DI with PhyP or 168PhyA significantly enhanced their catalytic efficiencies. This is the first report to elucidate the substrate specificity of the incomplete domain and the functional relationship of tandemly repeated domains in BPPs. We conjecture that dual-domain BPPs have succeeded evolutionarily because they can increase the amount of available phosphate by interacting together. Additionally, fusing PhyH-DI to a single-domain phytase appears to be an efficient way to improve the activity of the latter.
β-螺旋桨植酸酶（BPPs）具有串联重复的结构域，在自然界中非常丰富。以前的研究表明，完整的结构域负责植酸盐的水解，但其他结构域的功能相对未知。在这项研究中，从芽孢杆菌 HJB17 中鉴定出一种新的双结构域 BPP（PhyH），它含有一个不完整的 N 端 BPP 结构域（PhyH-DI，残基 41-318）和一个典型的 BPP 结构域（PhyH-DII，残基 319-644）在 C 端。纯化的重组 PhyH 和 PhyH-DII 需要 Ca(2+)才能发挥植酸酶活性，在低温（0-35°C）和 pH 6.0-8.0 下具有活性，在 60°C 和 pH 6.0-12.0 下孵育后仍保持活性（在 37°C）。与 PhyH-DII 相比，PhyH 对植酸盐的催化活性更高（催化常数为 27.72 与 4.17 s(-1)），这表明 PhyH-DI 在植酸盐降解中的重要性。发现 PhyH-DI 可以水解植酸盐中间产物 D-Ins(1,4,5,6)P(4)，并与 PhyH-DII、其他 BPPs（PhyP 和 168PhyA）和组氨酸酸性磷酸酶协同作用（释放的磷酸盐增加 1.2-2.5 倍）。此外，将 PhyH-DI 与 PhyP 或 168PhyA 融合可显著提高其催化效率。这是首次阐明 BPP 中不完整结构域的底物特异性和串联重复结构域的功能关系的报道。我们推测，双结构域 BPP 之所以能够成功进化，是因为它们可以通过相互作用来增加可用磷酸盐的数量。此外，将 PhyH-DI 融合到单结构域植酸酶上似乎是提高后者活性的有效方法。
