Expression and functional analysis of two NhaD type antiporters from the halotolerant and alkaliphilic Halomonas sp Y2
Expression and functional analysis of two NhaD type antiporters from the halotolerant and alkaliphilic Halomonas sp Y2
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耐盐嗜碱盐单胞菌 Y2 的两个 NhaD 型反向转运蛋白的表达和功能分析
DOI：
10.1007/s00792-016-0852-8
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发表时间：
2016-09-01
期刊：
影响因子：
2.9
通讯作者：
Yang, Chunyu
中科院分区：
文献类型：
--
作者：
Cui, Yanbing;Cheng, Bin;Yang, Chunyu
Na+/H+ 反向转运蛋白在离子和 pH 稳态中发挥重要作用。在本研究中，从 Halomonas sp. 中鉴定出两个可有效催化 Na+/H+ 逆向转运蛋白的 NhaD 同源物。 Y2，一种从富钠黑液中分离出来的耐盐、嗜碱菌株。它们表现出 72% 的高序列同一性以及对 Na+ 和 Li+ 易位相似的结合亲和力，同时具有不同的 pH 谱。 Ha-NhaD1 在 pH 6.0 时有活性，在 pH 8.0-8.5 时活性最强，而 Ha-NhaD2 在 pH 6.0 时缺乏活性，但在 pH 9.5 或更高时表现出最大活性。基于多重比对，选择了11个部分保守的残基，并为Ha-NhaD1生成了相应的突变体。正如预期的那样，大部分疏水残基的替换消除了阳离子交换活性。 Ha-NhaD1 中第 200、282 和 353 位的三个丝氨酸残基可被丙氨酸取代，并保留部分活性。与野生型Ha-NhaD1相比，S353A突变体表现出对Na+和Li+的结合亲和力显着降低，而S282突变体表现出约1.5pH单位的碱移。预计 282 位丝氨酸位于跨膜片段 VIII 中，并发现其与侧翼残基一起在调节 pH 敏感性方面发挥着重要作用。
Na+/H+ antiporters play important roles in ion and pH homeostasis. In this study, two NhaD homologues that effectively catalyze Na+/H+ antiporter were identified from Halomonas sp. Y2, a halotolerant and alkaliphilic strain isolated from sodium enriched black liquor. They exhibited high sequence identity of 72 % and similar binding affinities for Na+ and Li+ translocation, while having different pH profiles. Ha-NhaD1 was active at pH 6.0 and most active at pH 8.0-8.5, whereas Ha-NhaD2 lacked activity at pH 6.0 but exhibited maximum activity at pH 9.5 or higher. Based on multiple alignments, 11 partially conserved residues were selected and corresponding mutants were generated for Ha-NhaD1. As expected, replacement of most of the hydrophobic residues abolished the cation exchange activities. Three serine residues at positions 200, 282 and 353 in Ha-NhaD1 were replaceable by alanines with partial retention of activity. The S353A mutant exhibited significantly reduced binding affinity for Na+ and Li+, while S282 mutant exhibited an alkaline shift of about 1.5 pH units, as compared to the wild type Ha-NhaD1. Serine at position 282 was predicted to be located in transmembrane segment VIII and was found to be important in regulating pH sensitivity in concert with flanking residues.