Identification of the critical role of Tyr-194 in the catalytic activity of a novel N-acyl-homoserine lactonase from marine Bacillus cereus strain Y2
Identification of the critical role of Tyr-194 in the catalytic activity of a novel N-acyl-homoserine lactonase from marine Bacillus cereus strain Y2
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DOI：
10.1007/s00284-006-0224-1
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发表时间：
2006-10-01
影响因子：
2.6
通讯作者：
Zhou, S. -N.
中科院分区：
文献类型：
--
作者：
Lu, X.;Yuan, Y.;Zhou, S. -N.
病原生物群体感应（QS）途径的酶破坏是一种很有前途的抗感染治疗策略。ahl -内酯酶可以将QS信号分子n -酰基-高丝氨酸内酯（n -酰基-高丝氨酸内酯）水解为无活性产物，从而阻断QS系统，是一种有效的生物防治工具。蜡样芽孢杆菌一种海洋细菌分离物Y2，鉴定为蜡样芽孢杆菌亚种发现能够灭活ahl。从Y2菌株中克隆出编码ahl降解酶的aiiA基因，并在大肠杆菌中表达。纯化得到28 kda重组Y2-AiiA蛋白，该蛋白具有较强的ahl降解活性。Y2-aiiA与已知ahl内酯酶的序列比较显示，推断的氨基酸序列具有较高的一致性。用定点诱变法测定了ahl内酯酶的潜在催化残基Tyr-194的功能。ahl降解生物测定表明，与野生型（WT）重组Y2-AiiA相比，用Ala取代tyrr -194导致活性急剧下降，尽管突变的Y2-AiiA蛋白的表达水平与WT的Y2-AiiA相当。这些结果表明，保守残基Tyr-194对新型ahl内酯酶的催化功能至关重要。病原生物群体感应（QS）途径的酶破坏是一种很有前途的抗感染治疗策略。ahl -内酯酶可以将QS信号分子n -酰基-高丝氨酸内酯（n -酰基-高丝氨酸内酯）水解为无活性产物，从而阻断QS系统，是一种有效的生物防治工具。蜡样芽孢杆菌一种海洋细菌分离物Y2，鉴定为蜡样芽孢杆菌亚种发现能够灭活ahl。从Y2菌株中克隆出编码ahl降解酶的aiiA基因，并在大肠杆菌中表达。纯化得到28 kda重组Y2-AiiA蛋白，该蛋白具有较强的ahl降解活性。Y2-aiiA与已知ahl内酯酶的序列比较显示，推断的氨基酸序列具有较高的一致性。用定点诱变法测定了ahl内酯酶的潜在催化残基Tyr-194的功能。ahl降解生物测定表明，与野生型（WT）重组Y2-AiiA相比，用Ala取代tyrr -194导致活性急剧下降，尽管突变的Y2-AiiA蛋白的表达水平与WT的Y2-AiiA相当。这些结果表明，保守残基Tyr-194对新型ahl内酯酶的催化功能至关重要。
Enzymatic disruption of quorum-sensing ( QS) pathways in pathogenic organisms is a promising anti-infection therapeutic strategy. AHL-lactonase, a potent tool for biocontrol, can hydrolyze QS signal molecule N-acyl-homoserine lactones ( AHLs) into inactive products, thereby blocking the QS systems. A marine bacterial isolate Y2, identified as a Bacillus cereus subsp., was found capable of inactivating AHLs. The aiiA gene encoding the AHL-degrading enzyme from bacterial strain Y2 was cloned and expressed in Escherichia coli. The 28-kDa recombinant Y2-AiiA protein was purified and showed strong AHL-degrading activity. Sequence comparisons of Y2-aiiA with known AHL-lactonases revealed high identities in the deduced amino-acid sequences. Functional determination of a potential catalytic residue Tyr-194 of AHL-lactonases was performed by site-directed mutagenesis. As judged by AHL-degrading bioassay, substitution of Tyr-194 with Ala resulted in a dramatic decrease of activity compared with wild-type ( WT) recombinant Y2-AiiA, although the expression level of the mutated Y2-AiiA protein was equivalent to that of WT Y2-AiiA. These results suggested that the conserved residue Tyr-194 is critical for catalytic function of the novel AHL-lactonase. Enzymatic disruption of quorum-sensing (QS) pathways in pathogenic organisms is a promising anti-infection therapeutic strategy. AHL-lactonase, a potent tool for biocontrol, can hydrolyze QS signal molecule N-acyl-homoserine lactones (AHLs) into inactive products, thereby blocking the QS systems. A marine bacterial isolate Y2, identified as a Bacillus cereus subsp., was found capable of inactivating AHLs. The aiiA gene encoding the AHL-degrading enzyme from bacterial strain Y2 was cloned and expressed in Escherichia coli. The 28-kDa recombinant Y2-AiiA protein was purified and showed strong AHL-degrading activity. Sequence comparisons of Y2-aiiA with known AHL-lactonases revealed high identities in the deduced amino-acid sequences. Functional determination of a potential catalytic residue Tyr-194 of AHL-lactonases was performed by site-directed mutagenesis. As judged by AHL-degrading bioassay, substitution of Tyr-194 with Ala resulted in a dramatic decrease of activity compared with wild-type (WT) recombinant Y2-AiiA, although the expression level of the mutated Y2-AiiA protein was equivalent to that of WT Y2-AiiA. These results suggested that the conserved residue Tyr-194 is critical for catalytic function of the novel AHL-lactonase.