Directed Evolution of a Thermostable Quorum-quenching Lactonase from the Amidohydrolase Superfamily
Directed Evolution of a Thermostable Quorum-quenching Lactonase from the Amidohydrolase Superfamily
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DOI：
10.1074/jbc.m110.177139
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发表时间：
2010-12-24
影响因子：
4.8
通讯作者：
Yew, Wen Shan
中科院分区：
文献类型：
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作者：
Chow, Jeng Yeong;Xue, Bo;Yew, Wen Shan
以嗜酸地质杆菌HTA426(GI：56420041)的耐热群体猝灭内酯酶为模板，进行了体外定向进化实验。该酶属于氨基水解酶超家族中的磷酸三酯酶样内酯酶(PLL)家族，它能降解参与群体敏感致病菌毒力途径的N-酰基高丝氨酸内酯(AHLS)。在这里，我们测定了该酶催化失活的D266N突变体的N-丁酰基-L-高丝氨酸内酯配体结构，分辨率为1.6埃。使用可调的、基于生物发光的群体猝灭分子电路，通过在第三和第七条β链的C末端的环上的两个点突变，提高了催化效率，并增加了AHL底物的范围。该E101N/R230I突变体对3-氧代-N-十二酰基-L-高丝氨酸内酯的k(CAT)/K-m值提高了72倍。进化的突变体还显示出对N-丁酰基-L-高丝氨酸内酯的内酯酶活性，N-丁酰基-L-高丝氨酸内酯是一种以前不被野生型酶水解的AHL。纯化的野生型和突变酶都含有锌和铁的混合物，在高浓度下分别呈紫色和棕色。这种显色的起源被认为是由于涉及酶活性部位内的β-阳离子和Tyr-99的电荷转移络合物。电荷转移复合体的调制改变了突变酶的内酯酶活性，并反映在酶的着色性变化中。我们将观察到的进化酶催化活性的增强归因于对活性部位结构的有利调节，使其接近化学催化所需的生产几何形状。
A thermostable quorum-quenching lactonase from Geobacillus kaustophilus HTA426 (GI: 56420041) was used as an initial template for in vitro directed evolution experiments. This enzyme belongs to the phosphotriesterase-like lactonase (PLL) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acylhomoserine lactones (AHLs) that are involved in virulence pathways of quorum-sensing pathogenic bacteria. Here we have determined the N-butyryl-L-homoserine lactone-liganded structure of the catalytically inactive D266N mutant of this enzyme to a resolution of 1.6 angstrom. Using a tunable, bioluminescence-based quorum-quenching molecular circuit, the catalytic efficiency was enhanced, and the AHL substrate range increased through two point mutations on the loops at the C-terminal ends of the third and seventh beta-strands. This E101N/R230I mutant had an increased value of k(cat)/K-m of 72-fold toward 3-oxo-N-dodecanoyl-L-homoserine lactone. The evolved mutant also exhibited lactonase activity toward N-butyryl-L-homoserine lactone, an AHL that was previously not hydrolyzed by the wild-type enzyme. Both the purified wild-type and mutant enzymes contain a mixture of zinc and iron and are colored purple and brown, respectively, at high concentrations. The origin of this coloration is suggested to be because of a charge transfer complex involving the beta-cation and Tyr-99 within the enzyme active site. Modulation of the charge transfer complex alters the lactonase activity of the mutant enzymes and is reflected in enzyme coloration changes. We attribute the observed enhancement in catalytic reactivity of the evolved enzyme to favorable modulations of the active site architecture toward productive geometries required for chemical catalysis.