The quorum-quenching lactonase from Geobacillus caldoxylosilyticus: purification, characterization, crystallization and crystallographic analysis
The quorum-quenching lactonase from Geobacillus caldoxylosilyticus: purification, characterization, crystallization and crystallographic analysis
复制标题
DOI：
10.1107/s2053230x16011821
复制
发表时间：
2016-09-01
影响因子：
0.9
通讯作者：
Elias, Mikael
中科院分区：
文献类型：
--
作者：
Bergonzi, Celine;Schwab, Michael;Elias, Mikael
内酯酶是能够水解各种内酯的酶，例如脂肪族内酯或酰基高丝氨酸内酯（AHL），后者被许多革兰氏阴性细菌用作化学信号分子。因此，内酯酶能够抑制多种细菌的化学通讯，也称为群体感应，特别是抑制由该系统调节的行为，例如毒力因子的表达或生物膜的产生。从嗜热细菌 Geobacillus caldoxylosilyticus 中分离出一种金属-β-内酰胺酶超家族的新代表，称为 GcL。由于其嗜热起源，GcL 可能成为生物防治剂开发的一个有趣的候选者。在这里，我们证明 GcL 是一种热稳定性酶，在 75 摄氏度下的半衰期为 152.5 +/- 10 分钟。值得注意的是，还表明 GcL 是迄今为止最具活性的内酯酶之一，针对 AHL 的催化效率 (k(cat)/K-m) 大于 10(6) M-1 s(-1)。 GcL 的结构有望揭示酶的催化机制以及此类内酯酶底物特异性的分子决定因素。在此，报告了 1.6 埃分辨率的 GcL 的表达、纯化、表征、结晶和 X 射线衍射数据收集。
Lactonases are enzymes that are capable of hydrolyzing various lactones such as aliphatic lactones or acyl-homoserine lactones (AHLs), with the latter being used as chemical signaling molecules by numerous Gram-negative bacteria. Lactonases therefore have the ability to quench the chemical communication, also known as quorum sensing, of numerous bacteria, and in particular to inhibit behaviors that are regulated by this system, such as the expression of virulence factors or the production of biofilms. A novel representative from the metallo-beta-lactamase superfamily, dubbed GcL, was isolated from the thermophilic bacterium Geobacillus caldoxylosilyticus. Because of its thermophilic origin, GcL may constitute an interesting candidate for the development of biocontrol agents. Here, we show that GcL is a thermostable enzyme with a half-life at 75 degrees C of 152.5 +/- 10 min. Remarkably, it is also shown that GcL is among the most active lactonases characterized to date, with catalytic efficiencies (k(cat)/K-m) against AHLs of greater than 10(6) M-1 s(-1). The structure of GcL is expected to shed light on the catalytic mechanism of the enzyme and the molecular determinants for the substrate specificity in this class of lactonases. Here, the expression, purification, characterization, crystallization and X-ray diffraction data collection to 1.6 angstrom resolution of GcL are reported.