Montebello Aubrey N, Brecht Ryan M, Turner Remington D, Ghali Miranda, Pu Xinzhu, Nagarajan Rajesh
Department of Chemistry and Biochemistry, Boise State University , 1910 University Drive, Boise, Idaho 83725, United States.
Biochemistry. 2014 Oct 7;53(39):6231-42. doi: 10.1021/bi5009529. Epub 2014 Sep 24.
The acyl-homoserine lactone (AHL) autoinducer mediated quorum sensing regulates virulence in several pathogenic bacteria. The hallmark of an efficient quorum sensing system relies on the tight specificity in the signal generated by each bacterium. Since AHL signal specificity is derived from the acyl-chain of the acyl-ACP (ACP = acyl carrier protein) substrate, AHL synthase enzymes must recognize and react with the native acyl-ACP with high catalytic efficiency while keeping reaction rates with non-native acyl-ACPs low. The mechanism of acyl-ACP substrate recognition in these enzymes, however, remains elusive. In this study, we investigated differences in catalytic efficiencies for shorter and longer chain acyl-ACP substrates reacting with an octanoyl-homoserine lactone synthase Burkholderia mallei BmaI1. With the exception of two-carbon shorter hexanoyl-ACP, the catalytic efficiencies of butyryl-ACP, decanoyl-ACP, and octanoyl-CoA reacting with BmaI1 decreased by greater than 20-fold compared to the native octanoyl-ACP substrate. Furthermore, we also noticed kinetic cooperativity when BmaI1 reacted with non-native acyl-donor substrates. Our kinetic data suggest that non-native acyl-ACP substrates are unable to form a stable and productive BmaI1·acyl-ACP·SAM ternary complex and are thus effectively discriminated by the enzyme. These results offer insights into the molecular basis of substrate recognition for the BmaI1 enzyme.
酰基高丝氨酸内酯（AHL）自诱导物介导的群体感应调节多种致病细菌的毒力。高效群体感应系统的标志依赖于每种细菌产生信号的严格特异性。由于AHL信号特异性源自酰基-ACP（ACP=酰基载体蛋白）底物的酰基链，AHL合酶必须以高催化效率识别天然酰基-ACP并与之反应，同时保持与非天然酰基-ACP的反应速率较低。然而，这些酶中酰基-ACP底物识别的机制仍然不清楚。在本研究中，我们研究了较短和较长链酰基-ACP底物与辛酰高丝氨酸内酯合酶马鼻疽伯克霍尔德菌BmaI1反应时催化效率的差异。除了短两个碳的己酰-ACP外，与天然辛酰-ACP底物相比，丁酰-ACP、癸酰-ACP和辛酰-CoA与BmaI1反应的催化效率降低了20倍以上。此外，我们还注意到BmaI1与非天然酰基供体底物反应时的动力学协同性。我们的动力学数据表明，非天然酰基-ACP底物无法形成稳定且有活性的BmaI1·酰基-ACP·SAM三元复合物，因此该酶能有效区分它们。这些结果为BmaI1酶底物识别的分子基础提供了见解。
