Wong Wai-Ting, Au Ho-Wah, Yap Hong-Kin, Leung Yun-Chung, Wong Kwok-Yin, Zhao Yanxiang
Department of Applied Biology and Chemical Technology, Central Laboratory of Institute of Molecular Technology for Drug Discovery and Synthesis, The Hong Kong Polytechnic University, Hung Hom, Hong Hong, China.
BMC Struct Biol. 2011 Mar 28;11:15. doi: 10.1186/1472-6807-11-15.
β-lactamase conjugated with environment-sensitive fluorescein molecule to residue 166 on the Ω-loop near its catalytic site is a highly effective biosensor for β-lactam antibiotics. Yet the molecular mechanism of such fluorescence-based biosensing is not well understood.
Here we report the crystal structure of a Class A β-lactamase PenP from Bacillus licheniformis 749/C with fluorescein conjugated at residue 166 after E166C mutation, both in apo form (PenP-E166Cf) and in covalent complex form with cefotaxime (PenP-E166Cf-cefotaxime), to illustrate its biosensing mechanism. In the apo structure the fluorescein molecule partially occupies the antibiotic binding site and is highly dynamic. In the PenP-E166Cf-cefatoxime complex structure the binding and subsequent acylation of cefotaxime to PenP displaces fluorescein from its original location to avoid steric clash. Such displacement causes the well-folded Ω-loop to become fully flexible and the conjugated fluorescein molecule to relocate to a more solvent exposed environment, hence enhancing its fluorescence emission. Furthermore, the fully flexible Ω-loop enables the narrow-spectrum PenP enzyme to bind cefotaxime in a mode that resembles the extended-spectrum β-lactamase.
Our structural studies indicate the biosensing mechanism of a fluorescein-labelled β-lactamase. Such findings confirm our previous proposal based on molecular modelling and provide useful information for the rational design of β-lactamase-based biosensor to detect the wide spectrum of β-lactam antibiotics. The observation of increased Ω-loop flexibility upon conjugation of fluorophore may have the potential to serve as a screening tool for novel β-lactamase inhibitors that target the Ω-loop and not the active site.
与环境敏感型荧光素分子结合在其催化位点附近Ω环上第166位残基的β-内酰胺酶，是一种用于检测β-内酰胺类抗生素的高效生物传感器。然而，这种基于荧光的生物传感的分子机制尚不清楚。
在此，我们报道了地衣芽孢杆菌749/C中A类β-内酰胺酶PenP在E166C突变后第166位残基处连接荧光素的晶体结构，包括无配体形式（PenP-E166Cf）和与头孢噻肟的共价复合物形式（PenP-E166Cf-头孢噻肟），以阐明其生物传感机制。在无配体结构中，荧光素分子部分占据抗生素结合位点且高度动态。在PenP-E166Cf-头孢噻肟复合物结构中，头孢噻肟与PenP的结合及随后的酰化作用将荧光素从其原始位置取代，以避免空间冲突。这种取代导致折叠良好的Ω环变得完全灵活，且连接的荧光素分子重新定位到更易暴露于溶剂的环境中，从而增强其荧光发射。此外，完全灵活的Ω环使窄谱PenP酶能够以类似于超广谱β-内酰胺酶的模式结合头孢噻肟。
我们的结构研究揭示了荧光素标记的β-内酰胺酶的生物传感机制。这些发现证实了我们先前基于分子建模提出的观点，并为合理设计基于β-内酰胺酶的生物传感器以检测广谱β-内酰胺类抗生素提供了有用信息。观察到荧光团结合后Ω环灵活性增加，这可能有潜力作为一种筛选工具，用于筛选靶向Ω环而非活性位点的新型β-内酰胺酶抑制剂。
