Department of Molecular Medicine, University of South Florida College of Medicine, Tampa, FL, USA.
GMCA@APS, X-ray Science Division, Advanced Photon Source, Argonne National Laboratory, IL, USA.
FEBS J. 2018 Jan;285(1):87-100. doi: 10.1111/febs.14315. Epub 2017 Nov 20.
The β-lactam antibiotics inhibit penicillin-binding proteins (PBPs) by forming a stable, covalent, acyl-enzyme complex. During the evolution from PBPs to Class A β-lactamases, the β-lactamases acquired Glu166 to activate a catalytic water and cleave the acyl-enzyme bond. Here we present three product complex crystal structures of CTX-M-14 Class A β-lactamase with a ruthenocene-conjugated penicillin-a 0.85 Å resolution structure of E166A mutant complexed with the penilloate product, a 1.30 Å resolution complex structure of the same mutant with the penicilloate product, and a 1.18 Å resolution complex structure of S70G mutant with a penicilloate product epimer-shedding light on the catalytic mechanisms and product inhibition of PBPs and Class A β-lactamases. The E166A-penilloate complex captured the hydrogen bonding network following the protonation of the leaving group and, for the first time, unambiguously show that the ring nitrogen donates a proton to Ser130, which in turn donates a proton to Lys73. These observations indicate that in the absence of Glu166, the equivalent lysine would be neutral in PBPs and therefore capable of serving as the general base to activate the catalytic serine. Together with previous results, this structure suggests a common proton relay network shared by Class A β-lactamases and PBPs, from the catalytic serine to the lysine, and ultimately to the ring nitrogen. Additionally, the E166A-penicilloate complex reveals previously unseen conformational changes of key catalytic residues during the release of the product, and is the first structure to capture the hydrolyzed product in the presence of an unmutated catalytic serine.
Structural data are available in the PDB database under the accession numbers 5TOP, 5TOY, and 5VLE.
β-内酰胺类抗生素通过形成稳定的、共价的酰基-酶复合物来抑制青霉素结合蛋白（PBPs）。在 PBPs 向 A 类β-内酰胺酶进化的过程中，β-内酰胺酶获得了 Glu166，以激活催化水并切断酰基-酶键。在这里，我们展示了三种 CTX-M-14 类 A 型β-内酰胺酶与钌配合物结合的产物复合物的晶体结构，其中包括 E166A 突变体与 penilloate 产物的复合物的 0.85Å分辨率结构、相同突变体与 penicilloate 产物的复合物的 1.30Å分辨率结构以及 S70G 突变体与 penicilloate 产物差向异构体的复合物的 1.18Å分辨率结构，这些结构揭示了 PBPs 和 A 类β-内酰胺酶的催化机制和产物抑制的情况。E166A-penilloate 复合物捕获了离去基团质子化后的氢键网络，并且首次明确表明，环氮原子向 Ser130 提供质子，然后 Ser130 向 Lys73 提供质子。这些观察结果表明，在没有 Glu166 的情况下，等效的赖氨酸在 PBPs 中为中性，因此能够作为激活催化丝氨酸的通用碱。结合以前的结果，该结构表明 A 类β-内酰胺酶和 PBPs 之间存在一个共同的质子传递网络，从催化丝氨酸到赖氨酸，最终到环氮原子。此外，E166A-penicilloate 复合物揭示了产物释放过程中关键催化残基以前未见过的构象变化，并且是第一个在存在未突变的催化丝氨酸的情况下捕获水解产物的结构。
结构数据可在 PDB 数据库中以 5TOP、5TOY 和 5VLE 的访问号获得。
