Mechanisms of proton relay and product release by Class A β-lactamase at ultrahigh resolution.
Mechanisms of proton relay and product release by Class A β-lactamase at ultrahigh resolution.
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DOI：
10.1111/febs.14315
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发表时间：
2018-01
期刊：
影响因子：
--
通讯作者：
Chen Y
中科院分区：
文献类型：
--
作者：
Lewandowski EM;Lethbridge KG;Sanishvili R;Skiba J;Kowalski K;Chen Y
β-内酰胺类抗生素通过形成稳定的共价酰基酶复合物来抑制青霉素结合蛋白（PBPs）。在从PBPs到A类β-内酰胺酶的进化过程中，β-内酰胺酶获得Glu166来激活催化水并裂解酰基-酶键。本文研究了CTX-M-14 A类β-内酰胺酶与酪氨酸偶联青霉素的三种产物晶体结构——E166A突变体与青霉素偶联产物的0.85 Å分辨率结构，E166A突变体与青霉酸产物的1.30 Å分辨率结构，S70G突变体与青霉酸产物外聚体的1.18 Å分辨率结构——揭示了PBPs和A类β-内酰胺酶的催化机制和产物抑制作用。E166A-penilloate复合物捕获了离去基质子化后的氢键网络，并首次明确地表明，环氮给Ser130一个质子，而Ser130又给Lys73一个质子。这些观察结果表明，在没有Glu166的情况下，等效赖氨酸在PBPs中是中性的，因此能够作为激活催化丝氨酸的一般碱。结合先前的结果，该结构表明a类β-内酰胺酶和PBPs共享一个从催化丝氨酸到赖氨酸，最终到环氮的共同质子中继网络。此外，E166A-penicilloate复合物揭示了产物释放过程中关键催化残基的先前未见的构象变化，并且是第一个在未突变的催化丝氨酸存在的情况下捕获水解产物的结构。CTX-M-14 A类β-内酰胺酶与真核烯偶联青霉素产物配合物的亚埃分辨率晶体结构表明，Lys73在没有Glu166的情况下是中性的。另外两个晶体结构揭示了产品释放过程中活性位点的变化。这些结果对A类β-内酰胺酶和青霉素结合蛋白的催化机制有了新的认识。
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. A sub-Angstrom resolution crystal structure of CTX-M-14 Class A β-lactamase in complex with a ruthenocene-conjugated penicillin product reveals that Lys73 is neutral in the absence of Glu166. Two additional crystal structures shed light on changes in the active site during product release. These results give new insight into the catalytic mechanism of both Class A β-lactamases and penicillin-binding proteins.
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影响因子：
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作者：
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通讯作者：
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