Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA.
Stanford Synchrotron Radiation Lightsource, Stanford University, Menlo Park, California, USA.
Acta Crystallogr D Struct Biol. 2017 Aug 1;73(Pt 8):692-701. doi: 10.1107/S2059798317008671. Epub 2017 Jul 28.
Carbapenem-hydrolyzing class D β-lactamases (CHDLs) produce resistance to the last-resort carbapenem antibiotics and render these drugs ineffective for the treatment of life-threatening infections. Here, it is shown that among the clinically important CHDLs, OXA-143 produces the highest levels of resistance to carbapenems and has the highest catalytic efficiency against these substrates. Structural data demonstrate that acylated carbapenems entirely fill the active site of CHDLs, leaving no space for water molecules, including the deacylating water. Since the entrance to the active site is obstructed by the acylated antibiotic, the deacylating water molecule must take a different route for entry. It is shown that in OXA-143 the movement of a conserved hydrophobic valine residue on the surface opens a channel to the active site of the enzyme, which would not only allow the exchange of water molecules between the active site and the milieu, but would also create extra space for a water molecule to position itself in the vicinity of the scissile bond of the acyl-enzyme intermediate to perform deacylation. Structural analysis of the OXA-23 carbapenemase shows that in this enzyme movement of the conserved leucine residue, juxtaposed to the valine on the molecular surface, creates a similar channel to the active site. These data strongly suggest that all CHDLs may employ a mechanism whereupon the movement of highly conserved valine or leucine residues would allow a water molecule to access the active site to promote deacylation. It is further demonstrated that the 6α-hydroxyethyl group of the bound carbapenem plays an important role in the stabilization of this channel. The recognition of a universal deacylation mechanism for CHDLs suggests a direction for the future development of inhibitors and novel antibiotics for these enzymes of utmost clinical importance.
碳青霉烯水解酶 D 类β-内酰胺酶（CHDLs）可导致对最后一线碳青霉烯类抗生素的耐药性，使这些药物对危及生命的感染无效。在这里，研究表明在临床上重要的 CHDLs 中，OXA-143 对碳青霉烯类药物的耐药性最高，对这些底物的催化效率最高。结构数据表明，酰化碳青霉烯类完全填满 CHDLs 的活性位点，没有为水分子留出空间，包括脱酰化水。由于活性位点被酰化抗生素占据，脱酰化水分子必须通过不同的途径进入。研究表明，在 OXA-143 中，表面上保守的疏水性缬氨酸残基的运动打开了通向酶活性位点的通道，这不仅允许活性位点和环境之间水分子的交换，而且还为水分子在酰基-酶中间物的裂解键附近定位自身创造了额外的空间，以进行脱酰化。对 OXA-23 碳青霉烯酶的结构分析表明，在这种酶中，与分子表面上的缬氨酸相邻的保守亮氨酸残基的运动，在活性位点创造了一个类似的通道。这些数据强烈表明，所有 CHDLs 可能采用一种机制，其中高度保守的缬氨酸或亮氨酸残基的运动允许水分子进入活性位点以促进脱酰化。进一步证明，结合碳青霉烯的 6α-羟乙基基团在稳定该通道中起着重要作用。对 CHDLs 通用脱酰化机制的认识为这些对临床最重要的酶的抑制剂和新型抗生素的未来发展指明了方向。
