Fabiane S M, Sohi M K, Wan T, Payne D J, Bateson J H, Mitchell T, Sutton B J
The Randall Institute, King's College London, U.K.
Biochemistry. 1998 Sep 8;37(36):12404-11. doi: 10.1021/bi980506i.
The structure of the zinc-dependent beta-lactamase II from Bacillus cereus has been determined at 1.9 A resolution in a crystal form with two molecules in the asymmetric unit and 400 waters (space group P3121; Rcryst = 20.8%). The active site contains two zinc ions: Zn1 is tightly coordinated by His86, His88, and His149, while Zn2 is loosely coordinated by Asp90, Cys168, and His210. A water molecule (W1) lies between the two zinc ions but is significantly closer to Zn1 and at a distance of only 1.9 A is effectively a hydroxide moiety and a potential, preactivated nucleophile. In fact, Asp90 bridges W1 to Zn2, and its location is thus distinct from that of the bridging water molecules in the binuclear zinc peptidases or other binuclear zinc hydrolases. Modeling of penicillin, cephalosporin, and carbapenem binding shows that all are readily accommodated within the shallow active site cleft of the enzyme, and the Zn1-bound hydroxide is ideally located for nucleophilic attack at the beta-lactam carbonyl. This enzyme also functions with only one zinc ion present. The Zn1-Zn2 distances differ in the two independent molecules in the crystal (3.9 and 4.4 A), yet the Zn1-W1 distances are both 1.9 A, arguing against involvement of Zn2 in W1 activation. The role of Zn2 is unclear, but the B. cereus enzyme may be an evolutionary intermediate between the mono- and bizinc metallo-beta-lactamases. The broad specificity of this enzyme, together with the increasing prevalence of zinc-dependent metallo-beta-lactamases, poses a real clinical threat, and this structure provides a basis for understanding its mechanism and designing inhibitors.
蜡样芽孢杆菌锌依赖性β-内酰胺酶II的结构已在分辨率为1.9 Å的晶体中确定，该晶体的不对称单元中有两个分子和400个水分子（空间群P3121；Rcryst = 20.8%）。活性位点包含两个锌离子：Zn1由His86、His88和His149紧密配位，而Zn2由Asp90、Cys168和His210松散配位。一个水分子（W1）位于两个锌离子之间，但明显更靠近Zn1，距离仅为1.9 Å，实际上是一个氢氧根部分和一个潜在的预活化亲核试剂。事实上，Asp90将W1与Zn2桥连，因此其位置与双核锌肽酶或其他双核锌水解酶中桥连水分子的位置不同。青霉素、头孢菌素和碳青霉烯结合的模型表明，所有这些都很容易容纳在酶的浅活性位点裂隙中，并且与Zn1结合的氢氧根处于理想位置，可对β-内酰胺羰基进行亲核攻击。该酶在仅存在一个锌离子的情况下也能发挥作用。晶体中两个独立分子的Zn1-Zn2距离不同（3.9和4.4 Å），但Zn1-W1距离均为1.9 Å，这表明Zn2不参与W1的活化。Zn2的作用尚不清楚，但蜡样芽孢杆菌酶可能是单锌和双锌金属β-内酰胺酶之间的进化中间体。这种酶的广泛特异性，以及锌依赖性金属β-内酰胺酶的日益普遍，构成了真正的临床威胁，而该结构为理解其机制和设计抑制剂提供了基础。
