Shimamura Tatsuro, Ibuka Akiko, Fushinobu Shinya, Wakagi Takayoshi, Ishiguro Masaji, Ishii Yoshikazu, Matsuzawa Hiroshi
Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
J Biol Chem. 2002 Nov 29;277(48):46601-8. doi: 10.1074/jbc.M207884200. Epub 2002 Sep 8.
Bacterial resistance to beta-lactam antibiotics is a serious problem limiting current clinical therapy. The most common form of resistance is the production of beta-lactamases that inactivate beta-lactam antibiotics. Toho-1 is an extended-spectrum beta-lactamase that has acquired efficient activity not only to penicillins but also to cephalosporins including the expanded-spectrum cephalosporins that were developed to be stable in former beta-lactamases. We present the acyl-intermediate structures of Toho-1 in complex with cefotaxime (expanded-spectrum cephalosporin), cephalothin (non-expanded-spectrum cephalosporin), and benzylpenicillin at 1.8-, 2.0-, and 2.1-A resolutions, respectively. These structures reveal distinct features that can explain the ability of Toho-1 to hydrolyze expanded-spectrum cephalosporins. First, the Omega-loop of Toho-1 is displaced to avoid the steric contacts with the bulky side chain of cefotaxime. Second, the conserved residues Asn(104) and Asp(240) form unique interactions with the bulky side chain of cefotaxime to fix it tightly. Finally, the unique interaction between the conserved Ser(237) and cephalosporins probably helps to bring the beta-lactam carbonyl group to the suitable position in the oxyanion hole, thus increasing the cephalosporinase activity.
细菌对β-内酰胺类抗生素的耐药性是限制当前临床治疗的严重问题。最常见的耐药形式是产生使β-内酰胺类抗生素失活的β-内酰胺酶。Toho-1是一种超广谱β-内酰胺酶，它不仅对青霉素具有高效活性，而且对头孢菌素也有高效活性，包括那些被开发为对以前的β-内酰胺酶稳定的广谱头孢菌素。我们分别以1.8埃、2.0埃和2.1埃的分辨率展示了Toho-1与头孢噻肟（广谱头孢菌素）、头孢噻吩（非广谱头孢菌素）和苄青霉素形成复合物时的酰基中间体结构。这些结构揭示了不同的特征，这些特征可以解释Toho-1水解广谱头孢菌素的能力。首先，Toho-1的Ω环发生位移，以避免与头孢噻肟庞大的侧链发生空间接触。其次，保守残基Asn(104)和Asp(240)与头孢噻肟庞大的侧链形成独特的相互作用，从而将其紧密固定。最后，保守的Ser(237)与头孢菌素之间的独特相互作用可能有助于将β-内酰胺羰基带到氧阴离子孔中的合适位置，从而提高头孢菌素酶的活性。
