Goldberg Shalom D, Iannuccilli William, Nguyen Tuan, Ju Jingyue, Cornish Virginia W
Department of Chemistry, Columbia University, 3000 Broadway, New York, NY 10027, USA.
Protein Sci. 2003 Aug;12(8):1633-45. doi: 10.1110/ps.0302903.
Despite their clinical importance, the mechanism of action of the class C beta-lactamases is poorly understood. In contrast to the class A and class D beta-lactamases, which contain a glutamate residue and a carbamylated lysine in their respective active sites that are thought to serve as general base catalysts for beta-lactam hydrolysis, the mechanism of activation of the serine and water nucleophiles in the class C enzymes is unclear. To probe for residues involved in catalysis, the class C beta-lactamase from Enterobacter cloacae P99 was studied by combinatorial scanning mutagenesis at 122 positions in and around the active site. Over 1000 P99 variants were screened for activity in a high-throughput in vivo antibiotic resistance assay and sequenced by 96-capillary electrophoresis to identify residues that are important for catalysis. P99 mutants showing reduced capability to convey antibiotic resistance were purified and characterized in vitro. The screen identified an active-site hydrogen-bonding network that is key to catalysis. A second cluster of residues was identified that likely plays a structural role in the enzyme. Otherwise, residues not directly contacting the substrate showed tolerance to substitution. The study lends support to the notion that the class C beta-lactamases do not have a single residue that acts as the catalytic general base. Rather, catalysis is affected by a hydrogen-bonding network in the active site, suggesting a possible charge relay system.
尽管C类β-内酰胺酶具有临床重要性，但其作用机制却鲜为人知。与A类和D类β-内酰胺酶不同，A类和D类β-内酰胺酶在各自的活性位点含有一个谷氨酸残基和一个氨甲酰化赖氨酸，它们被认为是β-内酰胺水解的一般碱催化剂，而C类酶中丝氨酸和亲水核试剂的激活机制尚不清楚。为了探究参与催化的残基，通过对阴沟肠杆菌P99的C类β-内酰胺酶活性位点及其周围122个位置进行组合扫描诱变来进行研究。在高通量体内抗生素抗性试验中筛选了1000多个P99变体的活性，并通过96毛细管电泳进行测序，以鉴定对催化重要的残基。纯化并在体外表征了显示出传递抗生素抗性能力降低的P99突变体。该筛选确定了一个对催化至关重要的活性位点氢键网络。还鉴定出了第二组残基，它们可能在酶中起结构作用。否则，未直接与底物接触的残基对取代具有耐受性。该研究支持了C类β-内酰胺酶没有单一残基作为催化一般碱的观点。相反，催化作用受活性位点氢键网络的影响，这表明可能存在电荷中继系统。
