Docquier Jean-Denis, Pantanella Fabrizio, Giuliani Francesco, Thaller Maria Cristina, Amicosante Gianfranco, Galleni Moreno, Frère Jean-Marie, Bush Karen, Rossolini Gian Maria
Dipartimento di Biologia Molecolare, Sezione di Microbiologia, Università di Siena, 53100 Siena, Italy.
Antimicrob Agents Chemother. 2002 Jun;46(6):1823-30. doi: 10.1128/AAC.46.6.1823-1830.2002.
The sequenced chromosome of Caulobacter crescentus CB15 encodes a hypothetical protein that exhibits significant similarity (30 to 35% identical residues) to metallo-beta-lactamases of subclass B3. An allelic variant of this gene (divergent by 3% of its nucleotides) was cloned in Escherichia coli from C. crescentus type strain DSM4727. Expression studies confirmed the metallo-beta-lactamase activity of its product, CAU-1. The enzyme produced in E. coli was purified by two ion-exchange chromatography steps. CAU-1 contains a 29-kDa polypeptide with an alkaline isoelectric pH (> 9), and unlike the L1 enzyme of Stenotrophomonas maltophilia, the native form is monomeric. Kinetic analysis revealed a preferential activity toward penicillins, carbapenems, and narrow-spectrum cephalosporins, while oxyimino cephalosporins were poorly or not hydrolyzed. Affinities for the various beta-lactams were poor overall (K(m) values were always > 100 microM and often > 400 microM). The interaction with divalent ion chelators appeared to occur by a mechanism similar to that prevailing in other members of subclass B3. In C. crescentus, the CAU-1 enzyme is produced independently of beta-lactam exposure and, interestingly, the bla(CAU) determinant is bracketed by three other genes, including two genes encoding enzymes involved in methionine biosynthesis and a gene encoding a putative transcriptional regulator, in an operon-like structure. The CAU-1 enzyme is the first example of a metallo-beta-lactamase in a member of the alpha subdivision of the class Proteobacteria:
新月柄杆菌CB15的测序染色体编码一种假定蛋白，该蛋白与B3亚类金属β-内酰胺酶具有显著相似性（30%至35%的相同残基）。从新月柄杆菌模式菌株DSM4727中克隆了该基因的一个等位变体（其核苷酸差异为3%），并在大肠杆菌中进行表达研究，证实了其产物CAU-1具有金属β-内酰胺酶活性。在大肠杆菌中产生的该酶通过两步离子交换色谱法进行纯化。CAU-1包含一条29 kDa的多肽，其碱性等电点pH值大于9，与嗜麦芽窄食单胞菌的L1酶不同，其天然形式为单体。动力学分析表明，该酶对青霉素、碳青霉烯类和窄谱头孢菌素具有优先活性，而氧亚氨基头孢菌素的水解能力较差或根本不被水解。总体而言，该酶对各种β-内酰胺类的亲和力较差（米氏常数K(m)值始终大于100 μM，且经常大于400 μM）。与二价离子螯合剂的相互作用似乎通过一种与B3亚类其他成员中普遍存在的机制类似的机制发生。在新月柄杆菌中，CAU-1酶的产生与β-内酰胺的暴露无关，有趣的是，bla(CAU)决定簇被另外三个基因包围，包括两个编码参与甲硫氨酸生物合成的酶的基因和一个编码假定转录调节因子的基因，呈操纵子样结构。CAU-1酶是变形菌门α亚类成员中金属β-内酰胺酶的首个实例。
