Franceschini N, Caravelli B, Docquier J D, Galleni M, Frère J M, Amicosante G, Rossolini G M
Dipartimento di Scienze e Tecnologie Biomediche, Università degli Studi L'Aquila, I-67100 Coppito, L'Aquila, Italy.
Antimicrob Agents Chemother. 2000 Nov;44(11):3003-7. doi: 10.1128/AAC.44.11.3003-3007.2000.
VIM-1 is a new group 3 metallo-beta-lactamase recently detected in carbapenem-resistant nosocomial isolates of Pseudomonas aeruginosa from the Mediterranean area. In this work, VIM-1 was purified from an Escherichia coli strain carrying the cloned bla(VIM-1) gene by means of an anion-exchange chromatography step followed by a gel permeation chromatography step. The purified enzyme exhibited a molecular mass of 26 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an acidic pI of 5.1 in analytical isoelectric focusing. Amino-terminal sequencing showed that mature VIM-1 results from the removal of a 26-amino-acid signal peptide from the precursor. VIM-1 hydrolyzes a broad array of beta-lactam compounds, including penicillins, narrow- to expanded-spectrum cephalosporins, carbapenems, and mechanism-based serine-beta-lactamase inactivators. Only monobactams escape hydrolysis. The highest catalytic constant/K(m) ratios (>10(6) M(-1). s(-1)) were observed with carbenicillin, azlocillin, some cephalosporins (cephaloridine, cephalothin, cefuroxime, cefepime, and cefpirome), imipenem, and biapenem. Kinetic parameters showed remarkable variability with different beta-lactams and also within the various penam, cephem, and carbapenem compounds, resulting in no clear preference of the enzyme for any of these beta-lactam subfamilies. Significant differences were observed with some substrates between the kinetic parameters of VIM-1 and those of other metallo-beta-lactamases. Inactivation assays carried out with various chelating agents (EDTA, 1,10-o-phenanthroline, and pyridine-2,6-dicarboxylic acid) indicated that formation of a ternary enzyme-metal-chelator complex precedes metal removal from the zinc center of the protein and revealed notable differences in the inactivation parameters of VIM-1 with different agents.
VIM-1是一种新的3类金属β-内酰胺酶，最近在地中海地区耐碳青霉烯类的医院获得性铜绿假单胞菌分离株中被检测到。在这项研究中，通过阴离子交换色谱步骤，随后进行凝胶渗透色谱步骤，从携带克隆的bla(VIM-1)基因的大肠杆菌菌株中纯化出VIM-1。在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳中，纯化后的酶分子量为26 kDa，在分析等电聚焦中酸性pI为5.1。氨基末端测序表明，成熟的VIM-1是由前体去除26个氨基酸的信号肽后产生的。VIM-1能水解多种β-内酰胺化合物，包括青霉素、窄谱至广谱头孢菌素、碳青霉烯类以及基于机制的丝氨酸β-内酰胺酶失活剂。只有单环β-内酰胺类不被水解。在用羧苄西林、阿洛西林、一些头孢菌素（头孢菌素I、头孢噻吩、头孢呋辛、头孢吡肟和头孢匹罗）、亚胺培南和美罗培南时，观察到最高的催化常数/K(m)比值(>10(6) M(-1). s(-1))。动力学参数显示，不同的β-内酰胺类以及各种青霉烷、头孢烯和碳青霉烯化合物之间存在显著差异，导致该酶对这些β-内酰胺亚家族中的任何一种都没有明显偏好。在一些底物上，观察到VIM-1与其他金属β-内酰胺酶的动力学参数存在显著差异。用各种螯合剂（乙二胺四乙酸、1,10-邻菲啰啉和吡啶-2,6-二羧酸）进行的失活试验表明，三元酶-金属-螯合剂复合物的形成先于金属从蛋白质锌中心的去除，并揭示了VIM-1与不同试剂的失活参数存在显著差异。
