Simm Alan M, Higgins Catherine S, Carenbauer Anne L, Crowder Michael W, Bateson John H, Bennett Peter M, Clarke Anthony R, Halford Stephen E, Walsh Timothy R
Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom.
J Biol Chem. 2002 Jul 5;277(27):24744-52. doi: 10.1074/jbc.M201524200. Epub 2002 Apr 8.
The L1 metallo-beta-lactamase from Stenotrophomonas maltophilia is unique among this class of enzymes because it is tetrameric. Previous work predicted that the two regions of important intersubunit interaction were the residue Met-140 and the N-terminal extensions of each subunit. The N-terminal extension was also implicated in beta-lactam binding. Mutation of methionine 140 to aspartic acid results in a monomeric L1 beta-lactamase with a greatly altered substrate specificity profile. A 20-amino acid N-terminal deletion mutant enzyme (N-Del) could be isolated in a tetrameric form but demonstrated greatly reduced rates of beta-lactam hydrolysis and different substrate profiles compared with that of the parent enzyme. Specific site-directed mutations of individual N terminus residues were made (Y11S, W17S, and a double mutant L5A/L8A). All N-terminal mutant enzymes were tetramers and all showed higher K(m) values for ampicillin and nitrocefin, hydrolyzed ceftazidime poorly, and hydrolyzed imipenem more efficiently than ampicillin in contrast to wild-type L1. Nitrocefin turnover was significantly increased, probably because of an increased rate of breakdown of the intermediate species due to a lack of stabilizing forces. K(m) values for monomeric L1 were greatly increased for all antibiotics tested. A model of a highly mobile N-terminal extension in the monomeric enzyme is proposed to explain these findings. Tetrameric L1 shows negative cooperativity, which is not present in either the monomer or N-terminal deletion enzymes, suggesting that the cooperative effect is mediated via N-terminal intersubunit interactions. These data indicate that while the N terminus of L1 is not essential for beta-lactam hydrolysis, it is clearly important to its activity and substrate specificity.
嗜麦芽窄食单胞菌的L1金属β-内酰胺酶在这类酶中很独特，因为它是四聚体。先前的研究预测，亚基间重要相互作用的两个区域是第140位残基甲硫氨酸和每个亚基的N端延伸区。N端延伸区也与β-内酰胺结合有关。将甲硫氨酸140突变为天冬氨酸会产生一种单体L1β-内酰胺酶，其底物特异性谱有很大改变。一种20个氨基酸的N端缺失突变酶（N-Del）可以以四聚体形式分离出来，但与亲本酶相比，其β-内酰胺水解速率大大降低，底物谱也不同。对单个N端残基进行了特定位点定向突变（Y11S、W17S和双突变体L5A/L8A）。所有N端突变酶都是四聚体，与野生型L1相比，它们对氨苄西林和硝基头孢菌素的K(m)值都更高，对头孢他啶的水解能力很差，对亚胺培南的水解效率比对氨苄西林更高。硝基头孢菌素的周转显著增加，可能是由于缺乏稳定力导致中间物种分解速率增加。单体L1对所有测试抗生素的K(m)值都大大增加。提出了一个单体酶中高度可移动的N端延伸区模型来解释这些发现。四聚体L1表现出负协同性，单体或N端缺失酶中不存在这种协同性，这表明协同效应是通过N端亚基间相互作用介导的。这些数据表明，虽然L1的N端对β-内酰胺水解不是必需的，但对其活性和底物特异性显然很重要。
