THE AEROMONAS-HYDROPHILA CPHA GENE - MOLECULAR HETEROGENEITY AMONG CLASS-B METALLO-BETA-LACTAMASES
THE AEROMONAS-HYDROPHILA CPHA GENE - MOLECULAR HETEROGENEITY AMONG CLASS-B METALLO-BETA-LACTAMASES
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DOI：
10.1128/jb.173.15.4611-4617.1991
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发表时间：
1991-08-01
影响因子：
3.2
通讯作者：
SATTA, G
中科院分区：
文献类型：
--
作者：
MASSIDDA, O;ROSSOLINI, GM;SATTA, G
通过筛选气单胞菌基因组文库中能够在含亚胺培南培养基上生长的克隆，在大肠杆菌中克隆了编码碳青霉烯水解金属β-内酰胺酶的气单胞菌基因，命名为cphA。根据测序数据，克隆的cphA基因似乎能够编码254个氨基酸的多肽，其序列包括用于将蛋白质靶向至周质空间的潜在N-末端前导序列。这些数据与原始气单胞菌酶和在E.大肠杆菌中，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳的粗β-内酰胺酶制剂，然后通过对凝胶中分离的蛋白质进行复性处理和通过改良的碘量法技术定位显示碳青霉烯水解β-内酰胺酶活性的蛋白条带进行评价。推导的CphA酶的氨基酸序列显示出与蜡状芽孢杆菌的β-内酰胺酶II和脆弱拟杆菌的CfiA β-内酰胺酶都具有部分同源性的区域。序列同源性在包含B的酶中已知的氨基酸残基的区域中更明显。蜡状芽孢杆菌作为金属辅因子的配体结合残基发挥作用。然而，CphA酶似乎与其他两种酶的相似性较低，而这两种酶似乎彼此关系更密切。因此，这些结果表明在分子B类金属β-内酰胺酶中存在至少两个分子亚类。
An Aeromonas hydrophila gene, named cphA, coding for a carbapenem-hydrolyzing metallo-beta-lactamase, was cloned in Escherichia coli by screening an Aeromonas genomic library for clones able to grow on imipenem-containing medium. From sequencing data, the cloned cphA gene appeared able to code for a polypeptide of 254 amino acids whose sequence includes a potential N-terminal leader sequence for targeting the protein to the periplasmic space. These data were in agreement with the molecular mass of the original Aeromonas enzyme and of the recombinant enzyme produced in E. coli, evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude beta-lactamase preparations followed by renaturation treatment for proteins separated in the gel and localization of protein bands showing carbapenem-hydrolyzing beta-lactamase activity by a modified iodometric technique. The deduced amino acid sequence of the CphA enzyme showed regions of partial homology with both the beta-lactamase II of Bacillus cereus and the CfiA beta-lactamase of Bacteroides fragilis. Sequence homologies were more pronounced in the regions encompassing the amino acid residues known in the enzyme of B. cereus to function as ligand-binding residues for the metal cofactor. The CphA enzyme, however, appeared to share a lower degree of similarity with the two other enzymes, which, in turn, seemed more closely related to each other. These results, therefore, suggest the existence of at least two molecular subclasses within molecular class B metallo-beta-lactamases.