Recombinant expression and characterization of the major beta-lactamase of Mycobacterium tuberculosis.
Recombinant expression and characterization of the major beta-lactamase of Mycobacterium tuberculosis.
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结核分枝杆菌主要β-内酰胺酶的重组表达和表征。
DOI：
10.1128/aac.42.6.1375
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发表时间：
1998
影响因子：
4.9
通讯作者：
Kernodle,DS
中科院分区：
文献类型：
--
作者：
Voladri,RK;Lakey,DL;Hennigan,SH;Menzies,BE;Edwards,KM;Kernodle,DS
耐多药结核病的治疗需要新的抗生素方案，结核分枝杆菌具有一层厚的肽聚糖层，参与其生物合成的青霉素结合蛋白被临床相关浓度的β-内酰胺类抗生素抑制。β-内酰胺酶的产生可能是M.结核菌对β-内酰胺类抗生素耐药。从3- 4周龄的M.用连续凝胶过滤层析和聚焦层析对结核杆菌H37 Ra进行了部分纯化。鉴定出三个β-内酰胺酶活性峰，pI值分别为5.1、4.9和4.5，分别占总层析后聚焦β-内酰胺酶活性的10%、78%和12%。pI值为5.1和4.9的β-内酰胺酶在动力学上难以区分，并表现出主要的青霉素酶活性。相反，pI值为4.5的β-内酰胺酶显示出相对较高的头孢菌素酶活性。在M的DNA文库的粘粒Y 49中有一个开放的阅读框。从结核分枝杆菌染色体DNA中扩增出与已知A类β-内酰胺酶同源的H37 Rv。结核杆菌H37 Ra基因在大肠杆菌中高效表达。重组酶与直接从mM纯化的pI 5.1和4.9酶在动力学上相似。结核它表现出主要的青霉素酶活性，特别是对阿洛西林的活性。克拉维酸和间氨基苯硼酸对该酶有抑制作用，EDTA对该酶无抑制作用。结果表明，M.结核杆菌A类β-内酰胺酶以青霉素酶活性为主。还存在具有相对较大头孢菌素酶活性的第二种次要β-内酰胺酶。
New antibiotic regimens are needed for the treatment of multidrug-resistant tuberculosis.Mycobacterium tuberculosishas a thick peptidoglycan layer, and the penicillin-binding proteins involved in its biosynthesis are inhibited by clinically relevant concentrations of β-lactam antibiotics. β-Lactamase production appears to be the major mechanism by whichM. tuberculosisexpresses β-lactam resistance. β-Lactamases from the broth supernatant of 3- to 4-week-old cultures ofM. tuberculosisH37Ra were partially purified by sequential gel filtration chromatography and chromatofocusing. Three peaks of β-lactamase activity with pI values of 5.1, 4.9, and 4.5, respectively, and which accounted for 10, 78, and 12% of the total postchromatofocusing β-lactamase activity, respectively, were identified. The β-lactamases with pI values of 5.1 and 4.9 were kinetically indistinguishable and exhibited predominant penicillinase activity. In contrast, the β-lactamase with a pI value of 4.5 showed relatively greater cephalosporinase activity. An open reading frame in cosmid Y49 of the DNA library ofM. tuberculosisH37Rv with homology to known class A β-lactamases was amplified from chromosomal DNA ofM. tuberculosisH37Ra by PCR and was overexpressed inEscherichia coli. The recombinant enzyme was kinetically similar to the pI 5.1 and 4.9 enzymes purified directly fromM. tuberculosis. It exhibited predominant penicillinase activity and was especially active against azlocillin. It was inhibited by clavulanic acid andm-aminophenylboronic acid but not by EDTA. We conclude that the major β-lactamase ofM. tuberculosisis a class A β-lactamase with predominant penicillinase activity. A second, minor β-lactamase with relatively greater cephalosporinase activity is also present.