Identification, purification, and characterization of transpeptidase and glycosyltransferase domains of Streptococcus pneumoniae penicillin-binding protein 1a
Identification, purification, and characterization of transpeptidase and glycosyltransferase domains of Streptococcus pneumoniae penicillin-binding protein 1a
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DOI：
10.1128/jb.180.21.5652-5659.1998
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发表时间：
1998-11-01
影响因子：
3.2
通讯作者：
Vernet, T
中科院分区：
文献类型：
--
作者：
Di Guilmi, AM;Mouz, N;Vernet, T
肺炎链球菌对β -内酰胺类抗生素的耐药性是由于青霉素结合蛋白（pbp）的改变。肺炎链球菌PBP 1a属于A类高分子量PBP，具有转肽酶（TP）和糖基转移酶（GT）活性。PBP 1a的683个氨基酸的胞外区（PBP 1a*）在大肠杆菌中以GST融合蛋白的形式表达。纯化了GST-PBP 1a*可溶性蛋白，并通过有限的蛋白水解揭示了其结构域组织，一个跨越Ser 264至Arg 653的蛋白酶抗性片段对β -内酰胺和底物类似物的反应性与亲本蛋白相似，该蛋白片段代表TP结构域。GT结构域（Ser 37 ~ Lys 263）以重组GST融合蛋白表达，莫诺霉素对GT结构域抗胰蛋白酶降解的保护作用被解释为GT结构域与莫诺霉素的相互作用。
Resistance to beta-lactam antibiotics in Streptococcus pneumoniae is due to alteration of penicillin-binding proteins (PBPs). S. pneumoniae PBP 1a belongs to the class A high-molecular-mass PBPs, which harbor transpeptidase (TP) and glycosyltransferase (GT) activities. The GT active site represents a new potential target for the generation of novel nonpenicillin antibiotics, The 683-amino-acid extracellular region of PBP 1a (PBP 1a*) was expressed in Escherichia coli as a GST fusion protein. The GST-PBP 1a* soluble protein was purified, and its domain organization was revealed by limited proteolysis, A protease-resistant fragment spanning Ser 264 to Arg 653 exhibited a reactivity profile against both beta-lactams and substrate analogues similar to that of the parent protein, This protein fragment represents the TP domain. The GT domain (Ser 37 to Lys 263) was expressed as a recombinant GST fusion protein, Protection by moenomycin of the GT domain against trypsin degradation was interpreted as an interaction between the GT domain and the moenomycin.