Structure and function of penicillin-binding protein from methicillinresistans Staphylococcus aureus

耐甲氧西林金黄色葡萄球菌青霉素结合蛋白的结构和功能

• 批准号: 06454074
• 负责人: MATSUZAWA Hiroshi
• 金额: $ 4.74万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06454074/
• 关键词: beta-Lactam antibiotcs; MRSA; Penicillin-binding protein; MRSA-PBP; Expanded-specrum cephem antibiotics; beta-Lactamase; beta-Lactamase with extended substrate specificity; Protein engineering; メチシリン耐性黄色ブドウ球菌; PBP 2a; 水可溶性PBP

In order to understand the structure-function relationship through the crystal sturcture, we tried to purify and then aimed to crystallize methicillin-resistant Staphylococcus aureus penicillin-binding protein (MRSA-PBP). To make water-soluble MRSA-PBP which is membrane-bound originally, its N-terminal hydrophobic segment was truncated. We constructed fusion proteins of the truncated MRSA-PBP which was fued with the C-terminal side of His-tag and maltose-binding protein (MBP). The production of the His-tag fusion protein was not so much, and furthermore the fusion protein was unstable and unable to be purified. The MBP fusion protein was purified. After protease treatment ot remove MBP domain from the fusion protein. the truncated MRSA-PBP was also unstable and lost the penicillin-bindin activity, Thus, we had to give up the crystallization of MRSA-PBP.As well as MRSA-PBP,extended-spectrum beta-lactamases have attracted public attention. Recently, it was found that Enterobacteriaceae such as Klebsiella pneumoniae, Klebsiella oxytoca, and Escherichia coli acquire resistance aganist expanded-spectrum cephem antibiotics by producing the extended-spectrum beta-lactamases. Toho-1 beta-lactamase obtained from E.coli is one of the extended-spectrum enzymes, and its amino acid sequence homology with TEM-1 beta-lactamase (a class A enzyme) is about 60%. By comparing the amino acid sequences, it was presumed that mutations of amino acid replacements from usual class A beta-lactamases, Arg244 with Thr and Glu274- (Arg275) -Asn276 with Arg- (Arg) -Arg, were important for the Toho-1 enzyme to acquire the extension of the substrate specificity. Analyzes of mutant enzymesconstructed by site-directed mutagenesis suggested that both Arg274 and Arg276 were essential for its extended substrate specificity, and that Thr, instead of Arg, at position 244 was important for functioning of the both arginine residues.

为了通过晶体结构了解其结构-功能关系，我们尝试对耐甲氧西林金黄色葡萄球菌青霉素结合蛋白（MRSA-PBP）进行纯化和结晶。为了使原本是膜结合的MRSA-PBP具有水溶性，将其n端疏水段截断。我们构建了截断的MRSA-PBP融合蛋白，该融合蛋白与His-tag的c端和麦芽糖结合蛋白（MBP）结合。His-tag融合蛋白产量不高，且融合蛋白不稳定，无法纯化。纯化了MBP融合蛋白。经过蛋白酶处理后，从融合蛋白中去除MBP结构域。截断的MRSA-PBP也不稳定，失去了青霉素结合活性，因此我们不得不放弃MRSA-PBP的结晶。与MRSA-PBP一样，广谱β -内酰胺酶也引起了人们的广泛关注。近年来，人们发现肺炎克雷伯菌、氧化克雷伯菌和大肠杆菌等肠杆菌科细菌通过产生广谱β -内酰胺酶获得对广谱头孢类抗生素的耐药性。从大肠杆菌中获得的Toho-1 β -内酰胺酶是一种扩展谱酶，其氨基酸序列与TEM-1 β -内酰胺酶（a类酶）的同源性约为60%。通过对氨基酸序列的比较，我们推测，从常见的A类β -内酰胺酶中替换氨基酸的突变，即Arg244替换Thr和Glu274- (Arg275) - asn276替换Arg- (Arg) -Arg，对于Toho-1酶获得底物特异性的扩展是重要的。通过位点定向诱变构建的突变酶分析表明，Arg274和Arg276对其扩展的底物特异性都是必需的，并且位置244的Thr而不是Arg对这两个精氨酸残基的功能都很重要。