| Budget Amount *help |
¥7,400,000 (Direct Cost: ¥7,400,000)
Fiscal Year 1995: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1994: ¥6,000,000 (Direct Cost: ¥6,000,000)
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| Research Abstract |
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.
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