Suzuki Yutaka, Haruki Mitsuru, Takano Kazufumi, Morikawa Masaaki, Kanaya Shigenori
Department of Material and Life Science, Graduate School of Engineering, Osaka University, Japan.
Eur J Biochem. 2004 Apr;271(7):1372-81. doi: 10.1111/j.1432-1033.2004.04049.x.
A psychrotrophic bacterium Shewanella sp. strain SIB1 was grown at 4 and 20 degrees C, and total soluble proteins extracted from the cells were analyzed by two-dimensional polyacrylamide gel electrophoresis. Comparison of these patterns showed that the cellular content of a protein with a molecular mass of 28 kDa and an isoelectric point of four greatly increased at 4 degrees C compared to that at 20 degrees C. Determination of the N-terminal amino acid sequence, followed by the cloning and sequencing of the gene encoding this protein, revealed that this protein is a member of the FKBP family of proteins with an amino acid sequence identity of 56% to Escherichia coli FKBP22. This protein was overproduced in E. coli in a His-tagged form, purified, and analyzed for peptidyl-prolyl cis-trans isomerase activity. When this activity was determined by the protease coupling assay using N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide as a substrate at various temperatures, the protein exhibited the highest activity at 10 degrees C with a k(cat)/K(m) value of 0.87 micro m(-1) x s(-1). When the peptidyl-prolyl cis-trans isomerase activity was determined by the RNase T(1) refolding assay at 10 and 20 degrees C, the protein exhibited higher activity at 10 degrees C with a k(cat)/K(m) value of 0.50 micro m(-1) x s(-1). These k(cat)/K(m) values are lower but comparable to those of E. coli FKBP22. We propose that a FKBP family protein is involved in cold-adaptation of psychrotrophic bacteria.
嗜冷细菌希瓦氏菌属菌株SIB1在4℃和20℃下培养，从细胞中提取的总可溶性蛋白通过二维聚丙烯酰胺凝胶电泳进行分析。这些图谱的比较表明，与20℃相比，分子量为28 kDa、等电点为4的一种蛋白质的细胞含量在4℃时大幅增加。通过测定其N端氨基酸序列，随后对编码该蛋白质的基因进行克隆和测序，发现该蛋白质是FKBP蛋白家族的一员，与大肠杆菌FKBP22的氨基酸序列同一性为56%。该蛋白质以带有His标签的形式在大肠杆菌中过量表达、纯化，并分析其肽基脯氨酰顺反异构酶活性。当使用N-琥珀酰-Ala-Leu-Pro-Phe-对硝基苯胺作为底物，通过蛋白酶偶联测定法在不同温度下测定该活性时，该蛋白质在10℃时表现出最高活性，k(cat)/K(m)值为0.87 μM(-1)×s(-1)。当在10℃和20℃下通过RNase T(1)重折叠测定法测定肽基脯氨酰顺反异构酶活性时，该蛋白质在10℃时表现出更高活性，k(cat)/K(m)值为0.50 μM(-1)×s(-1)。这些k(cat)/K(m)值较低，但与大肠杆菌FKBP22的k(cat)/K(m)值相当。我们提出，一种FKBP家族蛋白质参与嗜冷细菌的冷适应过程。
