Enzymatic and biochemical properties of a novel human serine dehydratase isoform
Enzymatic and biochemical properties of a novel human serine dehydratase isoform
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DOI：
10.1016/j.bbapap.2006.02.010
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发表时间：
2006-05-01
影响因子：
3.2
通讯作者：
Kobayashi, M
中科院分区：
文献类型：
--
作者：
Ogawa, H;Gomi, T;Kobayashi, M
一个与人丝氨酸脱氢酶（SDH）相似的cDNA克隆已被存入GenBank/EMBL数据库，但其结构和功能基础仍不清楚。尽管存在mRNA，但在培养的细胞中发现预期的蛋白质水平较低。为了了解蛋白质的理化性质，我们在大肠杆菌中表达了cDNA，并将表达的蛋白质与肝SDH的蛋白质进行了比较。纯化的蛋白显示L-丝氨酸和L-苏氨酸脱氢酶活性，证明是SDH的同工型。但它们的K-m和V-max常数在二阶范围内不同。从由328个残基组成的肝SDH中去除Pro 128（其在由329个残基组成的同种型的相应位置中缺失）显著改变了5 '-磷酸吡哆醛的米氏常数和Kd值，而向同种型中添加脯氨酸残基则没有影响。这些发现表明两种酶的活性位点的结构的差异。另一个显著的特点是该异构体在E.大肠杆菌的SDH活性比肝SDH低7倍。在同种型中用瓦尔取代Leu 287显著增加了蛋白质水平。体外转录和翻译实验也证实了突变异构体的高产率。该亚型的低表达可能是由于其mRNA的二级结构比肝SDH mRNA的二级结构更稳定。目前的研究结果可能会提供一个线索，为什么在培养的细胞中的蛋白质水平低。(c)2006 Elsevier B. V.保留所有权利。
A cDNA clone similar to human serine dehydratase (SDH) is deposited in the GenBank/EMBL databases, but its structural and functional bases remain unknown. Despite the occurrence of mRNA, the expected protein level was found to be low in cultured cells. To learn about physicochemical properties of the protein, we expressed the cDNA in Escherichia coli, and compared the expressed protein with that of a hepatic SDH. The purified protein showed L-serine and L-threonine dehydratase activity, demonstrating to be an isoform of SDH. However, their K-m and V-max, constants were different in a range of two-order. Removal of Pro 128 from the hepatic SDH consisting of 328 residues, which is missing in the corresponding position of the isoform consisting of 329 residues, significantly changed the Michaelis constants and K-d value for pyridoxal 5'-phosphate, whereas addition of a proline residue to the isoform was without effect. These findings suggest the difference in the structures of the active sites of the two enzymes. Another striking feature was that the expressed level of the isoform in E. coli was 7-fold lower than that of the hepatic SDH. Substitution of Val for Leu287 in the isoform dramatically increased the protein level. The high yield of the mutated isoform was also confirmed by the in vitro transcription and translation experiment. The poor expression of the isoform could be explained by the more stable secondary structure of the mRNA than that of the hepatic SDH mRNA. The present findings may provide a clue as to why the protein level in cultured cells is low. (c) 2006 Elsevier B.V. All rights reserved.