CLONING AND EXPRESSION OF HUMAN DEOXYHYPUSINE SYNTHASE CDNA - STRUCTURE-FUNCTION STUDIES WITH THE RECOMBINANT ENZYME AND MUTANT PROTEINS
CLONING AND EXPRESSION OF HUMAN DEOXYHYPUSINE SYNTHASE CDNA - STRUCTURE-FUNCTION STUDIES WITH THE RECOMBINANT ENZYME AND MUTANT PROTEINS
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DOI：
10.1074/jbc.270.38.22386
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发表时间：
1995-09-22
影响因子：
4.8
通讯作者：
PARK, MH
中科院分区：
文献类型：
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作者：
JOE, YA;WOLFF, EC;PARK, MH
脱氧马尿苷合成酶催化马尿苷（N-ε-(4-氨基-2-羟丁基)赖氨酸）翻译后形成的第一步。从人 HeLa 细胞文库中分离出编码脱氧马尿苷合酶的 cDNA 克隆。分离了编码 369 个氨基酸蛋白质（计算分子量为 40,970 Da）的全长 cDNA 克隆和可能编码内部删除 56 个氨基酸的蛋白质（Asp（262）-Ser（317））的较短 cDNA 克隆。推导的人酶的氨基酸序列与酵母脱氧马尿苷合酶的氨基酸序列以及来自大鼠和粗糙脉孢菌酶的胰蛋白酶肽的已知序列具有高度的同一性。在大肠杆菌中表达后形成的重组酶有效地催化脱氧马尿苷合成。具有 (i) 48 或 97 个 NH2 末端氨基酸截短、(ii) 39 个 COOH 末端氨基酸截短或 (iii) 内部缺失（Asp(262)-Ser(317)）的变体人重组蛋白均无活性。由完整的人类序列和插入 Glu(193) 和 Gln(194) 之间的酵母序列的 16 个氨基酸（Gln(197)-Asn(212)，不存在于人类酶中）组成的嵌合蛋白表现出中等活性。
Deoxyhypusine synthase catalyzes the first step in the post-translational formation of hypusine (N-epsilon-(4-amino-2-hydroxybutyl)lysine). cDNA clones encoding deoxyhypusine synthase were isolated from a human HeLa cell library. Full-length cDNA clones encoding a 369-amino acid protein (calculated molecular mass of 40,970 Da) and a shorter cDNA clone that would potentially encode a protein with an internal deletion of 56 amino acids (Asp(262)-Ser(317)) were isolated. The deduced amino acid sequence of the human enzyme shows a high degree of identity to that of yeast deoxyhypusine synthase and to the known sequences of tryptic peptides from the rat and Neurospora crassa enzymes. The recombinant enzyme formed upon expression in Escherichia coli effectively catalyzed deoxyhypusine synthesis. Variant human recombinant proteins with (i) a truncation of 48 or 97 NH2-terminal amino acids, (ii) a truncation of 39 COOH-terminal amino acids, or (iii) an internal deletion (Asp(262)-Ser(317)) were inactive. A chimeric protein consisting of the complete human sequence and 16 amino acids of the yeast sequence (Gln(197)-Asn(212), not present in the human enzyme) inserted between Glu(193) and Gln(194) exhibited moderate activity.