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首页> 外文期刊>Journal of bacteriology >Purification and cloning of a proline 3-hydroxylase, a novel enzyme which hydroxylates free L-proline to cis-3-hydroxy-L-proline.
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Purification and cloning of a proline 3-hydroxylase, a novel enzyme which hydroxylates free L-proline to cis-3-hydroxy-L-proline.

机译：脯氨酸3-羟化酶的纯化和克隆，脯氨酸3-羟化酶是一种将游离的L-脯氨酸羟化为顺式-3-羟基-L-脯氨酸的新型酶。

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摘要

Proline 3-hydroxylase was purified from Streptomyces sp. strain TH1, and its structural gene was cloned. The purified enzyme hydroxylated free L-proline to cis-3-hydroxy-L-proline and showed properties of a 2-oxoglutarate-dependent dioxygenase (H. Mori, T. Shibasaki, Y. Uosaki, K. Ochiai, and A. Ozaki, Appl. Environ. Microbiol, 62:1903-1907, 1996). The molecular mass of the purified enzyme was 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was 4.3. The optimal pH and temperature were 7.0 and 35 degrees C, respectively. The K(m) values were 0.56 and 0.11 mM for L-proline and 2-oxoglutarate, respectively. The Kcat value of hydroxylation was 3.2 s-1. Determined N-terminal and internal amino acid sequences of the purified protein were not found in the SwissProt protein database. A DNA fragment of 74 bp was amplified by PCR with degenerate primers based on the determined N-terminal amino acid sequence. With this fragment as a template, a digoxigenin-labeled N-terminal probe was synthesized by PCR. A 6.5-kbp chromosome fragment was cloned by colony hybridization with the labeled probe. The determined DNA sequence of the cloned fragment revealed a 870-bp open reading frame (ORF 3), encoding a protein of 290 amino acids with a calculated molecular weight of 33,158. No sequence homolog was found in EMBL, GenBank, and DDBJ databases. ORF 3 was expressed in Escherichia coli DH1. Recombinants showed hydroxylating activity five times higher than that of the original bacterium, Streptomyces sp. strain TH1. It was concluded that the ORF 3 encodes functional proline 3-hydroxylase.
机译：脯氨酸3-羟化酶从链霉菌属物种中纯化。株TH1，并克隆其结构基因。纯化的酶将游离L-脯氨酸羟化为顺式3-羟基-L-脯氨酸，并显示了2-氧戊二酸酯依赖性双加氧酶的性质（H.Mori，T.Shibasaki，Y.Uosaki，K.Ochiai和A.Ozaki ，Appl.Environ.Microbiol，62：1903-1907，1996）。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定，纯化的酶的分子量为35kDa。酶的等电点为4.3。最佳pH和温度分别为7.0和35摄氏度。 L-脯氨酸和2-氧戊二酸的K（m）值分别为0.56和0.11 mM。羟基化的Kcat值为3.2s-1。在SwissProt蛋白质数据库中找不到纯化的蛋白质的已确定N末端和内部氨基酸序列。根据确定的N端氨基酸序列，用简并引物通过PCR扩增74 bp的DNA片段。以该片段为模板，通过PCR合成了洋地黄毒苷标记的N末端探针。通过与标记探针的菌落杂交克隆了一个6.5kbp的染色体片段。确定的克隆片段的DNA序列显示了一个870 bp的开放阅读框（ORF 3），编码290个氨基酸的蛋白质，计算的分子量为33,158。在EMBL，GenBank和DDBJ数据库中找不到序列同源物。 ORF 3在大肠杆菌DH1中表达。重组子的羟化活性是原始细菌链霉菌的五倍。 TH1株。结论是，ORF 3编码功能性脯氨酸3-羟化酶。

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