Characterization of a novel dye-linked l-proline dehydrogenase from an aerobic hyperthermophilic archaeon, Pyrobaculum calidifontis
Characterization of a novel dye-linked l-proline dehydrogenase from an aerobic hyperthermophilic archaeon, Pyrobaculum calidifontis
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DOI：
10.1007/s00253-010-2914-7
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发表时间：
2011-02
影响因子：
5
通讯作者：
T. Satomura;Xiaodong Zhang;Y. Hara;Katsumi Doi;H. Sakuraba;T. Ohshima
中科院分区：
文献类型：
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作者：
T. Satomura;Xiaodong Zhang;Y. Hara;Katsumi Doi;H. Sakuraba;T. Ohshima
染料-l-脯氨酸脱氢酶（dye-1-proDH）的活性在需氧超嗜热古菌Pyrobaculum calidifontis JCM 11548的粗提物中被发现，并且通过四个连续的色谱步骤被纯化163倍。该酶具有约108 kDa的分子量，并且是具有约46 kDa的亚基分子量的同源二聚体。该酶在100 °C（pH 7.5）下孵育120 min后或在50 °C下在pH 4.5-9.0下孵育30 min后保留超过90%的活性。该酶以2，6-二氯靛酚（DCIP）为电子受体，催化脯氨酸脱氢生成Δ1-吡咯啉-5-羧酸，其米氏常数分别为1.67和0.026 mM。通过高效液相色谱法鉴定酶的辅基为黄素腺嘌呤二核苷酸。亚基N-末端氨基酸序列为MYDYVVVGAG。使用该序列和之前报道的基因组信息，鉴定了编码该酶的基因（Pcal_1655）。然后克隆该基因并在大肠杆菌中表达，发现其编码415个氨基酸的多肽，计算分子量为46，259。染料-l-proDH基因在P. calidifontisinherently不同于那些在其他hyperthermophiles迄今报道。
The activity of a dye-linkedl-proline dehydrogenase (dye-l-proDH) was found in the crude extract of an aerobic hyperthermophilic archaeon,Pyrobaculum calidifontisJCM 11548, and was purified 163-fold through four sequential chromatography steps. The enzyme has a molecular mass of about 108 kDa and is a homodimer with a subunit molecular mass of about 46 kDa. The enzyme retained more than 90% of its activity after incubation at 100 °C for 120 min (pH 7.5) or after incubation at pHs 4.5–9.0 for 30 min at 50 °C. The enzyme catalyzedl-proline dehydrogenation to Δ1-pyroline-5-carboxylate using 2,6-dichloroindophenol (DCIP) as the electron acceptor and the Michaelis constants forl-proline and DCIP were 1.67 and 0.026 mM, respectively. The prosthetic group on the enzyme was identified as flavin adenine dinucleotide by high-performance liquid chromatography. The subunit N-terminal amino acid sequence was MYDYVVVGAG. Using that sequence and previously reported genome information, the gene encoding the enzyme (Pcal_1655) was identified. The gene was then cloned and expressed inEscherichia coliand found to encode a polypeptide of 415 amino acids with a calculated molecular weight of 46,259. The dye-l-proDH gene cluster inP. calidifontisinherently differs from those in the other hyperthermophiles reported so far.