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首页> 外文期刊>Journal of bacteriology >The First Archaeal ATP-Dependent Glucokinase, from the Hyperthermophilic Crenarchaeon Aeropyrum pernix, Represents a Monomeric, Extremely Thermophilic ROK Glucokinase with Broad Hexose Specificity
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The First Archaeal ATP-Dependent Glucokinase, from the Hyperthermophilic Crenarchaeon Aeropyrum pernix, Represents a Monomeric, Extremely Thermophilic ROK Glucokinase with Broad Hexose Specificity

机译：来自嗜热嗜热气单胞菌多年生菌的首个古细菌ATP依赖性葡糖激酶代表了具有广泛己糖特异性的单体，极其嗜热的韩国葡糖激酶。

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An ATP-dependent glucokinase of the hyperthermophilic aerobic crenarchaeon Aeropyrum pernix was purified 230-fold to homogeneity. The enzyme is a monomeric protein with an apparent molecular mass of about 36 kDa. The apparent Km values for ATP and glucose (at 90°C and pH 6.2) were 0.42 and 0.044 mM, respectively; the apparent Vmax was about 35 U/mg. The enzyme was specific for ATP as a phosphoryl donor, but showed a broad spectrum for phosphoryl acceptors: in addition to glucose, which showed the highest catalytic efficiency (kcat/Km), the enzyme also phosphorylates glucosamin, fructose, mannose, and 2-deoxyglucose. Divalent cations were required for maximal activity: Mg2+, which was most effective, could partially be replaced with Co2+, Mn2+, and Ni2+. The enzyme had a temperature optimum of at least 100°C and showed significant thermostability up to 100°C. The coding function of open reading frame (ORF) APE2091 (Y. Kawarabayasi, Y. Hino, H. Horikawa, S. Yamazaki, Y. Haikawa, K. Jin-no, M. Takahashi, M. Sekine, S. Baba, A. Ankai, H. Kosugi, A. Hosoyama, S. Fukui, Y. Nagai, K. Nishijima, H. Nakazawa, M. Takamiya, S. Masuda, T. Funahashi, T. Tanaka, Y. Kudoh, J. Yamazaki, N. Kushida, A. Oguchi, and H. Kikuchi, DNA Res. 6:83-101, 145-152, 1999), previously annotated as gene glk, coding for ATP-glucokinase of A. pernix, was proved by functional expression in Escherichia coli. The purified recombinant ATP-dependent glucokinase showed a 5-kDa higher molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but almost identical kinetic and thermostability properties in comparison to the native enzyme purified from A. pernix. N-terminal amino acid sequence of the native enzyme revealed that the translation start codon is a GTG 171 bp downstream of the annotated start codon of ORF APE2091. The amino acid sequence deduced from the truncated ORF APE2091 revealed sequence similarity to members of the ROK family, which comprise bacterial sugar kinases and transcriptional repressors. This is the first report of the characterization of an ATP-dependent glucokinase from the domain of Archaea, which differs from its bacterial counterparts by its monomeric structure and its broad specificity for hexoses.
机译：高温依赖的好氧性克氏杆菌 Aeropyrum pernix 的ATP依赖性葡萄糖激酶被纯化230倍至均质。该酶是具有约36kDa的表观分子量的单体蛋白。 ATP和葡萄糖（在90°C和pH 6.2时）的表观 K m 值分别为0.42和0.044 mM。表观 V max 约为35 U / mg。该酶对ATP作为磷酰基供体具有特异性，但对磷酰基受体表现出广谱性：除葡萄糖外，还显示出最高的催化效率（ k cat / < em> K m ），该酶还将磷酸氨基葡萄糖，果糖，甘露糖和2-脱氧葡萄糖磷酸化。最大活性需要二价阳离子：最有效的Mg 2 + 可以部分替换为Co 2 + ，Mn 2 + 和Ni 2 + 。该酶的最佳温度至少为100°C，在高达100°C的温度下显示出明显的热稳定性。开放阅读框（ORF）APE2091的编码功能（Y. Kawarabayasi，Y。Hino，H。Horikawa，S。Yamazaki，Y。Haikawa，K。Jin-no，M。Takahashi，M。Sekine，S。Baba， A.Ankai，H.Kosugi，A.Hosoyama，S.Fukui，Y.Nagai，K.Nishijima，H.Nakazawa，M.Takamiya，S.Masuda，T.Funahashi，T.Tanaka，Y.Kudoh，J。 Yamazaki，N.Kushida，A.Oguchi和H.Kikuchi，DNA Res.6：83-101，145-152，1999），以前被注释为基因 glk ，编码ATP的葡萄糖激酶 A。通过在大肠杆菌中的功能性表达证明了pernix 。纯化的重组ATP依赖性葡萄糖激酶在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上显示5 kDa的较高分子量，但与从 A纯化的天然酶相比，几乎具有相同的动力学和热稳定性。天然酶的N-末端氨基酸序列显示，翻译起始密码子是在ORF APE2091的注释起始密码子下游的GTG 171 bp。从截短的ORF APE2091推导的氨基酸序列揭示了与ROK家族成员的序列相似性，该家族成员包含细菌糖激酶和转录阻遏物。这是关于 Archaea 域中ATP依赖性葡萄糖激酶的特征的首次报道，该酶的区别在于其单体结构和对己糖的广泛特异性与细菌类似物不同。

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