Hansen Thomas, Reichstein Bianca, Schmid Roland, Schönheit Peter
Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität Kiel, D-24118 Kiel, Am Botanischen Garten 1-9, Germany.
J Bacteriol. 2002 Nov;184(21):5955-65. doi: 10.1128/JB.184.21.5955-5965.2002.
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 K(m) values for ATP and glucose (at 90 degrees C and pH 6.2) were 0.42 and 0.044 mM, respectively; the apparent V(max) 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 (k(cat)/K(m)), the enzyme also phosphorylates glucosamin, fructose, mannose, and 2-deoxyglucose. Divalent cations were required for maximal activity: Mg(2+), which was most effective, could partially be replaced with Co(2+), Mn(2+), and Ni(2+). The enzyme had a temperature optimum of at least 100 degrees C and showed significant thermostability up to 100 degrees 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.
对嗜热需氧泉古菌嗜酸热硫化叶菌（Aeropyrum pernix）的一种ATP依赖性葡萄糖激酶进行了纯化，纯化倍数达230倍，达到了均一性。该酶是一种单体蛋白，表观分子量约为36 kDa。在90℃和pH 6.2条件下，ATP和葡萄糖的表观K(m)值分别为0.42和0.044 mM；表观V(max)约为35 U/mg。该酶对作为磷酰基供体的ATP具有特异性，但对磷酰基受体具有广泛的作用谱：除了葡萄糖表现出最高的催化效率（k(cat)/K(m)）外，该酶还能使葡糖胺、果糖、甘露糖和2-脱氧葡萄糖磷酸化。二价阳离子是最大活性所必需的：最有效的Mg(2+)可部分被Co(2+)、Mn(2+)和Ni(2+)替代。该酶的最适温度至少为100℃，在高达100℃时表现出显著的热稳定性。开放阅读框（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和H. Kikuchi，DNA Res. 6:83 - 101, 145 - 152, 1999）先前被注释为编码嗜酸热硫化叶菌ATP-葡萄糖激酶的基因glk，通过在大肠杆菌中的功能表达证明了其编码功能。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分子量高5 kDa，但与从嗜酸热硫化叶菌纯化的天然酶相比，具有几乎相同的动力学和热稳定性特性。天然酶的N端氨基酸序列表明，翻译起始密码子是ORF APE2091注释起始密码子下游171 bp处的GTG。从截短的ORF APE2091推导的氨基酸序列显示与ROK家族成员具有序列相似性，ROK家族包括细菌糖激酶和转录阻遏物。这是古菌域中ATP依赖性葡萄糖激酶特性表征的首次报道，其单体结构和对己糖的广泛特异性不同于其细菌对应物。
