Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, pr. Nauki 5, 142290 Pushchino, Moscow Region, Russia.
Biochemistry (Mosc). 2012 Apr;77(4):372-7. doi: 10.1134/S0006297912040086.
In the cluster of genes for sucrose biosynthesis and cleavage in Methylomicrobium alcaliphilum 20Z, a gene whose encoded sequence showed high similarity to sugar kinases of the ribokinase family was found. By heterologous expression of this gene in Escherichia coli cells and following metal chelate affinity chromatography, the electrophoretically homogenous recombinant enzyme with six histidine residues on the C-end was obtained. The enzyme catalyzes ATP-dependent phosphorylation of fructose into fructose-6-phosphate but is not active with other sugars as phosphoryl acceptors. The fructokinase of M. alcaliphilum 20Z is most active in the presence of Mn(2+) at pH 9.0 and 60°C, being inhibited by ADP (K(i) = 2.50 ± 0.03 mM). The apparent K(m) values for fructose and ATP are 0.26 and 1.3 mM, respectively; the maximal activity is 141 U/mg protein. The enzyme shows the highest similarity of translated amino acid sequence with putative fructokinases of methylotrophic and autotrophic proteobacteria whose fruK gene is located in the gene cluster of sucrose biosynthesis. The involvement of fructokinase in sucrose metabolism in M. alcaliphilum 20Z and other methanotrophs and autotrophs is discussed.
在产碱甲基杆菌 20Z 中蔗糖生物合成和分解簇的基因中，发现了一个基因，其编码序列与核酮糖激酶家族的糖激酶具有高度相似性。通过在大肠杆菌细胞中异源表达该基因，并进行金属螯合亲和层析，获得了在 C 末端带有六个组氨酸残基的电泳均一的重组酶。该酶催化 ATP 依赖性磷酸化果糖生成果糖-6-磷酸，但不以其他糖作为磷酸受体而发挥活性。产碱甲基杆菌 20Z 的果糖激酶在 pH 9.0 和 60°C 时以 Mn(2+)存在时最活跃，被 ADP 抑制（K(i) = 2.50 ± 0.03 mM）。果糖和 ATP 的表观 K(m)值分别为 0.26 和 1.3 mM；最大活性为 141 U/mg 蛋白。该酶与甲基营养菌和自养菌的推定果糖激酶具有翻译氨基酸序列的最高相似性，其 fruK 基因位于蔗糖生物合成基因簇中。讨论了果糖激酶在产碱甲基杆菌 20Z 和其他甲烷营养菌和自养菌中的蔗糖代谢中的作用。
