Lonhienne T, Zoidakis J, Vorgias C E, Feller G, Gerday C, Bouriotis V
Laboratory of Biochemistry, Institute of Chemistry B6, University of Liege, Liege, B-4000, Belgium.
J Mol Biol. 2001 Jul 6;310(2):291-7. doi: 10.1006/jmbi.2001.4774.
The gene archb encoding for the cell-bound chitobiase from the Antarctic Gram-positive bacterium Arthrobacter sp. TAD20 was cloned and expressed in Escherichia coli in a soluble form. The mature chitobiase ArChb possesses four functionally independent domains: a catalytic domain stabilized by Ca(2+), a galactose-binding domain and an immunoglobulin-like domain followed by a cell-wall anchorage signal, typical of cell-surface proteins from Gram-positive bacteria. Binding of saccharides was analyzed by differential scanning calorimetry, allowing to distinguish unequivocally the catalytic domain from the galactose-binding domain and to study binding specificities. The results suggest that ArChb could play a role in bacterium attachment to natural hosts. Kinetic parameters of ArChb demonstrate perfect adaptation to catalysis at low temperatures, as shown by a low activation energy associated with unusually low K(m) and high k(cat) values. Thermodependence of these parameters indicates that discrete amino acid substitutions in the catalytic center have optimized the thermodynamic properties of weak interactions involved in substrate binding at low temperatures. Microcalorimetry also reveals that heat-lability, a general trait of psychrophilic enzymes, only affects the active site domain of ArChb.
编码来自南极革兰氏阳性菌节杆菌属TAD20的细胞结合型壳二糖酶的基因archb被克隆，并以可溶形式在大肠杆菌中表达。成熟的壳二糖酶ArChb具有四个功能独立的结构域：一个由Ca(2+)稳定的催化结构域、一个半乳糖结合结构域、一个免疫球蛋白样结构域，随后是一个细胞壁锚定信号，这是革兰氏阳性菌细胞表面蛋白的典型特征。通过差示扫描量热法分析糖类的结合情况，从而能够明确区分催化结构域和半乳糖结合结构域，并研究结合特异性。结果表明，ArChb可能在细菌附着于天然宿主的过程中发挥作用。ArChb的动力学参数表明其在低温下对催化具有完美的适应性，这表现为与异常低的K(m)值和高的k(cat)值相关的低活化能。这些参数的热依赖性表明，催化中心的离散氨基酸取代优化了低温下底物结合所涉及的弱相互作用的热力学性质。微量热法还表明，热不稳定性作为嗜冷酶的一个普遍特征，仅影响ArChb的活性位点结构域。
