The Norwegian Structural Biology Center (NorStruct), Department of Chemistry, University of Tromsø, N-9037 Tromsø, Norway.
Comp Biochem Physiol B Biochem Mol Biol. 2012 Jan;161(1):60-8. doi: 10.1016/j.cbpb.2011.09.007. Epub 2011 Sep 18.
Uracil-DNA N-glycosylase (UNG; EC 3.2.2.27) from Atlantic cod (cUNG) possesses cold adapted features like increased catalytic efficiency and reduced temperature optimum for activity compared to its warm-adapted homologue human UNG (hUNG). Here, we present the first thermal stability analysis of cUNG and hUNG by differential scanning calorimetry (DSC), and the results showed that cUNG is less stable than hUNG and unfolds at a melting temperature (T(m)) 9° lower than its warm-adapted homologue. In addition, an ion-pair (D183-K302) suggested to be crucial for global stability of hUNG was investigated by biochemical characterization and DSC of four mutants (cUNG G183D and cUNG G183D-R302K, hUNG D183G and hUNG D183G-K302R). The hUNG mutants with an expected disruption of the ion-pair showed a slight increase in stability with concomitant reduction in the enzyme activity, while the apparent introduction of the ion-pair in cUNG caused a reduction in the enzyme activity but no increase in stability. Because the mutants did not behave as expected, the phenomenon was further investigated by crystal structure determination. Indeed, the crystal structure of the hUNG D183G-K302R mutant revealed that compensating interactions for the loss of the ion-pair were generated close to and in regions distant from the mutation site. In conclusion, the reduced stability of cUNG supports the suggested requirement of a flexible structure for improved activity at low temperatures. Furthermore, the lack of a direct correlation between enzyme activity and global stability of the mutants supports the significance of distributing locally flexible and/or rigid regions for modulation of enzyme activity.
尿嘧啶-DNA N-糖基化酶（UNG；EC 3.2.2.27）来自大西洋鳕鱼（cUNG），与它的温暖适应同源物人 UNG（hUNG）相比，具有冷适应特征，如催化效率提高和最适温度降低。在这里，我们通过差示扫描量热法（DSC）首次对 cUNG 和 hUNG 进行了热稳定性分析，结果表明 cUNG 不如 hUNG 稳定，其解链温度（T(m)）比温暖适应的同源物低 9°C。此外，通过生化特性和四个突变体（cUNG G183D 和 cUNG G183D-R302K、hUNG D183G 和 hUNG D183G-K302R）的 DSC 研究了一个离子对（D183-K302），该离子对被认为对 hUNG 的整体稳定性至关重要。预计该离子对的破坏会导致 hUNG 突变体的稳定性略有增加，同时酶活性降低，而 cUNG 中引入该离子对会导致酶活性降低，但稳定性没有增加。由于突变体的行为不符合预期，因此通过晶体结构确定进一步研究了该现象。事实上，hUNG D183G-K302R 突变体的晶体结构表明，在靠近突变位点和远离突变位点的区域产生了补偿相互作用，以弥补离子对的损失。总之，cUNG 的稳定性降低支持了在低温下提高活性需要灵活结构的假设。此外，突变体的酶活性和整体稳定性之间缺乏直接相关性，支持了在局部灵活和/或刚性区域分布对于调节酶活性的重要性。
