Fedøy Anita-Elin, Yang Nannan, Martinez Aurora, Leiros Hanna-Kirsti S, Steen Ida Helene
Department of Biology, University of Bergen, P.O. Box 7800, Jahnebakken 5, N-5020 Bergen, Norway.
J Mol Biol. 2007 Sep 7;372(1):130-49. doi: 10.1016/j.jmb.2007.06.040. Epub 2007 Jun 19.
Isocitrate dehydrogenase (IDH) has been studied extensively due to its central role in the Krebs cycle, catalyzing the oxidative NAD(P)(+)-dependent decarboxylation of isocitrate to alpha-ketoglutarate and CO(2). Here, we present the first crystal structure of IDH from a psychrophilic bacterium, Desulfotalea psychrophila (DpIDH). The structural information is combined with a detailed biochemical characterization and a comparative study with IDHs from the mesophilic bacterium Desulfitobacterium hafniense (DhIDH), porcine (PcIDH), human cytosolic (HcIDH) and the hyperthermophilic Thermotoga maritima (TmIDH). DpIDH was found to have a higher melting temperature (T(m)=66.9 degrees C) than its mesophilic homologues and a suboptimal catalytic efficiency at low temperatures. The thermodynamic activation parameters indicated a disordered active site, as seen also for the drastic increase in K(m) for isocitrate at elevated temperatures. A methionine cluster situated at the dimeric interface between the two active sites and a cluster of destabilizing charged amino acids in a region close to the active site might explain the poor isocitrate affinity. On the other hand, DpIDH was optimized for interacting with NADP(+) and the crystal structure revealed unique interactions with the cofactor. The highly acidic surface, destabilizing charged residues, fewer ion pairs and reduced size of ionic networks in DpIDH suggest a flexible global structure. However, strategic placement of ionic interactions stabilizing the N and C termini, and additional ionic interactions in the clasp domain as well as two enlarged aromatic clusters might counteract the destabilizing interactions and promote the increased thermal stability. The structure analysis of DpIDH illustrates how psychrophilic enzymes can adjust their flexibility in dynamic regions during their catalytic cycle without compromising the global stability of the protein.
异柠檬酸脱氢酶（IDH）因其在三羧酸循环中的核心作用而受到广泛研究，它催化异柠檬酸依赖NAD(P)(+)的氧化脱羧反应生成α-酮戊二酸和CO(2)。在此，我们展示了来自嗜冷细菌嗜冷脱硫弧菌（DpIDH）的IDH的首个晶体结构。该结构信息与详细的生化特性分析以及与嗜温细菌哈氏脱硫肠状菌（DhIDH）、猪（PcIDH）、人胞质（HcIDH）和嗜热栖热菌（TmIDH）的IDH的比较研究相结合。发现DpIDH的解链温度（T(m)=66.9℃）高于其嗜温同源物，且在低温下催化效率欠佳。热力学活化参数表明其活性位点无序，这在高温下异柠檬酸的K(m)急剧增加时也有体现。位于两个活性位点之间二聚体界面处的甲硫氨酸簇以及靠近活性位点区域中一组不稳定的带电荷氨基酸可能解释了其对异柠檬酸亲和力较差的原因。另一方面，DpIDH针对与NADP(+)相互作用进行了优化，晶体结构揭示了与该辅因子的独特相互作用。DpIDH高度酸性的表面、不稳定的带电荷残基、较少的离子对以及减小的离子网络尺寸表明其整体结构具有灵活性。然而，稳定N和C末端的离子相互作用的策略性定位、扣环结构域中的额外离子相互作用以及两个扩大的芳香族簇可能抵消了不稳定相互作用并促进了热稳定性的提高。DpIDH的结构分析说明了嗜冷酶如何在催化循环过程中在动态区域调节其灵活性而不损害蛋白质的整体稳定性。
