DataSheet1_An insight into the role of the N-terminal domain of Salmonella CobB in oligomerization and Zn2+ mediated inhibition of the deacetylase activity.docx

Prokaryotic deacetylases are classified into nicotinamide adenine dinucleotide (NAD+)-dependent sirtuins and Zn2+-dependent deacetylases. NAD+ is a coenzyme for redox reactions, thus serving as an essential component for energy metabolism. The NAD+-dependent deacetylase domain is quite conserved and well characterized across bacterial species like CobB in Escherichia coli and Salmonella, Rv1151c in Mycobacterium, and SirtN in Bacillus subtilis. E. coli CobB is the only bacterial deacetylase with a known crystal structure (PDB ID: 1S5P), which has 91% sequence similarity with Salmonella CobB (SeCobB). Salmonella encodes two CobB isoforms, SeCobBS and SeCobBL, with a difference of 37 amino acids in its N-terminal domain (NTD). The hydrophobic nature of NTD leads to the stable oligomerization of SeCobBL. The homology modeling-based predicted structure of SeCobB showed the presence of a zinc-binding motif of unknown function. Tryptophan fluorescence quenching induced by ZnCl2 showed that Zn2+ has a weak interaction with SeCobBS but higher binding affinity toward SeCobBL, which clearly demonstrated the crucial role of NTD in Zn2+ binding. In the presence of Zn2+, both isoforms had significantly reduced thermal stability, and a greater effect was observed on SeCobBL. Dynamic light scattering (DLS) studies reflected a ninefold increase in the scattering intensity of SeCobBL upon ZnCl2 addition in contrast to an ∼onefold change in the case of SeCobBS, indicating that the Zn2+ interaction leads to the formation of large particles of SeCobBL. An in vitro lysine deacetylase assay showed that SeCobB deacetylated mammalian histones, which can be inhibited in the presence of 0.25–1.00 mM ZnCl2. Taken together, our data conclusively showed that Zn2+ strongly binds to SeCobBL through the NTD that drastically alters its stability, oligomeric status, and enzymatic activity in vitro.

原核生物脱乙酰酶可分为烟酰胺腺嘌呤二核苷酸（NAD+）依赖型脱乙酰酶与锌离子（Zn²+）依赖型脱乙酰酶两类。NAD+是氧化还原反应的辅酶，因此是能量代谢的必需组分。NAD+依赖型脱乙酰酶结构域在大肠杆菌（Escherichia coli）、沙门氏菌的CobB、分枝杆菌的Rv1151c以及枯草芽孢杆菌的SirtN等细菌物种中高度保守且研究较为透彻。大肠杆菌CobB是目前唯一已知晶体结构的细菌脱乙酰酶（PDB ID: 1S5P），其与沙门氏菌CobB（SeCobB）的序列相似性达91%。沙门氏菌编码两种CobB同工型：SeCobBS与SeCobBL，二者的N端结构域（NTD）存在37个氨基酸的差异。NTD的疏水特性使得SeCobBL能够稳定寡聚化。基于同源建模得到的SeCobB预测结构显示，其含有一个功能未知的锌结合基序。氯化锌（ZnCl₂）诱导的色氨酸荧光猝灭实验表明，Zn²+与SeCobBS的相互作用较弱，但对SeCobBL的结合亲和力更高，这明确证实了NTD在Zn²+结合过程中的关键作用。在Zn²+存在的条件下，两种同工型的热稳定性均显著降低，且对SeCobBL的影响更为显著。动态光散射（DLS）实验结果显示，添加氯化锌后，SeCobBL的散射强度提升了9倍，而SeCobBS的散射强度仅变化约1倍，这表明Zn²+的相互作用会促使SeCobBL形成更大的颗粒。体外赖氨酸脱乙酰酶活性测定实验表明，SeCobB能够使哺乳动物组蛋白发生去乙酰化，且该过程可被0.25~1.00 mM的氯化锌抑制。综上，本研究数据证实：Zn²+可通过NTD与SeCobBL强效结合，进而在体外显著改变其稳定性、寡聚状态与酶活性。