PDF(Pubmed)
Abstract：
The nickel-pincer nucleotide (NPN) cofactor discovered in lactate racemase from Lactiplantibacillus plantarum (LarALp) is essential for the activities of racemases/epimerases in the highly diverse LarA superfamily. Prior mechanistic studies have established a proton-coupled hydride-transfer mechanism for LarALp, but direct evidence showing that hydride attacks the C4 atom in the pyridinium ring of NPN has been lacking. Here, we show that sodium borohydride (NaBH4) irreversibly inactivates LarALp accompanied by a rapid color change of the enzyme. The altered ultraviolet-visible spectra during NaBH4 titration supported hydride transfer to C4 of NPN, and the concomitant Ni loss unraveled by mass spectrometry experiments accounted for the irreversible inactivation. High resolution structures of LarALp revealed a substantially weakened C-Ni bond in the metastable sulfite-NPN adduct where the NPN cofactor is in the reduced state. These findings allowed us to propose a mechanism of LarALp inactivation by NaBH4 that provides key insights into the enzyme-catalyzed reaction and sheds light on the reactivity of small molecule NPN mimetics.
摘要：
在植物乳杆菌（LarALp）的乳酸消旋酶中发现的镍钳核苷酸（NPN）辅因子对于高度多样化的LarA超家族中消旋酶/差向异构酶的活性至关重要。先前的机理研究已经为LarALp建立了质子耦合的氢化物转移机制，但是缺乏直接证据表明氢化物攻击NPN吡啶环上的C4原子。这里,我们表明硼氢化钠（NaBH4）不可逆地失活LarALp，伴随着酶的快速颜色变化。NaBH4滴定过程中改变的紫外可见光谱支持氢化物转移到NPN的C4，通过质谱实验揭示的伴随的Ni损失是不可逆失活的原因。LarALp的高分辨率结构揭示了亚稳态亚硫酸盐-NPN加合物中的C-Ni键明显减弱，其中NPN辅因子处于还原状态。这些发现使我们提出了NaBH4使LarALp失活的机制，该机制为酶催化反应提供了关键见解，并揭示了小分子NPN模拟物的反应性。
