Background Previous studies have highlighted the catalytic activity of Escherichia coli alcohol dehydrogenase YahK in the presence of coenzyme nicotinamide adenine dinucleotide (NAD) and metal zinc. Notably, competitive interaction between iron and zinc ligands has been shown to influence the catalytic efficiency of several key proteases. This study aims to unravel the intricate mechanisms underlying YahK’s catalytic action, with a particular focus on the pivotal roles played by metal ions zinc and iron. Methods The purified YahK protein from E. coli cells cultivated in LB medium was utilized to investigate its metal-binding properties through UV-visible absorption measurements and determination of metal content. Subsequently, the effects of excess zinc and iron on the metal-binding ability and alcohol dehydrogenase activity of the YahK protein were explored using M9 minimal medium. Furthermore, site-directed mutagenesis technology was employed to determine the iron-binding site location within the YahK protein. Polyacrylamide gel electrophoresis was conducted to examine the relationship between iron and zinc with respect to the YahK protein. Results The study confirmed the presence of iron and zinc in the YahK protein, with the zinc-bound form exhibiting enhanced catalytic activity in alcohol dehydrogenation reactions. Conversely, the presence of iron appears to play a pivotal role in maintaining overall stability of the YahK protein. Furthermore, experimental findings indicate that excessive zinc within M9 minimal medium can competitively bind to iron-binding sites on YahK, thereby augmenting its alcohol dehydrogenase activity. Conclusion The dynamic binding of YahK to iron and zinc unveils its intricate regulatory mechanism as an alcohol dehydrogenase, thereby highlighting the possible physiological role of YahK in E. coli and its significance in governing cellular metabolic processes. This discovery provides a novel perspective for further investigating the specific impact of metal ion binding on YahK and E. coli cell metabolism.

中文翻译：

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大肠杆菌乙醇脱氢酶 YahK 是一种结合铁和锌的蛋白质


背景 先前的研究强调了大肠杆菌醇脱氢酶 YahK 在辅酶烟酰胺腺嘌呤二核苷酸 (NAD) 和金属锌存在下的催化活性。值得注意的是，铁和锌配体之间的竞争性相互作用已被证明会影响几种关键蛋白酶的催化效率。本研究旨在揭示 YahK 催化作用的复杂机制，特别关注金属离子锌和铁所发挥的关键作用。方法利用LB培养基中培养的大肠杆菌细胞纯化的YahK蛋白，通过紫外-可见光吸收测量和金属含量测定来研究其金属结合特性。随后，使用 M9 基本培养基探讨了过量的锌和铁对 YahK 蛋白的金属结合能力和乙醇脱氢酶活性的影响。此外，采用定点诱变技术来确定 YahK 蛋白内铁结合位点的位置。进行聚丙烯酰胺凝胶电泳来检查铁和锌与 YahK 蛋白之间的关系。结果 研究证实了 YahK 蛋白中铁和锌的存在，锌结合形式在醇脱氢反应中表现出增强的催化活性。相反，铁的存在似乎在维持 YahK 蛋白的整体稳定性方面发挥着关键作用。此外，实验结果表明，M9 基本培养基中过量的锌可以竞争性地结合 YahK 上的铁结合位点，从而增强其乙醇脱氢酶活性。 结论 YahK 与铁和锌的动态结合揭示了其作为乙醇脱氢酶的复杂调节机制，从而凸显了 YahK 在大肠杆菌中可能的生理作用及其在控制细胞代谢过程中的重要性。这一发现为进一步研究金属离子结合对YahK和大肠杆菌细胞代谢的具体影响提供了新的视角。