Department of Microbiology and Molecular Genetics, 6193 Biomedical Physical Sciences, Michigan State University, East Lansing, MI 48824-4320, USA.
J Bacteriol. 2010 May;192(9):2294-304. doi: 10.1128/JB.01426-09. Epub 2010 Mar 5.
Assembly of the Klebsiella aerogenes urease metallocenter requires four accessory proteins, UreD, UreE, UreF, and UreG, to effectively deliver and incorporate two Ni2+ ions into the nascent active site of the urease apoprotein (UreABC). Each accessory protein has been purified and characterized with the exception of UreD due to its insolubility when it is overproduced in recombinant cells. In this study, a translational fusion was made between the maltose binding protein (MBP) and UreD, with the resulting MBP-UreD found to be soluble in Escherichia coli cell extracts and able to complement a DeltaureD-urease cluster in this host microorganism. MBP-UreD was purified as a large multimer (> 670 kDa) that bound approximately 2.5 Ni2+ ions (K(d) of approximately 50 microM, where K(d) is the dissociation constant) per UreD protomer according to equilibrium dialysis measurements. Zn2+ directly competes with 10-fold higher affinity (approximately 4 Zn2+ ions per protomer; K(d) of 5 microM) for the Ni2+ binding sites. MBP pulldown experiments demonstrated that the UreD domain of MBP-UreD formed in vivo complexes with UreF, UreG, UreF plus UreG, or UreABC when these proteins were overproduced in the same E. coli cells. In addition, a UreABC-(MBP-UreD)-UreFG complex was observed in cells producing all urease components. Comparative in vitro binding experiments with purified proteins demonstrated an approximate 1:1 binding ratio between the UreD domain of MBP-UreD and the UreF domain of the UreEF fusion, only weak or transient interaction between MBP-UreD and UreG, and no binding with UreABC. These studies are the first to describe the properties of purified UreD, and they extend our understanding of its binding partners both in vitro and in the cell.
产碱假单胞菌脲酶金属中心的组装需要四个辅助蛋白，即 UreD、UreE、UreF 和 UreG，以便有效地将两个 Ni2+离子递送到脲酶脱辅基蛋白（UreABC）的新生活性部位并将其掺入其中。除了 UreD 之外，所有辅助蛋白都已被纯化和表征，因为在重组细胞中过量表达时，UreD 是不溶的。在这项研究中，将麦芽糖结合蛋白（MBP）与 UreD 进行了翻译融合，结果发现 MBP-UreD 在大肠杆菌细胞提取物中是可溶性的，并能够在该宿主微生物中补充 DeltaureD-脲酶簇。MBP-UreD 作为一个大的多聚体（>670 kDa）被纯化，根据平衡透析测量，每个 UreD 原聚体结合约 2.5 Ni2+离子（K(d)约为 50 microM，其中 K(d)是解离常数）。Zn2+ 直接以高 10 倍的亲和力（每个原聚体约 4 Zn2+离子；K(d)为 5 microM）与 Ni2+结合位点竞争。MBP 下拉实验表明，当这些蛋白质在相同的大肠杆菌细胞中过量表达时，MBP-UreD 的 UreD 结构域在体内与 UreF、UreG、UreF 加 UreG 或 UreABC 形成复合物。此外，在产生所有脲酶成分的细胞中观察到 UreABC-(MBP-UreD)-UreFG 复合物。与纯化蛋白进行的比较体外结合实验表明，MBP-UreD 的 UreD 结构域与 UreEF 融合的 UreF 结构域之间的结合比约为 1:1，MBP-UreD 与 UreG 之间仅存在弱或瞬时相互作用，并且与 UreABC 没有结合。这些研究首次描述了纯化的 UreD 的性质，并扩展了我们对其在体外和细胞内结合伙伴的理解。
