STRUCTURE OF 5'-NUCLEOTIDASE FROM E. COLI CONTAINING A DIZINC ACTIVE SITE
Thomas Knöfel and Norbert Sträter
Institut für Kristallographie, Freie Universität Berlin, Takustr. 6, D-14195 Berlin, Germany knoefel@chemie.fu-berlin.de, strater@chemie.fu-berlin.de
The ushA gene of E. coli encodes a UDP-sugar hydrolase, which also possesses 5'-nucleotidase activity [1-3]. The zinc-containing enzyme is secreted and localised in the periplasm, and might catalyse the degradation of external UDP-glucose to uridine, glucose-1-phosphate and phosphate for utilization by the cell. The enzyme is widely distributed in bacteria and plant cells as well as in vertebrate tissues. Whereas cytosolic 5'-nucleotidase activity controls intracellular levels of nucleoside 5'-mononucleotides, the surface-located enzyme is a a major contributor to the cascade that hydrolyses extracellular ATP of pharmacological interest [4]. These extracellular enzyme forms have also been implicated in cell-matrix and cell-cell interactions and in transmembrane signalling.
We have crystallized the monomeric 58.1 kDa 5'-nucleotidase of E. coli following homologous overexpression and enzyme purification in an orthorhombic and a tetragonal lattice. The former crystals grew only in the presence of sodium tungstate and zinc sulfate. Structure determination of the orthorhombic crystal form has been carried out by multiple-wavelength anomalous-dispersion technique by using the anomalous scattering contribution of the tungsten and zinc sites. Five datasets were collected at different wavelength at beamline BM14 at the ESRF. In order to obtain interpretable maps the contribution from two relatively poor isomorphous heavy atom derivatives was included in the phasing procedure. The failure to obtain interpretable maps using the MAD-phases alone is attributed to a strong disorder of at least three tungstate binding sites in close proximity, thus limiting the phasing power of the high resolution data. The structure has been refined to 2.2 A resolution in both crystal forms.
5'-nucleotidase consists of two domains connected by an a-helix. The N-terminal domain bears the ligands to the zinc ions, which are about 3.2 A apart. This domain is distantly related to other phosphoesterases containing a dinuclear metal center [5], including the Ser/Thr protein phosphatases [6-9] and purple acid phosphatases [10] for which crystal structures have been determined. In contrast to these phosphatases which have broad active site pockets, access to the active site in the nucleotidase is limited to smaller substrates since the active site is located in a shallow cleft formed by both domains. Residues of the C-terminal domain probably also form part of the active site and contribute to substrate specificity.
Fig.: Fold of 5'-nucleotidase with the N-terminal domain in
blue, the C-terminal domain in green, and a connecting helix in
red. The two zinc ions in the active site are colored yellow.