Agnes Rinaldo-Matthis
PI: Nordlund (SU)
Project: Structural studies on protein phosphatases
Funding ended: 31 December, 2000
Background
Most organisms can synthesise nucleotides in sufficient amounts for their needs from low-molecular weight precursors, the so-called de novo pathway. Nucleotides can also be synthesised by the salvage pathway, where the nucleotides are made from nucleosides or nucleobases that becomes available via the diet or through enzymatic degradation. The substrates in the salvage pathway are the nucleosides or the nucleobases, which permeates the plasma membrane or is taken up by transporters, when the concentration in the cell is higher than outside the cell. When the concentration gradient is in the opposite direction the nucleosides enters the cell and is trapped by the phosphorylation of nucleoside kinase enzymes. The nucleotides are then phosphorylated by nucleoside monophosphate kinases and nucleoside diphosphate kinases to its triphosphate form.
In mammalian cells there is a pool of deoxyribonucleotide triphosphates (dNTPs) that are regulated according to the needs for DNA synthesis. In the S-phase the dNTP pool expand and during the interphase the dNTP pool is low. The anabolic and catabolic enzymes, the deoxynucleoside kinase and the nucleotidase regulate the regulation of the dNTP pool. The two enzymes reverses the effects of each other and both of the reactions are irreversible. The nucleotidase catalyses a reaction where the nucleoside is formed which can readily pass through the plasma membrane and be excreted when the concentration is higher in the cell and the reverse when it is higher outside the cell. As soon as the deoxyribonucleoside kinase catalysed the reaction where a phosphate is added, the mononucleotide is trapped in the cell. The nucleotidases are responsible for the dephosphorylation of nucleotides to nucleosides. It catalyses the hydrolysis of phosphate esterified at carbon 5 of the ribose or deoxyribose portions of the nucleotide molecules. Nucleotidases display significant differences in the range of substrate hydrolysed, the substrate specificity and also their presence in different cellular locations. Nucleotidases are found both in the soluble and in the membrane-bound form. The family of 5nucleotidases can hydrolyse the 5purine and 5pyrimidine mononucleotides, UDP-glucose and FAD but the common denominator is the ability to hydrolyse the 5nucleotides.
The 53deoxynucleotidase (53dNT) subject for this study is the only 5nucleotidase known that prefer the deoxy form of the 5nucleotides. It is believed that the 53dNT plays an important role in regulating the nucleotide pool in the cell during DNA-synthesis. Activity of 5-nucleotidases has been described for bacteria, plants and in vertebrate tissues. Relevant for this study is the mammalian and the bacterial nucleotidases.
In mammalian systems there has been described three major classes of 5nucleotidases, a membrane-bound nucleotidase called ecto-5-nucleotidase and two soluble 5nucleotidase. The most studied nucleotidase is the ecto-5-nucleotidase also called CD73. The ecto-5-nucleotidase plays an important role in T-lymphocyte differentiation. One of the soluble enzymes is known as the high Km nucleotidase. The high Km nucleotidase has phosphotransferase activity and transfers a phosphate group from a 5-nucleotidase to the 5 position of a nucleoside.
The third enzyme, called the 53-deoxyribonucleotidase, 53dNT dephosphorylates specifically the 5- and 2(3)-phosphates of uracil and thymine deoxyribonucleotides. The enzyme is thought to be important in regulating the pool of deoxyribonucleotides in the mitochondria. Deoxyribonucleotides are imported into the mitochondria and are further metabolised either to become DNA or degraded to deoxyribonucleosides where the 53dNT is involved. A deoxyribonucleotidase in the cytosol has also been discovered. They seem to act in parallel in different cell compartments and their sequences are 52% identical.
Project
Crystals of 53dNT were grown by hanging drop vapour diffusion by mixing 1 ul protein (13.2 mg/ml) with 1 ul reservoir solution containing 20% PEG 8000 and 0.05 M K2H2PO4 Ph 3.8. Crystals belong to the space-group of P43212 and with 1 molecule per asymmetric unit. Cell-dimensions a = 73.9 Å, b = 73.9 Å, c = 105.9 Å. Substrate soaks were also made with inhibitors. The phases were obtained by MAD phasing with protein containing Hg as anomalous scatterer. The structure was determined to a resolution of 1.8 Å. The final model contained 193 amino acids and it was refined to an R-free and R-factor of 23% and 21% respectively. The structure of the inhibitor was solved to a resolution of 1.4 Å using molecular replacement with phases from the native structure. It consists of 194 amino acids with R-factor of 20.9% and R-free of 22.4%.
Publications
* A Rinaldo-Matthis, C Rampazzo, V Bianchi & P Nordlund (2002). Crystal structures of the native and the inhibitor-bound forms of the human mitochondrial deoxyribonucleotidase. Manuscript in preparation.
* XD Su, ME Andersson, A Rinaldo-Matthis, W Blodig, BO Persson, BM Sjöberg & P Nordlund (2002). Protonation behaviour of carboxylate residues in the interior of proteins: Structural and mutagenesis studies of the carboxylate cluster of ribonucleotide reductase R2. Submitted.
Latest update at 29 March, 2001.