Expression, purification, crystallization and preliminary X-ray analysis of a nucleoside kinase from the hyperthermophile Methanocaldococcus jannaschii

Abstract

Methanocaldococcus jannaschii nucleoside kinase (MjNK) is an ATP-dependent non-allosteric phosphotransferase that shows high catalytic activity for guanosine, inosine and cytidine. MjNK is a member of the phosphofructokinase B family, but participates in the biosynthesis of nucleoside monophosphates rather than in glycolysis. MjNK was crystallized as the apoenzyme as well as in complex with an ATP analogue and Mg2+. The latter crystal form was also soaked with fructose-6-phosphate. Synchrotron-radiation data were collected to 1.70 A for the apoenzyme crystals and 1.93 A for the complex crystals. All crystals exhibit orthorhombic symmetry; however, the apoenzyme crystals contain one monomer per asymmetric unit whereas the complex crystals contain a dimer.

Figure 1

Orthorhombic crystals of M. jannaschii nucleoside kinase showing (a) the apo form (MjNK-apo) and (b) the enzyme in complex with the ATP analogue AMPPNP and MgCl₂. The crystal sizes for MjNK-apo and MjNK-A were 0.1 × 0.3 × 0.3 and 0.2 × 0.2 × 0.2 mm, respectively.

Figure 2

Stereographic plot of the self-rotation function at the section κ = 180° calculated from the MjNK-AF data. The integration radius is between 15 and 5 Å and the resolution limits are 10–4 Å. Contour lines are drawn in steps of 10%. Only high peaks arising from crystallographic symmetry can be seen, which indicates that the twofold non-crystallographic axis is parallel to a crystallographic axis. The plot was produced using POLARRFN (Collaborative Computational Project, Number 4, 1994 ▶).

Figure 3

Native Patterson function calculated from all data from the MjNK-AF complex. The plot shows fractional coordinates of u and w ranging from 0.0 to 0.5 at Harker section v = 0.5. An intense pseudo-origin peak appears at (u, w) = (0.0, 0.0). Grid lines are drawn every 0.10 units and contours are drawn every 0.5% of the origin peak hight, starting at 1.5%. The figure was prepared with the programs NPO and PLTDEV from the CCP4 suite (Collaborative Computational Project, Number 4, 1994 ▶).