Preliminary crystallographic studies of purine nucleoside phosphorylase from the cariogenic pathogen Streptococcus mutans

Abstract

The punA gene of the cariogenic pathogen Streptococcus mutans encodes purine nucleoside phosphorylase (PNP), which is a pivotal enzyme in the nucleotide-salvage pathway, catalyzing the phosphorolysis of purine nucleosides to generate purine bases and alpha-ribose 1-phosphate. In the present work, the PNP protein was expressed in Escherichia coli strain BL21 (DE3) in a soluble form at a high level. After purification of the PNP enzyme, the protein was crystallized using the sitting-drop vapour-diffusion technique; the crystals diffracted to 1.6 A resolution at best. The crystals belonged to space group H3, with unit-cell parameters a = b = 113.0, c = 60.1 A.

Figure 1

Chromatogram elution profile of S. mutans PNP (SmPNP) on a Superdex 75 gel-filtration column. The absorption curves of gel-filtration standard proteins (Bio-Rad Laboratories, USA) and SmPNP are shown as dashed and solid lines, respectively.

Figure 2

Crystals of S. mutans PNP protein grown by the sitting-drop vapour-diffusion method using a reservoir solution containing 25%(w/v) PEG 3350, 0.15 M ammonium sulfate and 0.1 M bis-tris pH 6.3.