Neutron diffraction studies towards deciphering the protonation state of catalytic residues in the bacterial KDN9P phosphatase

Abstract

The enzyme 2-keto-3-deoxy-9-O-phosphonononic acid phosphatase (KDN9P phosphatase) functions in the pathway for the production of 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid, a sialic acid that is important for the survival of commensal bacteria in the human intestine. The enzyme is a member of the haloalkanoate dehalogenase superfamily and represents a good model for the active-site protonation state of family members. Crystals of approximate dimensions 1.5 × 1.0 × 1.0 mm were obtained in space group P2(1)2(1)2, with unit-cell parameters a = 83.1, b = 108.9, c = 75.7 Å. A complete neutron data set was collected from a medium-sized H/D-exchanged crystal at BIODIFF at the Heinz Maier-Leibnitz Zentrum (MLZ), Garching, Germany in 18 d. Initial refinement to 2.3 Å resolution using only neutron data showed significant density for catalytically important residues.

Keywords: KDN9P phosphatase; neutron diffraction; protonation states.

Figure 1

Active site of the KDN9PP–Mg²⁺–VO₃ ⁻–neuramic acid complex with the ligands and catalytic residues shown in stick representation and the Mg²⁺ cofactor shown as a magenta sphere. The X-ray coordinates are from Lu et al. (2009 ▶).

Figure 2

Photograph of an H/D-exchanged large single KDN9PP crystal grown at pH 8.5 (dimensions of ∼1.5 × 1.0 × 1.0 mm).

Figure 3

Representative neutron diffraction images collected from H/D-exchanged KDN9PP crystals. (a) Neutron Laue time-of-flight diffraction test image for KDN9PP collected at the Protein Crystallography Station at LANL and (b) monochromatic neutron diffraction pattern collected at BIODIFF at FRM II.

Figure 4

Representative nuclear density maps of His44 and Trp29 after rigid-body refinement against neutron data only. The exchanged D atoms are shown in white; 2F _o − F _c nuclear density is shown as a blue mesh and is contoured at 1σ.