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

Bryan T, Gonzalez J, Bacik J, DeNunzio N, Unkefer C, Schrader T, Ostermann A, Dunaway-Mariano D, Allen KN, Fisher Z (2013) Acta Crystallogr Sect F F69, 1015–1019. PMCID: PMC3758152

In this collaboration between the NIH's Neutron Diffraction Center at Los Alamos National Laboratories and the EFI's HAD Bridging Project, the protonation states of residues that form the conserved catalytic motif of a HAD phosphatase, 2-keto-3-deoxy-9-O-phosphonononic acid phosphatase, were examined by neutron diffraction. The data collected will enable discovery of ionization states of specific residues and hydrogen bonding patterns in the active site. This knowledge will facilitate the development of superior docking protocols for computationally-based substrate (and therefore function) prediction.

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 P21212, 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.

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2013 Bryan 01Figure 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[Lu, Z., Wang, L., Dunaway-Mariano, D. & Allen, K. N. (2009). J. Biol. Chem. 284, 1224-1233.]).

2013 Bryan 02Figure 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).

2013 Bryan 03Figure 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.

2013 Bryan 04Figure 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; 2Fo - Fc nuclear density is shown as a blue mesh and is contoured at 1[sigma].

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