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.

Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix

“…As the neutron scattering cross-section varies by element (or isotope) in a nonlinear fashion, the scattering lengths of light atoms such as hydrogen and deuterium (D) atoms are similar to those of the heavier atoms (C, O and N). It is therefore possible to locate H (or D) atoms and to deduce their protonation states; this knowledge helps in understanding catalytic mechanisms and ligand binding (Yamaguchi et al, 2009;Bryan et al, 2013;Knihtila et al, 2015). Furthermore, the neutron scattering length can be positive or negative (it is always positive for X-rays).…”

Order–Disorder Phenomenon of Nematogens at Molecular Level—A Computational Approach

“…It is well known that subtle alternations in molecular structure can have profound effects on the stability and properties of mesophases. The molecular geometries of nOCAC (n = 2,4) have been constructed on the basis of published crystallographic data [11] with the standard values of bond lengths and bond angles. The structures of the systems have the all-trans extended conformation, and the molecules exist in the crystals as planar hydrogen-bonded dimers.…”

“…The configurationally probability and entropy distribution picture based on the interaction energy results will provide valuable information in this respect. The molecules 2OCAC and 4OCAC show nematic to isotropic transition at 471 K, and 460.5 K, respectively [11].…”

Constant-pH Molecular Dynamics Simulations of Closed and Open States of a Proton-gated Ion Channel

“…This problem is not limited to GLIC, but applies to pH-dependent macromolecular systems in general. Even with methods such as neutron diffraction that theoretically can resolve protons [26], the local electrostatic environment in a crystal or on a grid is not guaranteed to correspond to the cellular environment. Given these experimental limitations, computational methods such as molecular dynamics (MD) simulations may provide deeper insight.…”