Hen egg-white lysozyme is an enzyme that cleaves the 1,4-β-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglycan. Its enzymatic mechanism has been studied for many decades, and it has become a model system in crystallographic studies. Despite the plethora of atomic-resolution crystal structures of lysozyme obtained by X-ray diffraction over the years, its enzymatic mechanism remains controversial. There is ambiguity regarding the interaction between the catalytic residue Asp52 and the intermediate species formed after cleaving the substrate’s glycosidic bond. To answer this question, it is crucial to define lysozyme’s active site in near-physiological conditions, including protonation states of the catalytic residues Glu35 and Asp52, and hydrogen-bond interactions with nearby residues and water molecules. In this regard, neutron diffraction provides unique and complementary information compared to X-ray diffraction.
X-ray diffraction is relatively insensitive to hydrogen atoms because of their low electron density. On the other hand, neutron diffraction provides information on hydrogen positions, particularly for its isotope deuterium which has a neutron coherent scattering length comparable to carbon, nitrogen and oxygen atoms. The low flux of neutron beam sources compared to those of X-ray sources requires the use of large protein crystals in neutron diffraction. Additionally, due to the high background from the neutron incoherent scattering of hydrogen, its partial or complete substitution for deuterium is necessary.
Ramos et al [1] produced recombinant perdeuterated lysozyme (where most protein hydrogen atoms were replaced by deuterium) and obtained large crystals at the Deuteration Laboratory of the ILL. At the ILL’s D19 instrument, the authors collected neutron diffraction data at room temperature from a 4 mm3 perdeuterated lysozyme crystal, and from a 6 mm3 crystal of native lysozyme soaked in deuterated solution (replacing about 25% of all protein hydrogens by deuterium). The authors also collected X-ray diffraction data at the ESRF’s MASSIF-1 beamline for comparative analyses.
The atomic-resolution crystal structures of lysozyme obtained by neutron and X-ray diffraction represent the active state of the enzyme since crystallization and data collection were performed at pH optimal for activity and room temperature. The structures from neutron diffraction enabled the observation of hydrogen positions in the active site. Glu35 is protonated while Asp52 is deprotonated (Figure 1), as suggested by the proposed reaction mechanisms. These observations agree with early neutron diffraction studies, while more recent studies reported alternative Glu35 protonation. The intricate hydrogen-bond interactions involving Asp52 suggest it has a somewhat restricted position (Figure 2), enabling the interaction with the intermediate species but not enough distortion to form a covalent bond. Therefore, the authors argue that the mechanism proposed by Phillips et al.[2], of an ionic pair interaction between Asp52 and the reaction’s intermediate, is more plausible than that of a short-lived covalent interaction, proposed by Koshland [3]. Although further structural studies of lysozyme bound to substrate are required to resolve its mechanism, this work will be helpful to guide future crystallographic studies. This study also highlights the exceptional details available from atomic-resolution neutron crystallography which can be crucial to decipher complex questions in enzymology and structural biology.
J. Ramos (ILL)
[1] Ramos, J. et al. (2025) Structure. 33, 136-148.
[2] Blake, C. C., et al. (1967). Proc. R. Soc. Lond. B. Biol. Sci. 167, 378-388.
[3] Koshland, D.E., Jr. (1953). Biol. Rev. 28, 416–436.
Figure 1: Protonation states of the catalytic residues based on the perdeuterated lysozyme crystal structure from neutron diffraction. 3Fo-2Fc density map contoured at 1σ. Carbon atoms colored in purple, oxygen in red, nitrogen in blue, deuterium in orange and hydrogen in yellow.
Figure 2: Hydrogen-bond interactions involving Asp52 according to the perdeuterated lysozyme crystal structure from neutron diffraction. 3Fo-2Fc density map contoured at 1σ. Carbon atoms colored in purple, oxygen in red, nitrogen in blue, deuterium in orange and hydrogen in yellow. Hydrogen bonds are represented as dashed lines.