Preparation and crystallization of perdeuterated T4 phage lysozyme for neutron diffraction

Hiromoto, Takeshi; Adachi, Motoyasu; Shibazaki, Chie; Schrader, T. E.*; Ostermann, A.*; Kuroki, Ryota

T4 phage lysozyme (T4L) is an endoacetylmuramidase that degrades the murein of the bacterial cell wall by cleaving the -1,4-glycosidic bond between -acetylmuramic acid and -acetylglucosamine. We previously reported that the substitution of the catalytic Thr26 with the nucleophilic His converts the wild-type (WT) T4L from an inverting to a retaining glycosidase, in which the -configuration of the substrate is retained in the product. We also found that the Thr26His (T26H) mutant can catalyze a transglycosylation reaction more effectively than hydrolysis, although the WT-T4L has no transglycosidase activity. To clarify the role of the substituted His26 in transglycosylation and to investigate its relationship to the neighboring acidic residue Asp20 using neutron crystallography, a perdeuterated recombinant protein of the T26H mutant (d-T26H) was prepared for crystallization. The perdeuterated form was produced in cells cultured in deuterated-rich media. After purification, the d-T26H mutant was crystallized under deuterated conditions and grown to a volume of 0.12 mm using a macroseeding technique. A preliminary neutron-diffraction experiment at 100 K at the FRM II research reactor (Munich, Germany) gave diffraction spots of up to 2.8 resolution after a 1.5 h exposure.