Ryota Kuroki

A Covalent Enzyme-Substrate Adduct in a Mutant Hen Egg White Lysozyme (D52E)

A mutant hen egg white lysozyme, D52E, was designed to correspond to the structure of the mutant T4 lysozyme T26E (Kuroki, R., Weaver, L. H., and Matthews B. W. (1993)Science 262, 2030–2033) to investigate the role of the catalytic residue on the α-side of the saccharide in these enzymes. The D52E mutant forms a covalent enzyme-substrate adduct, which was detected by electron ion spray mass spectrometry. X-ray crystallographic analysis showed that the covalent adduct was formed between Glu-52 and the C-1 carbon of theN-acetylglucosamine residue in subsite D of the saccharide binding site. It suggests that the catalytic mechanism of D52E mutant lysozyme proceeds through a covalent enzyme-substrate intermediate indicating a different catalytic mechanism from the wild type hen egg white lysozyme. It was confirmed that the substitution of Asp-52 with Glu is structurally and functionally equivalent to the substitution of Thr-26 with Glu in T4 lysozyme. Although the position of the catalytic residue on the β-side of the saccharide is quite conserved among hen egg white lysozyme, goose egg white lysozyme, and T4 phage lysozyme, the adaptability of the side chain on the α-side of the saccharide is considered to be responsible for the functional variation in their glycosidase and transglycosidase activities.

Evaluation of the Resolvable Capacity of Bragg Reflections for a New Diffractometer at J-PARC/MLF Designed for Protein Crystals With Large Unit Cells

We aim to build a high-resolution neutron time-of-flight diffractometer for biomacromolecules at the Materials and Life Science Experimental Facility (MLF) at the Japan Proton Accelerator Research Complex (J-PARC) that allows the collection of neutron diffraction data from crystals with unit cells of ≈250 A. Considering both the flux and pulse width necessary to realize data collection covering a minimum d-spacing of 2.0 A and with a unit cell constant of ≈250 A, we chose a decoupled moderator (DM) as the appropriate source for this high-resolution diffractometer. We considered a simple instrumentation model that includes a moderator, neutron guide, sample size, and neutron detector; we then investigated its spot separation performance and estimated the instrumental parameters for the design of a new diffractometer for protein crystals with large unit cells at J-PARC/MLF.

Preparation and Crystallization of Perdeuterated T4 Phage Lysozyme for Neutron Diffraction Study

T4 phage lysozyme (T4L) is an endoacetylmuramidase that degrades the murein of the bacterial cell wall by cleaving the β-1,4-glycosidic bond between N-acetylmuramic acid and N-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 β-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 perdeuteration would reduce the crystal-size requirement by one order of magnitude and enable better visualization of the protonation and hydration states of the catalytic residues. The perdeuterated form was produced in Escherichia coli 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 A resolution after a 1.5 h exposure.