Biochemical, spectroscopic and X-ray structural analysis of deuterated multicopper oxidase CueO prepared from a new expression construct for neutron crystallography
| Autor: | Yoshiki Higuchi, Mahfuza Akter, Takeshi Sakurai, Hirofumi Komori, Kunishige Kataoka, Naoki Shibata, Nana Matsuda, Chika Inoue |
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| Rok vydání: | 2016 |
| Předmět: |
Models
Molecular Protein Conformation alpha-Helical 0301 basic medicine Reaction mechanism Astrophysics::High Energy Astrophysical Phenomena Amino Acid Motifs Neutron diffraction Biophysics Physics::Optics Gene Expression Crystal structure Crystallography X-Ray Multicopper oxidase Biochemistry Research Communications Substrate Specificity 03 medical and health sciences Structural Biology Oxidoreductase Condensed Matter::Superconductivity Escherichia coli Genetics Protein Interaction Domains and Motifs Benzothiazoles Cloning Molecular chemistry.chemical_classification biology Escherichia coli Proteins Deuterium Exchange Measurement Active site Electron acceptor Deuterium Condensed Matter Physics Recombinant Proteins Crystallography 030104 developmental biology chemistry X-ray crystallography biology.protein Protein Conformation beta-Strand Sulfonic Acids Oxidoreductases Oxidation-Reduction Copper Plasmids |
| Zdroj: | Acta Crystallographica Section F Structural Biology Communications. 72:788-794 |
| ISSN: | 2053-230X |
| Popis: | Multicopper oxidases oxidize various phenolic and nonphenolic compounds by using molecular oxygen as an electron acceptor to produce water. A multicopper oxidase protein, CueO, fromEscherichia coliis involved in copper homeostasis in the bacterial cell. Although X-ray crystallographic studies have been conducted, the reduction mechanism of oxygen and the proton-transfer pathway remain unclear owing to the difficulty in identifying H atoms from X-ray diffraction data alone. To elucidate the reaction mechanism using neutron crystallography, a preparation system for obtaining large, high-quality single crystals of deuterated CueO was developed. Tiny crystals were obtained from the deuterated CueO initially prepared from the original construct. The X-ray crystal structure of the deuterated CueO showed that the protein contained an incompletely truncated signal sequence at the N-terminus, which resulted in the heterogeneity of the protein sample for crystallization. Here, a new CueO expression system that had an HRV3C cleavage site just after the signal sequence was constructed. Deuterated CueO from the new construct was expressed in cells cultured in deuterated algae-extract medium and the signal sequence was completely eliminated by HRV3C protease. The deuteration level of the purified protein was estimated by MALDI-TOF mass spectrometry to be at least 83.2% compared with nondeuterated protein. Nondeuterated CueO crystallized in space groupP21, with unit-cell parametersa= 49.51,b= 88.79,c = 53.95 Å, β = 94.24°, and deuterated CueO crystallized in space groupP212121, with unit-cell parametersa= 49.91,b= 106.92,c= 262.89 Å. The crystallographic parameters for the crystals of the new construct were different from those previously reported for nondeuterated crystals. The nondeuterated and deuterated CueO from the new construct had similar UV–Vis spectra, enzymatic activities and overall structure and geometry of the ligands of the Cu atoms in the active site to those of previously reported CueO structures. These results indicate that the CueO protein prepared using the new construct is suitable for further neutron diffraction studies. |
| Databáze: | OpenAIRE |
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