Structural and mechanistic roles of three consecutive Pro residues of porcine NADH-cytochrome b_5 reductase for the binding of β-NADH(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

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概要

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• Well-conserved three consecutive Pro residues (Pro247-249) in the NADH-binding subdomain of NADH-cytochrome b_5 reductase were proposed to form a basal part of the NADH-binding site. To investigate the structural and mechanistic roles of these residues, we expressed site-directed mutants for a soluble domain of the porcine enzyme where each of the residues was replaced with either Ala or Leu residue, respectively, using a heterologous expression system in Escherichia coll. Six mutants (P247A, P247L, P248A, P248L, P249A, and P249L) were produced as a fusion protein containing a 6×His-tag sequence at the NH_2-terminus and were purified to homogeneity with a stoichiometric amount of bound FAD. Mutations were each confirmed for the purified proteins by MALDI-TOF mass spectrometry. Steady-state kinetic analyses for NADH: ferricyanide reductase and NADH: cytochrome b_5 reductase acitivities were conducted for all the mutants. Substitution of Pro247 with Leu residue was found to significantly decrease k_<cat> with slight increase in K_m for the physiological electron donor NADH. However, K_m values for the electron acceptors (both cytochrome b_5 and ferricyanide) of P247L were found to be decreased significantly. Such changes were not observed for P247A or other four mutants. These results suggested that Pro247 among the three consecutive Pro residues has the most important role for the formation of a binding site cavity and that only a slight change in the side-chain volume at this residue from Ala to Leu residue affected the electron transfer reaction from NADH and, further, on the recognition of ferricytochrome b_5.

• 社団法人日本生物工学会の論文

著者

• TSUBAKI Motonari

  Department of Life Science, Faculty of Science, Himeji Institute of Technology

• Takeuchi Fusako

  Department of Pharmacy, College of Pharmaceutical Sciences, Ritsumeikan University

• Shibuya Maiko

  Department Of Molecular Science And Material Engineering Graduate School Of Science And Technology K

• Nishimura Yuka

  Department of Molecular Science and Material Engineering, Graduate School of Science and Technology,

• Muraki Aya

  Department of Chemistry, Graduate School of Science, Kobe University

• Park Sam-Yong

  Division of Science of Biological Supramolecular Systems, Graduate School of Integrated Science, Yok

• Park Sam-yong

  Protein Design Lab. Yokohama City Univ.

• Muraki Aya

  Department Of Chemistry Graduate School Of Science Kobe University

• Nishimura Yuka

  Department Of Molecular Science And Material Engineering Graduate School Of Science And Technology K

• Tsubaki Motonari

  Department Of Life Science Faculty Of Science Himeji Institute Of Technology

• Takeuchi Fusako

  Department Of Pharmacy College Of Pharmaceutical Sciences Ritsumeikan University

• Takeuchi Fusako

  Department Of Life Science Graduate School Of Science Himeji Institute Of Technology

• Tsubaki Motonari

  Department Of Chemistry Graduate School Of Science Kobe University

• Park Sam-yong

  Protein Design Laboratory Department Of Supramolecular Biology Graduate School Of Nanobioscience Yok

• Tsubaki Motonari

  Departmemt of Chemistiy, Graduate School of Science, Kobe University

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