tailieunhanh - Báo cáo khoa học: Coexpression, purification and characterization of the E and S subunits of coenzyme B12 and B6 dependent Clostridium sticklandii D-ornithine aminomutase in Escherichia coli

D-Ornithine aminomutase from Clostridium sticklandii comprises two strongly associating subunits, OraS and OraE, with molecular masses of 12 800 and 82 900 Da. Previous studies have shown that inEscherichia colithe recombinant OraS protein is synthesized in the soluble form and OraE as inclusion bodies. Refolding experiments also indicate that the interactions between OraS and OraE and the binding of either pyridoxal phosphate (PLP) or aden-osylcobalamin (AdoCbl) play important roles in the refolding process | Eur. J. Biochem. 271 4293-4297 2004 FEBS 2004 doi Coexpression purification and characterization of the E and S subunits of coenzyme B12 and B6 dependent Clostridiumsticklandii D-ornithine aminomutase in Escherichia coli Hao-Ping Chen1 Fang-Ciao Hsui1 Li-Ying Lin1 Chien-Tai Ren2 and Shih-Hsiung Wu2 1 Institute of Biotechnology and Department of Chemical Engineering National Taipei University of Technology Taipei Taiwan 2Institute of Biological Chemistry Academia Sinica Nankang Taipei Taiwan D-Ornithine aminomutase from Clostridium sticklandii comprises two strongly associating subunits OraS and OraE with molecular masses of 12 800 and 82 900 Da. Previous studies have shown that in Escherichia coli the recombinant OraS protein is synthesized in the soluble form and OraE as inclusion bodies. Refolding experiments also indicate that the interactions between OraS and OraE and the binding of either pyridoxal phosphate PLP or adenosylcobalamin AdoCbl play important roles in the refolding process. In this study the DNA fragment containing both genes was cloned into the same expression vector and coexpression of the oraE and oraS genes was carried out in E. coli. The solubility of the coexpressed OraS and OraE increases with decreasing isopropyl thio-b-D-galactoside induction temperature. Among substrate analogues tested only 2 4-diamino-n-butyric acid displays competitive inhibition of the enzyme with a Ki of 96 14 M. Lys629 is responsible for the binding of PLP. The apparent Kd for coenzyme B6 binding to D-ornithine aminomutase is 224 41 nM as measured by equilibrium dialysis. The mutant protein OraSE-K629M is successfully expressed. It is catalytically inactive and unable to bind PLP. Because no coenzyme is involved in protein folding during in vivo translation of OraSE-K629M in E. coli in vitro refolding of the enzyme employs a different folding mechanism. In both cases the association of the S and E subunit is important for D-ornithine

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