Coexpression, purification and characterization of the E and S subunits of coenzyme B(12) and B(6) 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 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-beta-D-galactoside induction temperature. Among substrate analogues tested, only 2,4-diamino-n-butyric acid displays competitive inhibition of the enzyme with a K(i) of 96 +/- 14 microm. Lys629 is responsible for the binding of PLP. The apparent K(d) for coenzyme B(6) 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 aminomutase to maintain an active conformation.
Chen
Hao-Ping
HP
Institute of Biotechnology and Department of Chemical Engineering, National Taipei University of Technology, Taipei, Taiwan. hpchen@ntut.edu.tw
Hsui
Fang-Ciao
FC
Lin
Li-Ying
LY
Ren
Chien-Tai
CT
Wu
Shih-Hsiung
SH
D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D047991:Acetoanaerobium sticklandii / Q000201:enzymology*; D003038:Cobamides / Q000737:chemistry*; D004247:DNA / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D005822:Genetic Vectors; D019751:Intramolecular Transferases / Q000737:chemistry* / Q000302:isolation & purification*; D007700:Kinetics; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D010769:Phosphothreonine / Q000031:analogs & derivatives* / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011732:Pyridoxal Phosphate / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012270:Ribosomes / Q000648:ultrastructure; D013696:Temperature; D013862:Thiogalactosides / Q000737:chemistry; D014466:Ultraviolet Rays
D003038:Cobamides; D009841:Oligonucleotides; D011994:Recombinant Proteins; D013862:Thiogalactosides; C049349:7-mercaptoheptanoylthreonine phosphate; D010769:Phosphothreonine; D011732:Pyridoxal Phosphate; C031803:thiodigalactoside; D004247:DNA; D019751:Intramolecular Transferases; C021913:ornithine 5,4-aminomutase; C000913:cobamamide