Coexpression, purification and characterization of the E and S subunits of coenzyme B12 and B6 dependentClostridium sticklandiid-ornithine aminomutase in.

d-Ornithine aminomutase fromClostridium sticklandiicomprises 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 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 theoraEandoraSgenes was carried out inE. coli. The solubility of the coexpressed OraS and OraE increases with decreasing isopropyl thio-β-d-galactoside induction temperature. Among substrate analogues tested, only 2,4-diamino-n-butyric acid displays competitive inhibition of the enzyme with aKi of 96 ± 14 µm. Lys629 is responsible for the binding of PLP. The apparentKd for coenzyme B6 binding tod-ornithine aminomutase is 224 ± 41 nmas 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 duringin vivotranslation of OraSE–K629M inE. coli,in vitrorefolding of the enzyme employs a different folding mechanism. In both cases, the association of the S and E subunit is important ford-ornithine aminomutase to maintain an active conformation. [ABSTRACT FROM AUTHOR]