Maize Uroporphyrinogen Methyltransferase: Overexpression Of Different Gene Fragments, Purification And Partial Properties

S-adenosyl-L-methionine:uroporphyrinogen methyltransferase(SUMT), a key enzyme in the biosynthetic pathways of siroheme, vitamin B12, heme d1 and F430 factor, catalyzes the S-adenosyl-L-methionine-dependent bismethylation of its substrate uroporphyrinogen III at C-2 and C-7, resulting in the formation of precorrin-2. In this study, we overexpressed different fragment of maize SUMT fused with hexahistidine tag in Escherichia Coli using the pQE-His expression vector including mature enzyme(L92-W423), truncated enzyme 1 without 51 ammo acids at C terminus (L92-S372) and truncated enzyme 2 without 36 amino acids, SAM binding domain at N terminus (G93-W423) . The colonies carrying expressing vector with the mature enzyme and truncated enzyme 1 respectively displayed bright fluorescent when illuminated with UV light. The transformants harboring expressing vector with truncated enzyme 2 developed no fluorescence upon UV illumination and expressed in inclusion. The mature enzyme and truncated enzyme 1 were purified by a single-step purification procedure on Ni-NTA affinity chromatography respectively. The purified both proteins has respective molecular weights of 34Ku and 33Ku as shown on SDS-PAGE. The relative molecular mass of mature enzyme was approximately 66KD by Sephacryl S-200 gel filtration, indicating that the enzyme is homodimer. Co-purified red fluorescent compounds were found associate uncovalently with the purified the enzyme. The main component of this pigments freed from enzyme was identified to trimethylpyrrocorphin by UV/visible spectra, fluorescent spectra and MS. The CD spectra of enzyme removed pigments revealed a slight increase for p sheets content compared with wild type enzyme. Dynamic light scartering( DLS) analysis presented that the enzyme removed pigments has more aggregation tendency than the wild type enzyme.