Purification, cloning, and functional expression of dihydroneopterin triphosphate 2'-epimerase from Escherichia coli

Dihydroneopterin triphosphate (H2NTP) 2'-epimerase from Escherichia coli catalyzes the epimerization of H,NTP to dihydromonapterin triphosphate (H,MTP). The enzyme was purified 954-fold to apparent]homogeneity by a combination of ammonium sulfate fractionation and column chromatography of Cibacron blue 3GA dye ligand, phenyl-Sepharose CL-4B, methotrexate-agarose, and Superdex 200 HR 10/30 FPLC column. The molecular mass of the epimerase determined on a Superdex column was 82.6 kDa, while the subunit molecular mass determined on SDS-polyacrylamide gel electrophoresis was 13.7 kDa, This implies that the epimerase most probably exists as homohexamer, The 20-amino acid sequence from the N terminus was determined (AQPAAIIRIKNLRLRTFIGI). Based on this sequence, the gene encoding the epimerase was cloned using a simple polymerase chain reaction approach, Translation of the nucleotide sequence of the cloned gene revealed the presence of an open reading frame containing 120 amino acids with a predicted molecular mass of 13,993 Da, The epimerase gene located in a 2.3-kilobase BamHI-EcoRI fragment from Kohara's clone 406 was overexpressed 300 fold, which was confirmed by the prominent increase in the 14-kDa protein band on SDS-polyacrylamide electrophoresis gels, It showed no homology with the sequences of isomerases or other enzymes in GenBanK/EMBL data bases.