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 H2NTP to dihydromonapterin triphosphate (H2MTP). 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.
Ahn
C
C
Department of Microbiology, College of Natural Sciences, Seoul National University, Seoul 151-742, Korea.
Byun
J
J
Yim
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D001708:Biopterins / Q000031:analogs & derivatives / Q000302:isolation & purification; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D015698:Genomic Library; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019798:Neopterin; D016133:Polymerase Chain Reaction; D019998:Racemases and Epimerases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D004274:DNA, Recombinant; D029968:Escherichia coli Proteins; D001708:Biopterins; D019798:Neopterin; D019998:Racemases and Epimerases; C023128:folX protein, E coli