PMID:9182560
| Citation |
Ahn, C, Byun, J and Yim, J (1997) Purification, cloning, and functional expression of dihydroneopterin triphosphate 2'-epimerase from Escherichia coli. J. Biol. Chem. 272:15323-8 |
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| Abstract |
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. |
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| Keywords |
Amino Acid Sequence; Base Sequence; Biopterin/analogs & derivatives; Biopterin/isolation & purification; Chromatography, Ion Exchange; Cloning, Molecular; DNA, Recombinant; Escherichia coli/genetics; Escherichia coli Proteins; Genomic Library; Molecular Sequence Data; Molecular Weight; Neopterin; Polymerase Chain Reaction; Racemases and Epimerases/chemistry; Racemases and Epimerases/genetics; Racemases and Epimerases/isolation & purification; Racemases and Epimerases/metabolism |
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