The nucleotide sequence of porcine formiminotransferase cyclodeaminase. Expression and purification from Escherichia coli.
We have isolated and characterized cDNA clones encoding the porcine liver octameric enzyme, 5-formiminotetrahydrofolate:L-glutamate N-formiminotransferase (EC 2.1.2.5)-formiminotetrahydrofolate cyclodeaminase (EC 4.3.1.4). The cDNA encodes a novel amino acid sequence of 541 residues which contains exact matches to two sequences derived by automated sequence analysis of CNBr cleavage fragments isolated from the porcine enzyme. The recombinant enzyme has been expressed as a soluble protein in Escherichia coli at levels 4-fold higher than those observed in liver, and is bifunctional, displaying both transferase and deaminase activities. With a calculated subunit molecular mass of 58,926 Da, it is similar in size to the enzyme isolated from porcine liver. Purification of the enzyme from E. coli involves chromatography on a novel polyglutamate column which might interact with the folylpolyglutamate binding site of the protein. The purified recombinant enzyme has a transferase specific activity of 39-41 units/mg/min.
Murley
L L
LL
Department of Biochemistry, McGill University, Montréal, Quebec, Canada.
Mejia
N R
NR
MacKenzie
R E
RE
D000595:Amino Acid Sequence; D000642:Ammonia-Lyases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D000818:Animals; D001483:Base Sequence; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli; D015870:Gene Expression; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D011061:Poly A / Q000302:isolation & purification / Q000378:metabolism; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D013552:Swine
D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D011061:Poly A; D000642:Ammonia-Lyases; C020148:formiminotetrahydrofolate cyclodeaminase