Characterization of molecularly cloned human 5-aminoimidazole-4-carboxamide ribonucleotide transformylase.
The cDNA encoding human 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase has been cloned from a placenta cDNA library, utilizing a PCR-derived probe. It encodes a peptide of 592 amino acids. The amino (N)-terminal sequence of this enzyme, purified from HeLa cells and CCRF-CEM cells, was found to be APGQLALF-. Both sequencing results revealed a difference of six N-terminal residues when compared to the reported sequence of cloned cDNA from a hepatoma cDNA library. Northern-blot analysis of human AICAR transformylase mRNA showed the expression of a single 2.0 kb mRNA in all tissues examined. With the cloned cDNA fragment, we constructed expression vectors for mature and GST-fused AICAR transformylase. Both recombinant molecules possessing AICAR transformylase activity were overproduced in Escherichia coli. GST-AICAR transformylase can be purified to homogeneity by a single-step affinity procedure with glutathione Sepharose. Mutational analysis, utilizing this expression system, showed that His213 and His267 were essential for AICAR transformylase activity.
Sugita
T
T
Lead Generation Research Laboratory, Tanabe Seiyaku Co., Ltd., Osaka. t-sugita@tanabe.co.jp
Aya
H
H
Ueno
M
M
Ishizuka
T
T
Kawashima
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006367:HeLa Cells; D006639:Histidine / Q000502:physiology; D006801:Humans; D019877:Hydroxymethyl and Formyl Transferases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009928:Organ Specificity; D051027:Phosphoribosylaminoimidazolecarboxamide Formyltransferase; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins; D017421:Sequence Analysis; D013601:T-Lymphocytes / Q000201:enzymology
D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D006639:Histidine; D019877:Hydroxymethyl and Formyl Transferases; D051027:Phosphoribosylaminoimidazolecarboxamide Formyltransferase