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, u tilizing a PCR-derived probe, It encodes a peptide of 592 amino acids, The amino (N)-terminal sequence of this enzyme, purified from HeLa ce lls and CCRF-CEM cells, was found to be APGQLALF-. Both sequencing res ults revealed a difference of six N-terminal residues when compared to the reported sequence of cloned cDNA from a hepatoma cDNA library, No rthern-blot analysis of human AICAR transformylase mRNA showed the exp ression of a single 2.0 kb mRNA in all tissues examined, With the clon ed cDNA fragment, we constructed expression vectors for mature and GST -fused AICAR transformylase, Both recombinant molecules possessing AIC AR transformylase activity were overproduced in Escherichia coli, GST- AICAR transformylase can be purified to homogeneity by a single-step a ffinity procedure with glutathione Sepharose, Mutational analysis, uti lizing this expression system, showed that His213 and His287 were esse ntial for AICAR transformylase activity.