Cloning and molecular characterization of the Nicotiana tabacum purH cDNA encoding 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase

Here we report on the cloning and molecular analysis of a N. tabacum purH c;DNA, encoding AICAR transformylase/IMP cyclohydrolase (ATIC). The enzyme catalyzes the penultimate and final steps of the,de novo, purine biosynthesis. Throughout prokaryotes and eukaryotes investigated thus far, purH encodes a bifunctional enzyme catalyzing the formylation of AICAR and the formation of inosine 5'-monophosphate (IMP) as the first product of the purine biosynthesis. The NtpurH1 cDNA encoding ATIC was isolated from a N. tabacum leaf cDNA library. NtpurH1 encodes a protein of 612 amino acids with a calculated molecular mass of 66.4 kDa. The deduced amino acid sequence of the N. tabacum purH c;DNA shares high homologies to ATICs from other organisms and includes a N-terminal extension of 68 amino acids, which is predicted to be the chloroplast transit peptide. The expression of purH in N. tabacum is not restricted to meristematic tissues, but shows a rather constitutive expression. To verify the enzyme activity of the tobacco ATIC, a S. cerevisiae ade 16 ade 17 mutant was generated and used for functional analysis.