Characterization of an aspartate transcarbamoylase cDNA from pea (Pisum sativum L.).

ATCase carries out the first committed step in de novo pyrimidine nucleotide biosynthesis in plants, and its activity is regulated via classical end-product inhibition by UMP or its products (Lovatt and Cheng, 1984). As an important first step in understanding the mechanisms by which plant ATCase expression is regulated, we have cloned a pea (Pisum sativum L.) ATCase gene. This appears to be the first plant ATCase gene to have been isolated. With the exception of a recent report conceming the cloning of an alfalfa carbamoylP synthetase gene encoding a protein similar to the Glnutilizing pyrimidine pathway carbamoyl-P synthetase I1 found in eukaryotic cells (Maley et al., 1992), no other plant genes encoding enzymes of the pyrimidine pathway appear to have been isolated. The pea leaf ATCase was isolated by functional complementation of uracil auxotrophy in Escherichia coli strain TB2, in which part or a11 of the pyrB1 operon encoding the catalytic and regulatory subunits of ATCase has been deleted (Roof et al., 1982). We have isolated cDNAs representing at least two distinct pyrB genes in pea, as detennined by restriction mapping and sequencing (Table I). We do not presently know whether these genes are allelic. The first gene, described here and designated pyrB1 (a second pea ATCase gene, pyrB2, is presently being characterized), has been completely sequenced and exhibits a single open reading frame of 386 amino acids. Residues 1 to 55 exhibit features characteristic of a chloroplast transit peptide, consistent with reports that ATCase is a chloroplast-localized enzyme (Doremus and Jagendorf, 1985). Mature pea ATCase is labeled by wheat germ ATCase antibody and exhibits an M, of 37,000 on immunoblots, similar to that of the catalytic subunits of several bacterial ATCases, and does not appear to be part of a multifunctional complex like the CAD protein (Davidson et al., 1990). A comparison of the primary structures of prokaryotic and eukaryotic ATCases with that of pea indicates that several domains are highly conserved, especially those comprising the carbamoyl-P and L-Asp-binding sites (Kantrowitz and Lipscomb, 1988). In addition, the last 50 residues at the Cterminal end of most other eukaryotic ATCases are highly homologous to that of the pea enzyme. Pea ATCase has a putative pyrimidine-binding site that strongly resembles part of the pyrimidine-binding site on the regulatory subunit of the E. coli ATCase (Goruaux et al., 1990). Pea ATCase also