Molecular cloning and characterization of the pyrB1 and pyrB2 genes encoding aspartate transcarbamoylase in pea (Pisum sativum L.).
We cloned cDNAs encoding two different pea (Pisum sativum L.) aspartate transcarbamoylases (ATCases) by complementation of an Escherichia coli delta pyrB mutant. The two cDNAs, designated pyrB1 and pyrB2, encode polypeptides of 386 and 385 amino acid residues, respectively, both of which exhibit typical chloroplast transit peptide sequences. Wheat germ ATCase antibody recognizes a 36.5-kD polypeptide in pea leaf and root tissues that is similar in size to other plant ATCase polypeptides and to the catalytic polypeptides of bacterial ATCases. Northern analyses indicate that the pyrB1 and pyrB2 transcripts are 1.6 kb in size and are differentially expressed in pea tissues. The small transcript size and data from biochemical studies indicate that plant ATCases are simple homotrimers of 36- to 37-kD catalytic subunits, rather than part of a multifunctional enzyme containing glutamine-dependent carbamoylphosphate synthetase and dihydroorotase activities, as is seen in other eukaryotes. In the pea ATCases, the carbamoylphosphate- and aspartate-binding domains are highly homologous to those of other prokaryotic and eukaryotic ATCases and critical active-site residues are completely conserved. The pea ATCases also exhibit a putative pyrimidine-binding site, consistent with the known allosteric regulation of plant ATCases by UMP in vitro.
Williamson
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Department of Biological Sciences, Goucher College, Baltimore, Maryland 21204-2794.
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D000595:Amino Acid Sequence; D001221:Aspartate Carbamoyltransferase / Q000096:biosynthesis / Q000235:genetics*; D001412:Bacillus subtilis / Q000201:enzymology; D015152:Blotting, Northern; D015139:Blotting, Southern; D002736:Chloroplasts / Q000201:enzymology; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis; D004926:Escherichia coli / Q000201:enzymology; D007887:Fabaceae / Q000201:enzymology* / Q000235:genetics*; D015870:Gene Expression; D017343:Genes, Plant; D006801:Humans; D008969:Molecular Sequence Data; D010946:Plants, Medicinal; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014908:Triticum / Q000201:enzymology
D012333:RNA, Messenger; D004247:DNA; D001221:Aspartate Carbamoyltransferase