A multisubunit acetyl coenzyme A carboxylase from soybean

A multisubunit form of acetyl coenzyme A (CoA) carboxylase (ACCase) from so ybean (Glycine max) was characterized. The enzyme catalyzes the formation o f malonyl CoA from acetyl CoA, a rate-limiting step in fatty acid biosynthe sis. The four known components that constitute plastid ACCase are biotin ca rboxylase (BC), biotin carboxyl carrier protein (BCCP), and the alpha- and beta-subunits of carboxyltransferase (alpha- and beta-CT). At least three d ifferent cDNAs were isolated from germinating soybean seeds that encode BC, two that encode BCCP, and four that encode alpha-CT. Whereas BC, BCCP, and LU-CT are products of nuclear genes, the DNA that encodes soybean beta-CT is located in chloroplasts. Translation products from cDNAs for BC, BCCP, a nd alpha-CT were imported into isolated pea (Pisum sativum) chloroplasts an d became integrated into ACCase. Edman microsequence analysis of the subuni ts after import permitted the identification of the amino-terminal sequence of the mature protein after removal of the transit sequences. Antibodies s pecific for each of the chloroplast ACCase subunits were generated against products from the cDNAs expressed in bacteria. The antibodies permitted com ponents of ACCase to be followed during fractionation of the chloroplast st roma. Even in the presence of 0.5 M KCI, a complex that contained BC plus B CCP emerged from Sephacryl 400 with an apparent molecular mass greater than about 800 kD. A second complex, which contained alpha- and beta-CT, was al so recovered from the column, and it had an apparent molecular mass of grea ter than about 600 kD. By mixing the two complexes together at appropriate ratios, ACCase enzymatic activity was restored. Even higher ACCase activiti es were recovered by mixing complexes from pea and soybean. The results dem onstrate that the active form of ACCase can be reassembled and that it coul d form a high-molecular-mass complex.