Lozoya E, Hoffmann H, Douglas C, Schulz W, Scheel D, Hahlbrock K
Max-Planck-Institut für Züchtungsforschung, Abteilung Biochemie, Köln, Federal Republic of Germany.
Eur J Biochem. 1988 Oct 1;176(3):661-7. doi: 10.1111/j.1432-1033.1988.tb14328.x.
We have determined the primary structures of two 4-coumarate: CoA ligase (4CL) isoenzymes in parsley (Petroselinum crispum) by sequencing near full-length cDNAs corresponding to the two 4CL genes, Pc4CL-1 and Pc4CL-2, present in this plant. Comparison of the cDNA and genomic nucleotide sequences showed that each 4CL gene is organized in five exons separated by introns of varying lengths. The positions of introns are the same in both genes and 97-99% of the corresponding nucleotide sequences are identical. The two isoenzymes, which are nearly identical in their primary structures, were separated by ion-exchange chromatography, and were found to be indistinguishable with regard to substrate specificity. Assignment to Pc4CL-1 and Pc4CL-2 was achieved by comparison with catalytically active 4CL proteins, isolated from Escherichia coli cells which had been transformed with plasmids harboring the corresponding cDNAs.
我们通过对与该植物中存在的两个4-香豆酸：辅酶A连接酶（4CL）基因Pc4CL-1和Pc4CL-2相对应的近全长cDNA进行测序，确定了欧芹（Petroselinum crispum）中两种4CL同工酶的一级结构。cDNA和基因组核苷酸序列的比较表明，每个4CL基因由五个外显子组成，外显子被不同长度的内含子隔开。两个基因中内含子的位置相同，相应核苷酸序列的97-99%是相同的。这两种一级结构几乎相同的同工酶通过离子交换色谱法分离，并且发现它们在底物特异性方面没有区别。通过与从用携带相应cDNA的质粒转化的大肠杆菌细胞中分离出的具有催化活性的4CL蛋白进行比较，确定了Pc4CL-1和Pc4CL-2。
