PURIFICATION, CHARACTERIZATION, AND CLONING OF CINNAMYL ALCOHOL-DEHYDROGENASE IN LOBLOLLY-PINE (PINUS-TAEDA L)

被引:102
作者
OMALLEY, DM
PORTER, S
SEDEROFF, RR
机构
[1] Department of Forestry, North Carolina State University, Raleigh
关键词
D O I
10.1104/pp.98.4.1364
中图分类号
Q94 [植物学];
学科分类号
071001 ;
摘要
Cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) has been purified to homogeneity from differentiating xylem tissue and developing seeds of loblolly pine (Pinus taeda L.). The enzyme is a dimer with a native molecular weight of 82,000 and a subunit molecular weight of 44,000, and is the only form of CAD involved in lignification in differentiating xylem. High levels of loblolly pine CAD enzyme were found in nonlignifying seed tissue. Characterization of the enzyme from both seeds and xylem demonstrated that the enzyme is the same in both tissues. The enzyme has a high affinity for coinferaldehyde (K(m) = 1.7 micromolar) compared with sinapaldehyde (K(m) in excess of 100 micromolar). Kinetic data strongly suggest that coniferin is a noncompetitive inhibitor of CAD enzyme activity. Protein sequences were obtained for the N-terminus (28 amino acids) and for two other peptides. Degenerate oligonucleotide primers based on the protein sequences were used to amplify by polymerase chain reaction a 1050 base pair DNA fragment from xylem cDNA. Nucleotide sequence from the cloned DNA fragment coded for the N-terminal protein sequence and an internal peptide of CAD. The N-terminal protein sequence has little similarity with the lambda-CAD4 clone isolated from bean (MH Walter, J Grima-Pettenati, C Grand, AM Boudet, CJ Lamb [1988] Proc Natl Acad Sci USA 86:5546-5550), which has homology with malic enzyme.
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页码:1364 / 1371
页数:8
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