Purification, characterization, and cloning of cinnamyl alcohol dehydrogenase in loblolly pine (Pinus taeda L.)

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...

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Bibliographic Details
Published in:Plant physiology (Bethesda) 1992-04, Vol.98 (4), p.1364-1371
Main Authors: O'Malley, D.M. (North Carolina State University, Raleigh, NC), Porter, S, Sederoff, R.R
Format: Article
Language:English
Subjects:
ACTIVIDAD ENZIMATICA
ACTIVITE ENZYMATIQUE
Agronomy. Soil science and plant productions
ALCOHOL DESHIDROGENASA
Alcohols
ALCOOL DESHYDROGENASE
Biological and medical sciences
COMPOSE AROMATIQUE
COMPOSICION QUIMICA
COMPOSITION CHIMIQUE
COMPUESTOS AROMATICOS
Computer aided design
Dehydrogenases
Elution
Enzymes
Fundamental and applied biological sciences. Psychology
Gels
GRAINE
Lignin
MEDIO DE CULTIVO
Metabolism
MILIEU DE CULTURE
PESO MOLECULAR
Pine trees
PINUS TAEDA
Plant physiology and development
POIDS MOLECULAIRE
Proteins
PURIFICACION
PURIFICATION
SEMILLA
XILEMA
Xylem
XYLEME
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Summary: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 coniferaldehyde (Km = 1.7 micromolar) compared with sinapaldehyde (Km 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
ISSN:0032-0889
1532-2548
DOI:10.1104/pp.98.4.1364