Purification and characterization of cinnamyl alcohol dehydrogenase from tobacco stems

Claire Halpin; Mary E. Knight; Jacqueline Grima-Pettenati; Deborah Goffner; Alain Boudet; Wolfgang Schuch

doi:10.1104/pp.98.1.12

Purification and characterization of cinnamyl alcohol dehydrogenase from tobacco stems

Claire Halpin, Mary E. Knight, Jacqueline Grima-Pettenati, Deborah Goffner, Alain Boudet, Wolfgang Schuch

Plant Sciences

Research output: Contribution to journal › Article › peer-review

49 Citations (Scopus)

Abstract

Cinnamyl alcohol dehydrogenase (CAD) is an enzyme involved in lignin biosynthesis. In this paper, we report the purification of CAD to homogeneity from tobacco (Nicotiana tabacum) stems. The enzyme is low in abundance, comprising approximately 0.05% of total soluble cell protein. A simple and efficient purification procedure for CAD was developed. It employs three chromatography steps, including two affinity matrices, Blue Sepharose and 2′5′ADP-Sepharose. The purified enzyme has a specific cofactor requirement for NADP and has high affinity for coniferyl alcohol (K_m = 12 micromolar) and coniferaldehyde (K_m = 0.3 micromolar). Two different sized polypeptide subunits of 42.5 and 44 kilodaltons were identified and separated by reverse-phase HPLC. Peptide mapping and amino acid composition analysis of the polypeptides showed that they are closely related, although not identical.

Original language	English
Pages (from-to)	12-16
Number of pages	5
Journal	Plant Physiology
Volume	98
Issue number	1
DOIs	https://doi.org/10.1104/pp.98.1.12
Publication status	Published - Jan 1992

ASJC Scopus subject areas

Physiology
Genetics
Plant Science

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abstract = "Cinnamyl alcohol dehydrogenase (CAD) is an enzyme involved in lignin biosynthesis. In this paper, we report the purification of CAD to homogeneity from tobacco (Nicotiana tabacum) stems. The enzyme is low in abundance, comprising approximately 0.05% of total soluble cell protein. A simple and efficient purification procedure for CAD was developed. It employs three chromatography steps, including two affinity matrices, Blue Sepharose and 2′5′ADP-Sepharose. The purified enzyme has a specific cofactor requirement for NADP and has high affinity for coniferyl alcohol (Km = 12 micromolar) and coniferaldehyde (Km = 0.3 micromolar). Two different sized polypeptide subunits of 42.5 and 44 kilodaltons were identified and separated by reverse-phase HPLC. Peptide mapping and amino acid composition analysis of the polypeptides showed that they are closely related, although not identical.",

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AU - Halpin, Claire

AU - Knight, Mary E.

AU - Grima-Pettenati, Jacqueline

AU - Goffner, Deborah

AU - Boudet, Alain

AU - Schuch, Wolfgang

PY - 1992/1

Y1 - 1992/1

N2 - Cinnamyl alcohol dehydrogenase (CAD) is an enzyme involved in lignin biosynthesis. In this paper, we report the purification of CAD to homogeneity from tobacco (Nicotiana tabacum) stems. The enzyme is low in abundance, comprising approximately 0.05% of total soluble cell protein. A simple and efficient purification procedure for CAD was developed. It employs three chromatography steps, including two affinity matrices, Blue Sepharose and 2′5′ADP-Sepharose. The purified enzyme has a specific cofactor requirement for NADP and has high affinity for coniferyl alcohol (Km = 12 micromolar) and coniferaldehyde (Km = 0.3 micromolar). Two different sized polypeptide subunits of 42.5 and 44 kilodaltons were identified and separated by reverse-phase HPLC. Peptide mapping and amino acid composition analysis of the polypeptides showed that they are closely related, although not identical.

AB - Cinnamyl alcohol dehydrogenase (CAD) is an enzyme involved in lignin biosynthesis. In this paper, we report the purification of CAD to homogeneity from tobacco (Nicotiana tabacum) stems. The enzyme is low in abundance, comprising approximately 0.05% of total soluble cell protein. A simple and efficient purification procedure for CAD was developed. It employs three chromatography steps, including two affinity matrices, Blue Sepharose and 2′5′ADP-Sepharose. The purified enzyme has a specific cofactor requirement for NADP and has high affinity for coniferyl alcohol (Km = 12 micromolar) and coniferaldehyde (Km = 0.3 micromolar). Two different sized polypeptide subunits of 42.5 and 44 kilodaltons were identified and separated by reverse-phase HPLC. Peptide mapping and amino acid composition analysis of the polypeptides showed that they are closely related, although not identical.

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Purification and characterization of cinnamyl alcohol dehydrogenase from tobacco stems

Abstract

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