Enzymic Synthesis of Lignin Precursors

Abstract

Isoenzyme 2 of cinnamyl‐alcohol dehydrogenase from soybean suspension cultures was purified about 3800‐fold to apparent homogeneity by an improved purification procedure involving biospecific elution of the enzyme from a NADP+‐agarose column. On sodium dodecylsulfate gels the dehydrogenase showed only one protein band with Mr 40000 ± 500. The enzyme is strongly inhibited by thiol reagents. Various metal chelators as well as the non‐chelating 7,8‐benzoquinoline also inhibited enzyme activity. Inhibition by 10 mM 1,10‐phenanthroline could be partially reversed by addition of Zn2+. 1,10‐Phenanthroline and 7,8‐benzoquinoline are non‐competitive inhibitors with respect to NADP+. The presence of zinc in the dehydrogenase was proved by atomic absorption spectroscopy and by specific incorporation of 65Zn into the enzyme. In steady‐state kinetics inhibition patterns were obtained which are consistent with an ordered bi‐bi mechanism in which NADP(H) is the first substrate to bind and the last product released. The cinnamyl‐alcohol dehydrogenase belongs to the A‐specific dehydrogenases and removes the pro‐R hydrogen from coniferyl alcohol. The enzyme shows many similarities with alcohol dehydrogenases from horse and rat liver and from yeast.

Structural and Kinetic Basis for Substrate Selectivity in Populus tremuloides Sinapyl Alcohol Dehydrogenase

“…The A-side of the nicotinamide ring is exposed to the solvent, confirming that SAD catalyzes A-side-specific hydride transfer, as reported for soybean CAD (Wyrambik and Grisebach, 1979).…”

“…Although originally reported to bind only one Zn 2þ ion per monomer (Wyrambik and Grisebach, 1979), the SAD structure contains both catalytic and structural Zn 2þ ions, which are characteristically coordinated to zinc-dependent MDR family enzymes. The structural zinc is coordinated by four Cys residues (Cys-103, Cys-106, Cys-109, and Cys-117) and localized to a protrusion (residues 102 to 120) from the core of the substrate binding domain.…”

Cinnamyl-alcohol dehydrogenase, a molecular marker specific for lignin synthesis: cDNA cloning and mRNA induction by fungal elicitor.

“…CAD has cofactor requirements similar to alcohol dehydrogenase (26). The deduced bean CAD polypeptide contains several glycine residues at the same position as strictly conserved glycines of alcohol dehydrogenases in the fold involved in coenzyme binding (27) (5 ,ug) from bean leaves were digested with the restriction enzymes indicated, and the products were separated by electrophoresis on 0.9%o agarose gels.…”

Properties of Sorbitol Dehydrogenase and Characterization of a Reactive Cysteine Residue Reveal Unexpected Similarities to Alcohol Dehydrogenases

“…Zinc occurs at the active site of yeast [27] and liver [2S] alcohol dehydrogenases, and is present in soybean cinnamyl alcohol dehydrogenase [29] (but probably not in the alcohol dehydrogenase of Drosophrla [30]). Magnesium activates glucose-6-phosphate dehydrogenase [31] and 6-phosphogluconate dehydrogenase [32] though it is not strictly essential for activity [33,34], and NAD-linked isocitrate dehydrogenase probably requires bivalent metal (magnesium, manganese, or zinc) [35] because the true substrate is the metal complex of isocitrate [36].…”