Polygalacturonic acid trans-eliminase in the osmotic shock fluid of Erwinia rubrifaciens: characterization of the purified enzyme and its effect on plant cells

Abstract

An endopolygalacturonic acid transeliminiase (EC 4.2.2.2), released by osmotic shock of E. rubrifaciens cells, was purified to near homogeneity (3100-fold) by column chromatography on DEAE-cellulose, phosphocellulose, and hydroxyapatite-cellulose followed by isoelectric focusing. It has a MW of 41,000 s20,W of 3.09S, isoelectric point of pH 6.25, pH optimum of 9.5 and a temperature optimum of 37.degree. C and requires Ca2+ with an optimum concentration of 0.5-1.0 mM. Mg2+ could not substitute for Ca2+. Tyrosinyl residues seem essential for enzyme catalysis based on rapid inactivation by tetranitromethane. The enzyme prefers unmethylated polygalacturonic acid as the substrate, cleaving .alpha.-1,4-glycosidic linkages randomly to form unsaturated galacturonides at a Vmax of 1166 .mu.mol of product/min per mg of protein and a Km of 5 mg of polygalacuturonic acid/ml. Over 90% of the enzyme activity is released from osmotically shocked E. rubrifaciens cells. Unlike E. rubrifaciens, traneliminse is not released from E. carotovora cells by osmotic shock treatment, but enzyme activity is detected in the culture medium. The release of the enzyme is reduced 5-fold by the addition of dibutyryl cyclic AMP. The hypersensitive reaction in tobacco leaves was induced within 60 min after injection of < 1 .mu.g of purified E. rubrifaciens transeliminase. Single cells of tobacco in suspension culture are readily killed by the enzyme, but tobacco protoplasts remain unaffected when treated in the same manner. Endopolygalacturonic acid transeliminase is a constitutive enzyme possibly located in the periplasmic space of the E. rubrifaciens cell and releases enzyme into the culture medium in the presence of substrate. The release of the enzyme in tobacco tissue and the transeliminative cleavage of plant cell wall components may be steps leading to hypersensitivity of the tobacco tissue.