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 trans-eliminase (EC 4.2.2.2), released by osmotic shock of Erwinia rubrifaciens cells, has been purified to near homogeneity (3, 100-fold) by column chromatography on diethylaminoethyl-cellulose, phosphocellulose, and hydroxyapatite-cellulose followed by isoelectric focusing. It has a molecular weight of 41,000, s20,w of 3.09S, an isoelectric point of pH 6.25, pH optimum of 9.5, and a temperature optimum of 37 C and requires Ca2+ with an optimum concentration of 0.5 to 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 1,166 mumol of product/min per mg of protein and a Km of 5 mg of polygalacturonic acid per ml. Over 90% of the enzyme activity is released from osmotically shocked E. rubrifaciens cells. Unlike E. rubrifaciens, trans-eliminase is not released from Erwinia carotovora cells by osmotic shock treatment, but enzyme activity is detected in the culture medium. The release of the enzyme is reduced fivefold by the addition of dibutyryl cyclic adenosine 5'-monophosphate. The hypersensitive reaction in tobacco leaves was induced within 60 min after injection of less than 1 mug of purified E. rubrifaciens trans-eliminase. Single cells of tobacco in suspension culture are readily killed by the enzyme, whereas tobacco protoplasts remain unaffected when treated in the same manner. These results indicate that endopolygalacturonic acid trans-eliminase 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 trans-eliminative cleavage of plant cell wall components may be steps leading to hypersensitivity of the tobacco tissue.