MOLECULAR-CLONING OF A CDNA-ENCODING A (1-]4)-BETA-MANNAN ENDOHYDROLASE FROM THE SEEDS OF GERMINATED TOMATO (LYCOPERSICON-ESCULENTUM)

Mannose-containing polysaccharides are widely distributed in cell wall s of higher plants. During endosperm mobilization in germinated tomato seeds (1-->4)-beta-mannan endohydrolases (EC 3.2.1.78) participate in the enzymic depolymerization of these cell wall polysaccharides. A cD NA encoding a (1-->4)-beta-mannanase from the endosperm of germinated tomato (Lycopersicon esculentum Mill.) seeds has been isolated and cha racterized. The amino acid sequence deduced from the 5'-region of the cDNA exactly matches the sequence of the 65 NH2-terminal amino acids d etermined directly from the purified enzyme. The mature enzyme consist s of 346 amino acid residues, it has a calculated M-r of 38 950 and an isoelectric point of 5.3. Overall, the enzyme exhibits only 28-30% se quence identity with fungal (1-->4)-beta-mannanases, but more highly c onserved regions, which may represent catalytic and substrate-binding domains, can be identified. Based on classification of the tomato (1-- >4)-beta-mannanase as a member of the family 5 group of glycosyl hydro lases, Glu-148 and Glu-265 would be expected to be the catalytic acid and the catalytic nucleophile, respectively. Southern hybridization an alyses indicate that the enzyme is derived from a family of about four genes. Expression of the genes, as determined by the presence of mRNA transcripts in Northern hybridization analyses, occurs in the endospe rm of germinated seeds; no transcripts are detected in hypocotyls, cot yledons, roots or leaves.