Molecular cloning of a cDNA encoding a (1→4)-β-mannan endohydrolase from the seeds of germinated tomato (Lycopersicon esculentum)
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Mannose-containing polysaccharides are widely distributed in cell walls of higher plants. During endosperm mobilization in germinated tomato seeds (1→4)-β-mannan endohydrolases (EC 220.127.116.11) participate in the enzymic depolymerization of these cell wall polysaccharides. A cDNA encoding a (1→4)-β-mannanase from the endosperm of germinated tomato (Lycopersicon esculentum Mill.) seeds has been isolated and characterized. The amino acid sequence deduced from the 5′-region of the cDNA exactly matches the sequence of the 65 NH2-terminal amino acids determined directly from the purified enzyme. The mature enzyme consists of 346 amino acid residues, it has a calculated Mr of 38 950 and an isoelectric point of 5.3. Overall, the enzyme exhibits only 28–30% sequence identity with fungal (1→4)-β-mannanases, but more highly conserved regions, which may represent catalytic and substrate-binding domains, can be identified. Based on classification of the tomato (1→4)-β-mannanase as a member of the family 5 group of glycosyl hydrolases, Glu-148 and Glu-265 would be expected to be the catalytic acid and the catalytic nucleophile, respectively. Southern hybridization analyses 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 endosperm of germinated seeds; no transcripts are detected in hypocotyls, cotyledons, roots or leaves.
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