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 walls 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 cDNA encoding a (1-->4)-beta-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 M(r) of 38,950 and an isoelectric point of 5.3. Overall, the enzyme exhibits only 28-30% sequence identity with fungal (1-->4)-beta-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)-beta-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.
Bewley
J D
JD
Department of Botany, University of Guelph, Ontario, Canada. dbewley@uoguelph.ca
Burton
R A
RA
Morohashi
Y
Y
Fincher
G B
GB
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D018076:DNA, Complementary; D018744:DNA, Plant; D018525:Germination; D018551:Solanum lycopersicum / Q000201:enzymology* / Q000235:genetics; D008361:Mannosidases / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D012639:Seeds / Q000201:enzymology; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D018744:DNA, Plant; D008361:Mannosidases; C041867:endo-1,4-beta-D-mannanase