Purification of (1 leads to 3)-beta-glucan endohydrolase isoenzyme II from germinated barley and determination of its primary structure from a cDNA clone
1989
Hoj, P.B. | Hartman, D.J. | Morrice, N.A. | Doan, D.N.P. | Fincher, G.B.
A (1 leads to 3)-beta-D-glucan 3-glucanohydrolase (EC 3.2.1.39) of apparent Mr 32 000, designated GII, has been purified from germinated barley grain and characterized. The isoenzyme is resolved from a previously purified isoenzyme (GI) on the basis of differences in their isoelectric points; (1 leads to 3)-beta-glucanases GI and GII have pI values of 8.6 and greater than or equal 10.0, respectively. Comparison of the sequences of their 40 NH2-terminal amino acids reveals 68% positional identity. A 1265 nucleotide pair cDNA encoding (1 leads to 3)-beta-glucanase isoenzyme GII has been isolated from a library prepared with mRNA of 2-day germinated barley scutella. Nucleotide sequence analysis of the cDNA has enabled the complete primary structure of the 306 amino acid (1 leads to 3)-beta-glucanase to be deduced, together with that of a putative NH2-terminal signal peptide of 28 amino acid residues. The (1 leads to 3)-beta-glucanase cDNA is characterized by a high (G + C) content, which reflects a strong bias for the use of G or C in the wobble base position of codons. The amino acid sequence of the (1 leads to 3)-beta-glucanase shows highly conserved internal domains and 52% overall positional identity with barley (1 leads to 3, 1 leads to 4)-beta-glucanase isoenzyme EII, an enzyme of related but quite distinct substrate specificity. Thus, the (1 leads to 3)-beta-glucanases, which may provide a degree of protection against microbial invasion of germinated barley grain through their ability to degrade fungal cell wall polysaccharides, appear to share a common evolutionary origin with the (1 leads to 3, 1 leads to 4)-beta-glucanases, which function to depolymerize endosperm cell walls in the germinated grain.
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