Purification of (1-->3)-beta-glucan endohydrolase isoenzyme II from germinated barley and determination of its primary structure from a cDNA clone.
A (1-->3)-beta-D-glucan 3-glucanohydrolase (EC 3.2.1.39) of apparent M(r) 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-->3)-beta-glucanases GI and GII have pI values of 8.6 and > or = 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-->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-->3)-beta-glucanase to be deduced, together with that of a putative NH2-terminal signal peptide of 28 amino acid residues. The (1-->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-->3)-beta-glucanase shows highly conserved internal domains and 52% overall positional identity with barley (1-->3, 1-->4)-beta-glucanase isoenzyme EII, an enzyme of related but quite distinct substrate specificity. Thus, the (1-->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-->3, 1-->4)-beta-glucanases, which function to depolymerize endosperm cell walls in the germinated grain.
Høj
P B
PB
Commonwealth Special Research Centre for Protein and Enzyme Technology, La Trobe University, Bundoora, Victoria, Australia.
Hartman
D J
DJ
Morrice
N A
NA
Doan
D N
DN
Fincher
G B
GB
D000595:Amino Acid Sequence; D001483:Base Sequence; D005075:Biological Evolution; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D017343:Genes, Plant; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001467:Hordeum / Q000201:enzymology*; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D007527:Isoenzymes; D004247:DNA; D004693:Glucan Endo-1,3-beta-D-Glucosidase