Barley beta-D-glucan exohydrolases with beta-D-glucosidase activity. Purification, characterization, and determination of primary structure from a cDNA clone.
Two beta-glucan exohydrolases of apparent molecular masses 69,000 and 71,000 Da have been purified from extracts of 8-day germinated barley grains and are designated isoenzymes ExoI and ExoII, respectively. The sequences of their first 52 NH2-terminal amino acids show 64% positional identity. Both enzymes hydrolyze the (1,3)-beta-glucan, laminarin, but also hydrolyze (1,3;1,4)-beta-glucan and 4-nitrophenyl beta-D-glucoside. The complete sequence of 602 amino acid residues of the mature beta-glucan exohydrolase isoenzyme ExoII has been deduced by nucleotide sequence analysis of a near full-length cDNA. Two other enzymes of apparent molecular mass 62,000 Da, designated betaI and betaII, were also purified from the extracts. Their amino acid sequences are similar to enzymes classified as beta-glucosidases and although they hydrolyze 4-nitrophenyl beta-glucoside, their substrate specificities and action patterns are more typical of polysaccharide exohydrolases of the (1,4)-beta-glucan glucohydrolase type. Both the beta-glucan exohydrolase isoenzyme ExoI and the beta-glucosidase isoenzyme betaII release single glucosyl residues from the nonreducing ends of substrates and proton-NMR shows that anomeric configurations are retained during hydrolysis by both classes of enzyme. These results raise general questions regarding the distinction between polysaccharide exohydrolases and glucosidases, together with more specific questions regarding the functional roles of the two classes of enzyme in germinating barley grain.
Hrmova
M
M
Department of Plant Science, University of Adelaide, Waite Campus, Glen Osmond, S.A. 5064, Australia.
Harvey
A J
AJ
Wang
J
J
Shirley
N J
NJ
Jones
G P
GP
Stone
B A
BA
Høj
P B
PB
Fincher
G B
GB
D000595:Amino Acid Sequence; D001483:Base Sequence; D002240:Carbohydrate Sequence; D043366:Cellulose 1,4-beta-Cellobiosidase; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015723:Gene Library; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D001467:Hordeum / Q000201:enzymology*; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D018076:DNA, Complementary; D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D009844:Oligosaccharides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D001617:beta-Glucosidase; D043366:Cellulose 1,4-beta-Cellobiosidase