Biochemical characterization of wild-type and mutant isoamylases of Chlamydomonas reinhardtii supports a function of the multimeric enzyme organization in amylopectin maturation.
Chlamydomonas reinhardtii mutants of the STA8 gene produce reduced amounts of high amylose starch and phytoglycogen. In contrast to the previously described phytoglycogen-producing mutants of C. reinhardtii that contain no residual isoamylase activity, the sta8 mutants still contained 35% of the normal amount of enzyme activity. We have purified this residual isoamylase and compared it with the wild-type C. reinhardtii enzyme. We have found that the high-mass multimeric enzyme has reduced its average mass at least by one-half. This coincides with the disappearance of two out of the three activity bands that can be seen on zymogram gels. Wild-type and mutant enzymes are shown to be located within the plastid. In addition, they both act by cleaving off the outer branches of polysaccharides with no consistent difference in enzyme specificity. Because the mutant enzyme was demonstrated to digest phytoglycogen to completion in vitro, we propose that its inability to do so in vivo supports a function of the enzyme complex architecture in the processing of pre-amylopectin chains.
Dauvillée
D
D
Laboratoire de Chimie Biologique, Unité Mixte de Recherche du Centre National de la Recherche Scientifique, No. 8576, Université des Sciences et Technologies de Lille, 59655 Villeneuve d'ascq cedex, France.
Colleoni
C
C
Mouille
G
G
Morell
M K
MK
d'Hulst
C
C
Wattebled
F
F
Liénard
L
L
Delvallé
D
D
Ral
J P
JP
Myers
A M
AM
Ball
S G
SG
D000687:Amylopectin / Q000096:biosynthesis*; D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000235:genetics*; D002736:Chloroplasts / Q000201:enzymology; D017343:Genes, Plant; D007517:Isoamylase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D016254:Mutagenesis, Insertional; D011134:Polysaccharides / Q000096:biosynthesis
D007527:Isoenzymes; D011134:Polysaccharides; D000687:Amylopectin; D007517:Isoamylase