The 88 KD DEBRANCHING ENZYME MISSING IN GLYCOGEN ACCUMULATING MUTANTS OF CLAMYDOMONAS REINHARDTII DISPLAYS AN ISOAMYLASE-TYPE SPECIFICITY

Abstract

International audienceTo investigate the functions of debranching enzymes in starch biosynthesis, we have partially purified and characterized these activities from wild-type and mutant sta7 Clamydomonas Reinhardtii. Two distinct debranching enzymes of 95 and 88 kD were detected. The 88 kD enzyme behaved as a part of a very large homo and heteromultimeric complex containing a minimum of 4 subunits. The 95 kD debranching enzyme cleaved -1,6 linkages separated by as few as 3 glucose residues while the multimeric complex containing the 88 kD hydrolase was unable to do so. Both enzymes were able to debranch amylopectin efficiently while the -1,6 linkages of glycogen were completely debranched by the 88 kD hydrolase only. Therefore the 95 and 88 kD debranching enzymes display respectively the limit-dextrinase (pullulanase) and isoamylase-type specificities. Various mutations in the STA7 locus caused the loss of the 88 kD isoamylase. At variance with the results obtained from maize and rice, however, the isoamylase deficiency did not result in any qualitative or quantitative difference in pullulanase activity. Morever, because the isoamylase activity accounted for over 95% of the total debranching enzyme activity we believe that the relative abundance of both types of debranching enzymes differs markedly from that found in vascular plants. The consequences of these findings with respect to the recently proposed mechanism for plant amylopectin synthesis are discussed