The debranching enzyme complex missing in glycogen accumulating mutants ofChlamydomonas reinhardtii displays an isoamylase-type specificity

To investigate the functions of debranching enzymes in starch biosynthesis, we have partially purified and characterized these activities from wild ty pe and mutant sta7 Chlamydomonas reinhardtii. Mutants of the STA7 locus sub stitute synthesis of insoluble granular starch by that of small amounts of glycogen-like material. The mutants were previously shown to lack an 88 kDa debranching enzyme. Two distinct debranching activities were detected in w ild-type strains. The 88 kDa debranching enzyme subunit missing in glycogen -producing mutants (CIS1) is shown to be part of a multimeric enzyme comple x. A monomeric 95 kDa debranching enzyme (CLD1) cleaved alpha-1,6 linkages separated by as few as three glucose residues while the multimeric complex was unable to do so. Both enzymes were able to debranch amylopectin while t he alpha-1,6 linkages of glycogen were completely debranched by the multime ric complex only. Therefore CLD1 and the multimeric debranching enzyme disp lay respectively the limit-dextrinase (pullulanase) and isoamylase-type spe cificities. Various mutations in the STA7 locus caused the loss of both CIS 1 and of the multimeric isoamylase complex. In contrast to rice and maize m utants that accumulate phytoglycogen owing to mutation of an isoamylase-typ e DBE, isoamylase depletion in Chlamydomonas did not result in any qualitat ive or quantitative difference in pullulanase activity. (C) 2000 Elsevier S cience Ireland Ltd. All rights reserved.