Received July 25, 2002
Accepted October 24, 2002
Three Isoforms of Isoamylase Contribute Different Catalytic Properties for the Debranching
of Potato Glucans
Hasnain Hussain 1, Alexandra Mant 2, Robert Seale 1, Sam Zeeman 1, Edward Hinchliffe 3, Anne Edwards 1, Christopher Hylton 1, Stephen Bornemann 1, Alison M. Smith 1, Cathie Martin 1*, and Regla Bustos 1
1
Departments of Cell and Developmental Biology, Metabolic Biology, and Biological
Chemistry, John Innes Centre, Colney, Norwich NR4 7UH, United Kingdom
2
Plant Biochemistry Laboratory, Department of Plant Biology, The Royal Veterinary
and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark
3
Astrazeneca 14.20 CTL, Alderley Park, Macclesfield, Cheshire SK10 4TJ, UK
* To whom correspondence should be addressed. E-mail: cathie.martin{at}bbsrc.ac.uk.
Isoamylases are debranching enzymes that hydrolyze 
-1,6 linkages in -1,4/-1,6-linked
glucan polymers. In plants, they have been shown to be required for the normal synthesis
of amylopectin, although the precise manner in which they influence starch synthesis
is still debated. cDNA clones encoding three distinct isoamylase isoforms (Stisa1,
Stisa2, and Stisa3) have been identified from potato. The expression patterns of
the genes are consistent with the possibility that they all play roles in starch
synthesis. Analysis of the predicted sequences of the proteins suggested that only
Stisa1 and Stisa3 are likely to have hydrolytic activity and that there probably
are differences in substrate specificity between these two isoforms. This was confirmed
by the expression of each isoamylase in Escherichia coli and characterization
of its activity. Partial purification of isoamylase activity from potato tubers showed
that Stisa1 and Stisa2 are associated as a multimeric enzyme but that Stisa3 is not
associated with this enzyme complex. Our data suggest that Stisa1 and Stisa2 act
together to debranch soluble glucan during starch synthesis. The catalytic specificity
of Stisa3 is distinct from that of the multimeric enzyme, indicating that it may
play a different role in starch metabolism.
