The Multiple Forms of Starch‐Branching Enzyme I in Solanum Tuberosum

Abstract

Western blot analysis showed the presence of three forms of starch-branching enzyme (SBE), with apparent molecular masses of 103, 97 and 80 kDa, in extracts of leaves and stored tubers of Solanum tuberosurn. The 80-kDa form was absent in extracts of fresh tuber. Active 80-kDa enzyme was partially purified from stored tubers and sequence analysis showed that it, similar to the two larger enzyme forms, was an SBE-I isoform. Limited proteolysis of isolated 103-kDa SBE-I under native conditions removed approximately 200 amino acid residues from the carboxy terminus. A stable intermediate with an apparent molecular mass of approximately 80 kDa was formed. Since the 80-kDa form displayed full enzymatic activity and its circular-dichroism spectrum did not differ significantly from that of the 103-kDa enzyme, the carboxy-terminal portion of the enzyme was suggested to have an extended, unordered structure and therefore to be easily accessible to proteolysis. A cDNA sequence encoding a mature SBE-I was amplified from tuber mRNA of S. tuberosum by means of PCR. The 3' end of this sequence differed significantly from that of previously published data. PCR amplification and DNA sequencing of the 3' ends of the sbel gene showed that four sbel alleles exist in the cultivar studied. Two of these four alleles, sbela and sbelb, had slightly longer 3' ends compared with the other two, sbelc and sbeld. The difference between the two groups of alleles was due to a partial deletion in sbelc and sbeld of a segment duplicated in all alleles. All four alleles were expressed in leaf and tuber.Keywords: starch ; branching enzyme ; allele ; Solanum tuberosum ; Solanurn commersonii.Amylose and amylopectin, the two main components of starch in higher plants, are two a-glucan polysaccharides with different structures and molecular masses. Amylose is essentially a linear (a-1,4 linked) helical molecule with a variable molecular mass (=105-106 Da), while amylopectin, the branched component of the starch, has a significantly higher molecular mass (=107-109 Da) and is the major compound of the starch (Manners, 1985).Starch-branching enzyme (SBE), one of the key enzymes in starch biosynthesis, catalyzes hydrolysis of a-l,4-glucosidic bonds with concomitant condensation of a-1,6-linkages in the amylopectin molecule. Biosynthesis of starch in all plants studied is catalyzed by the same type of enzymes , a number of which, such as starch synthases and branching enzymes, exist as different isoforms, as shown for Zea mays (maize; Ozbun et al

Molecular evolution accompanying functional divergence of duplicated genes along the plant starch biosynthesis pathway

“…Using the beb method we were able to highlight 17 and 32 sites in branches D1a and D1b, respectively (Figure 5B). The structure of the branching enzyme family and the presence of five catalytic sites included in conserved domains has been well described in Pisum sativum [31], Solanum tuberosum [32], Oryza sativa [33] and Sorghum bicolor [34]. However, none of the sites that we detected under selection matched to these catalytic domains.…”

Isolation of a cDNA Encoding a Granule-Bound 152-Kilodalton Starch-Branching Enzyme in Wheat

“…This loop differs in length between SBEI and SBEII isoforms (Burton et al, 1995) and has been suggested to determine spacing between the branches (Jespersen et al, 1993). The lack of the 163-amino acid segment may not impair the ability of domain 1 to bind glucan polymers, since removal of a similar approximately 20-kD C-terminal peptide from a 103-kD potato SBEI did not alter its activity (Khoshnoodi et al, 1996). However, how the missing segments on domain 1 may affect the interactions between domain 1 and glucan polymers or starch biosynthetic enzymes cannot be predicted until further investigations have been done.…”

Starch branching enzyme IIb in wheat is expressed at low levels in the endosperm compared to other cereals and encoded at a non-syntenic locus

“…E14723), pot be I (Potato SBE I, Khoshnoodi et al 1996), pot be II (potato SBE II, Cangiano et al 1993), pea be I and be II (Burton et al1995), E.coli be (Baecker et al 1986), and Bacillus (Kiel et al 1992). Note that pea SBEI and pea SBEII sequences correspond to maize SBEII and SBEI, respectively, through differences in nomenclature conventions wSBE II-DB1.…”