Specificity of starch synthase isoforms from potato

Abstract

In higher plants several isoforms of starch synthase contribute to the extension of glucan chains in the synthesis of starch. Different isoforms are responsible for the synthesis of essentially linear amylose chains and branched, amylopectin chains. The activity of granule-bound starch synthase I from potato has been compared with that of starch synthase II from potato following expression of both isoforms in Escherichia coli. Significant differences in their activities are apparent which may be important in determining their specificities in vivo. These differences include affinities for ADPglucose and glucan substrates, activation by amylopectin, response to citrate, thermosensitivity and the processivity of glucan chain extension. To define regions of the isoforms determining these characteristic traits, chimeric proteins have been produced by expression in E. coli. These experiments reveal that the C-terminal region of granule-bound starch synthase I confers most of the specific properties of this isoform, except its processive elongation of glucan chains. This region of granule-bound starch synthase I is distinct from the C-terminal region of other starch synthases. The specific properties it confers may be important in defining the specificity of granule-bound starch synthase I in producing amylose in vivo.Keywords: amylopectin; amylose; starch synthase.In plants, starch synthases form starch by catalysing the transfer of the glucosyl moiety from ADPglucose to the nonreducing ends of glucan chains through an a1±4 linkage. Starch is composed of two types of glucan polymer: amylose, a predominantly linear a1±4-linked glucan and amylopectin, in which the a1±4 linked chains are highly branched by a1±6 linkages, which are formed by starch branching enzymes. Several isoforms of starch synthase are involved in starch formation and these can be classified according to their localization within the plastid; one isoform, granule-bound starch synthase I (GBSSI), is bound exclusively to the starch granule and others are localized predominantly in the soluble phase (stroma) of the plastid. In general, GBSSI is a protein of < 60 kDa. Through mutant analysis GBSSI is known to be solely responsible for the synthesis of amylose, because the waxy mutants of cereals, the amf mutant of potato, the lam mutants of pea and GBSSI antisense potato lines all involve the reduction or elimination of GBSSI activity and a specific reduction or elimination of amylose in the starch of the storage organs of these species [1±4].Among the starch synthase isoforms found in the plastid stroma of higher plants, starch synthase II (SSII) is also found bound to starch granules in significant amounts [5±9]. However, SSII cannot compensate for the effects of loss of GBSSI on amylose synthesis in amf mutant potatoes, indicating that SSII is not involved in amylose synthesis in vivo [8]. In the storage organs of different higher plants, SSII may make quite different quantitative contributions to total starch synthase activity. Mutant analysis in pea...

Discrete Forms of Amylose Are Synthesized by Isoforms of GBSSI in Pea[W]

“…The two mature peptide sequences are 73% identical (80% similar). Both possess the N-terminal KTGGL motif thought to be responsible for binding ADP/ADP-Glc and the C-terminal KTGGL "look-alike" motif that has been shown to influence the kinetic properties of potato GBSSI in vitro (Edwards et al, 1999a).…”

“…The mechanisms by which different isoforms of starch synthase catalyze the same reaction and yet generate polymer variation are not fully understood, but there is increasing evidence to suggest that isoform localization within the plastid and fundamental differences in kinetics and reaction mechanisms are important determinants of product specificity Denyer et al, 1999;Edwards et al, 1999a;Imparl-Radosevich et al, 1999;Commuri and Keeling, 2001). Furthermore, recent work has suggested that GBSSI, like the soluble starch synthases, may exist as more than one isoform.…”

Interaction with amylopectin influences the ability of granule-bound starch synthase I to elongate malto-oligosaccharides

“…The fact that amylopectin is an effector of GBSSI with respect to its activity on malto-oligosaccharide raises the possibility that it acts as both a substrate and an effector in assays which contain amylopectin alone. We are investigating this further (see [20]). …”

“…The activity of the enzyme is not stimulated by amylopectin and its distributive mode of action on malto-oligosaccharides is not altered by the addition of amylopectin. Further work is in progress to define the structural features of GBSSI and SSII responsible for their differing modes of elongation of malto-oligosaccharides and their interaction with amylopectin (see [20]). …”

“…However, use of the Michaelis-Menten equation to calculate a K m value for amylopectin may not be appropriate as, at least under some conditions, amylopectin can act as an effector as well as a substrate of GBSSI (see below). In addition, changes in the physical properties of solutions of amylopectin with increasing concentration can lead to activation of GBSSI [20].…”