Chain‐length specificities of maize starch synthase I enzyme: studies of glucan affinity and catalytic properties

Abstract

SummaryIt is widely known that some of the starch synthases and starch-branching enzymes are trapped inside the starch granule matrix during the course of starch deposition in amyloplasts. The objective of this study was to use maize SSI to further our understanding of the protein domains involved in starch granule entrapment and identify the chain-length speci®cities of the enzyme. Using af®nity gel electrophoresis, we measured the dissociation constants of maize SSI and its truncated forms using various glucans. The enzyme has a high degree of speci®city in terms of its substrate±enzyme dissociation constant, but has a greatly elevated af®nity for increasing chain lengths of a-1, 4 glucans. Deletion of the N-terminal arm of SSI did not affect the K d value. Further small deletions of either N-or C-terminal domains resulted in a complete loss of any measurable af®nity for its substrate, suggesting that the starch-af®nity domain of SSI is not discrete from the catalytic domain. Greater af®nity was displayed for the amylopectin fraction of starch as compared to amylose, whereas glycogen revealed the lowest af®nity. However, when the outer chain lengths (OCL) of glycogen were extended using the phosphorylase enzyme, we found an increase in af®nity for SSI between an average OCL of 7 and 14, and then an apparently exponential increase to an average OCL of 21. On the other hand, the catalytic ability of SSI was reduced several-fold using these glucans with extended chain lengths as substrates, and most of the label from [ 14 C]ADPG was incorporated into shorter chains of dp < 10. We conclude that the rate of catalysis of SSI enzyme decreases with the OCL of its glucan substrate, and it has a very high af®nity for the longer glucan chains of dp »20, rendering the enzyme catalytically incapable at longer chain lengths. Based on the observations in this study, we propose that during amylopectin synthesis shorter A and B 1 chains are extended by SSI up to a critical chain length that soon becomes unsuitable for catalysis by SSI and hence cannot be elongated further by this enzyme. Instead, SSI is likely to become entrapped as a relatively inactive protein within the starch granule. Further glucan extension for continuation of amylopectin synthesis must require a handover to other SS enzymes which can extend the glucan chains further or for branching by branching enzymes. If this is correct, this proposal provides a biochemical basis to explain how the speci®cities of various SS enzymes determine and set the limitations on the length of A, B, C chains in the starch granule.

Starch accumulation, activities of key enzyme and gene expression in starch synthesis of wheat endosperm with different starch contents

“…It is interesting that, among the SS genes, SSII appeared to be the most vigorously expressed, more than SSI or SSIII in E28 (high starch content), whereas SSI and SSIII are known to account for the major SS activities in cereal endosperm (Ball and Morell 2003). SS genes are exclusively involved in amylopectin biosynthesis, and their distribution within the plastid between the stroma and starch granules varies between species, tissue, and developmental stages (Commuri and Keeling 2001). Biochemical evidence suggests that SS-I is primarily responsible for the synthesis of the shortest glucan chains with a degree of polymerization (DP) of 10 glucosyl units or less (Li et al 2000), and further extension of longer chains is achieved by the activities of SS-II and SS-III isoforms (Li et al 2003).…”

Function and Characterization of Starch Synthase I Using Mutants in Rice

“…The N-terminal extension of SSI was thought to be important for its binding with starch granules (ImparlRadosevich et al, 1998). However, Commuri and Keeling (2001) found that the mobility of both fulllength SSI and N-terminally truncated SSI are similarly retarded on native gels containing 2% soluble starch, indicating that the N-terminal extension of SSI is not important for SSI interaction with glucans. To understand the function of SSI, Guan and Keeling (1998) analyzed the chain-length distribution of polyglucans synthesized by different combinations of recombinant maize SS and maize BE isoforms in E. coli and found that SSI preferentially synthesized short chains (degree of polymerization [DP] 6-15).…”

“…To understand the function of SSI, Guan and Keeling (1998) analyzed the chain-length distribution of polyglucans synthesized by different combinations of recombinant maize SS and maize BE isoforms in E. coli and found that SSI preferentially synthesized short chains (degree of polymerization [DP] 6-15). Commuri and Keeling (2001) reported that maize SSI enzyme, despite having the greatest affinity for longer glucans (DP . 20) based on the measurement of dissociation constants, preferentially elongates shorter glucans with DP , 10.…”

Recent progress toward understanding the role of starch biosynthetic enzymes in the cereal endosperm

“…SSI appears to be primarily responsible for the synthesis of short glucan chains, as evidenced from SSI mutants in Arabidopsis [52]. Further investigation of the chain-length specificities of maize endosperm SSI has suggested that SSI prefers the shortest amylopectin chains as substrates [53]. The isoforms of SS and other enzymes often exhibit a DP range where they dominate over other isoforms, but it is important to be aware that "dominate" does not mean that other isoforms have no effects in that range.…”