Phylogenetic analysis of ADP-glucose pyrophosphorylase subunits reveals a role of subunit interfaces in the allosteric properties of the enzyme

Abstract

ADP-glucose pyrophosphorylase (AGPase) catalyzes a rate-limiting step in glycogen and starch synthesis in bacteria and plants, respectively. Plant AGPase consists of two large and two small subunits that were derived by gene duplication. AGPase large subunits have functionally diverged, leading to different kinetic and allosteric properties. Amino acid changes that could account for these differences were identified previously by evolutionary analysis. In this study, these large subunit residues were mapped onto a modeled structure of the maize (Zea mays) endosperm enzyme. Surprisingly, of 29 amino acids identified via evolutionary considerations, 17 were located at subunit interfaces. Fourteen of the 29 amino acids were mutagenized in the maize endosperm large subunit (SHRUNKEN-2 [SH2]), and resulting variants were expressed in Escherichia coli with the maize endosperm small subunit (BT2). Comparisons of the amount of glycogen produced in E. coli, and the kinetic and allosteric properties of the variants with wild-type SH2/BT2, indicate that 11 variants differ from the wild type in enzyme properties or in vivo glycogen level. More interestingly, six of nine residues located at subunit interfaces exhibit altered allosteric properties. These results indicate that the interfaces between the large and small subunits are important for the allosteric properties of AGPase, and changes at these interfaces contribute to AGPase functional specialization. Our results also demonstrate that evolutionary analysis can greatly facilitate enzyme structure-function analyses.

Figure 1.

Amino acid alignment between maize endosperm (SH2) and potato tuber large subunit. Red boxes indicate sites that make direct contact with the small subunit as determined by Tuncel et al. (2008). Blue and orange arrows indicate type II and positively selected sites, respectively. Sites subjected to mutagenesis in this study are marked with asterisks.

Figure 2.

AGPase subunit interactions. The white structures correspond to the resolved structure of potato tuber small subunit homodimers. Cyan and magenta modeled structures of the small (BT2) and large (SH2) subunits of maize endosperm, respectively, are superimposed on the structure of potato tuber small subunit homodimers. Red circles indicate the candidate Pi-binding sites. A, Head-to-head subunit interaction. B, Tail-to-tail subunit interaction.

Figure 3.

Superimposition of maize endosperm large subunit (SH2) modeled structure (magenta) on the potato tuber large subunit modeled structure (white). Red areas indicate sites in the potato tuber large subunit that are proposed to make direct contact with the small subunit (Tuncel et al., 2008).

Figure 4.

Placement of all type II and positively selected amino acids on the subunit interfaces of maize endosperm large subunit (SH2). Type I sites 149 and 361 that were changed by site-directed mutagenesis are also placed on the structure of SH2. SH2 modeled structure (green) was superimposed on potato tuber large subunit modeled structure (white). Red areas indicate sites in the potato tuber large subunit that are proposed to make direct contact with the small subunit (Tuncel et al., 2008). Type I, type II, and positively selected sites detected by Georgelis et al. (2008) are shown in yellow. A, Areas that participate in tail-to-tail interactions. B, Areas that participate in head-to-head interactions.

Figure 5.

Glycogen produced by SH2 wild type and variants expressed in E. coli along with BT2. Asterisks indicate significant differences compared with wild-type BT2/SH2 at P = 0.05 (Student's t test; n = 4). [See online article for color version of this figure.]

Figure 6.

Heat stability of SH2 wild type and variants in a complex with BT2. The asterisk indicates a significant difference compared with wild-type BT2/SH2 at P = 0.05 (Student's t test; n = 6). [See online article for color version of this figure.]

Figure 7.

Western blot of protein extracts from E. coli cells expressing SH2, V502T, and A508S along with BT2. [See online article for color version of this figure.]