Arabidopsis β-Amylase2 Is a K+-Requiring, Catalytic Tetramer with Sigmoidal Kinetics

Abstract

The Arabidopsis (Arabidopsis thaliana) genome contains nine β-amylase (BAM) genes, some of which play important roles in starch hydrolysis. However, little is known about BAM2, a plastid-localized enzyme reported to have extremely low catalytic activity. Using conservation of intron positions, we determined that the nine Arabidopsis BAM genes fall into two distinct subfamilies. A similar pattern was found in each major lineage of land plants, suggesting that these subfamilies diverged prior to the origin of land plants. Moreover, phylogenetic analysis indicated that BAM2 is the ancestral member of one of these subfamilies. This finding, along with the conservation of amino acids in the active site of BAM2, suggested that it might be catalytically active. We then identified KCl as necessary for BAM2 activity. Unlike BAM1, BAM3, and BAM5, three Arabidopsis BAMs that all exhibited hyperbolic kinetics, BAM2 exhibited sigmoidal kinetics with a Hill coefficient of over 3. Using multi-angle light scattering, we determined that BAM2 was a tetramer, whereas BAM5 was a monomer. Conserved residues from a diverse set of BAM2 orthologs were mapped onto a homology model of the protein, revealing a large, conserved surface away from the active site that we hypothesize is a secondary carbohydrate-binding site. Introduction of bulky methionine for glycine at two points on this surface reduced catalytic activity significantly without disrupting the tetrameric structure. Expression analysis indicated that BAM2 is more closely coexpressed with other starch degradation enzymes than any other BAM, suggesting that BAM2 may play an important role in starch degradation in plants.

Figure 1.

Conserved intron positions and evolutionary history of Arabidopsis BAM genes. A, Black lines represent aligned amino acid sequences, and white breaks indicate intron positions. Vertical gray lines represent conserved intron positions within multiple genes. B, Dots represent the presence of BAM genes within the genomes of species listed in the text. BAM genes in a single row were judged to be orthologous based on sequence alignments and domain structure. Predictions of catalytic activity or inactivity were based on the conservation of active site residues.

Figure 2.

Conservation in the nine Arabidopsis BAMs of 15 starch-binding active site residues identified in soybean BAM5 (BMY1) by Laederach et al. (1999). Subsites 1 to 4 refer to the four Glc residues at the nonreducing end of the substrate. Residues in each Arabidopsis BAM that differ from the corresponding residues in the soybean BAM are shaded gray.

Figure 3.

Effect of salts on the activity of purified BAM2 using 100 mg mL⁻¹ soluble starch as the substrate. A, Effect of KCl on the specific activity of BAM2. B, Effect of 100 mm salts on the activity of BAM2 using 100 mg mL⁻¹ soluble starch. Activity is relative to that with KCl, and columns represent means ± sd (n = 3).

Figure 4.

Effect of soluble starch concentration on the activity of purified Arabidopsis BAM1, BAM2, BAM3, and BAM5. All points are relative to the highest rate measured for each enzyme and represent duplicate assays.

Figure 5.

SEC-MALS elution profiles (representative) and masses of BAM2 and BAM5 from Arabidopsis. Purified proteins were in 10 mm MOPS, pH 7, and 250 mm KCl. kDa-C is the calculated mass from the mature amino acid sequence of the expressed proteins, and kDa-M is the average mass of the active protein measured using MALS ± sd. For BAM2, n = 6; for BAM5, n = 4.

Figure 6.

Homology structures of Arabidopsis BAM2 and BAM5 illustrating conserved, surface-exposed residues. A, Structure of BAM2, viewing the active site in which nonconserved residues are colored gray and conserved residues are colored red. B, Same structure as in A but rotated 90°. Two conserved Gly residues that were mutated to Met are colored yellow. C, Structure of BAM5, viewing the active site colored as in A. D, Same structure as in C but rotated 90°.

Figure 7.

Catalytic activity of wild-type (WT) BAM2 and two mutants of BAM2 generated by site-directed mutagenesis in which Gly residues at positions 335 and 446 were changed to Met. Assays were conducted with 100 mg mL⁻¹ soluble starch and 250 mm KCl. Bars represent activity ± sd (n = 3).

Figure 8.

Coexpression analysis from the ATTED-II database (http://atted.jp/; Aoki et al., 2016) for each Arabidopsis BAM gene with 12 genes involved in starch degradation. Significance levels as provided by the ATTED-II database are P < 0.001 (***) and P < 0.01 (**).