Dataset Information

Quaternary Structure, Salt Sensitivity, and Allosteric Regulation of ?-AMYLASE2 From Arabidopsis thaliana.

ABSTRACT: The ?-amylase family in Arabidopsis thaliana has nine members, four of which are both plastid-localized and, based on active-site sequence conservation, potentially capable of hydrolyzing starch to maltose. We recently reported that one of these enzymes, BAM2, is catalytically active in the presence of physiological levels of KCl, exhibits sigmoidal kinetics with a Hill coefficient of over 3, is tetrameric, has a putative secondary binding site (SBS) for starch, and is highly co-expressed with other starch metabolizing enzymes. Here we generated a tetrameric homology model of Arabidopsis BAM2 that is a dimer of dimers in which the putative SBSs of two subunits form a deep groove between the subunits. To validate this model and identify key residues, we generated a series of mutations and characterized the purified proteins. (1) Three point mutations in the putative subunit interfaces disrupted tetramerization; two that interfered with the formation of the starch-binding groove were largely inactive, whereas a third mutation prevented pairs of dimers from forming and was active. (2) The model revealed that a 30-residue N-terminal acidic region, not found in other BAMs, appears to form part of the putative starch-binding groove. A mutant lacking this acidic region was active and did not require KCl for activity. (3) A conserved tryptophan residue in the SBS is necessary for activation and may form ?-bonds with sugars in starch. (4) Sequence alignments revealed a conserved serine residue next to one of the catalytic glutamic acid residues, that is a conserved glycine in all other active BAMs. The serine side chain points away from the active site and toward the putative starch-binding groove. Mutating the serine in BAM2 to a glycine resulted in an enzyme with a VMax similar to that of the wild type enzyme but with a 7.5-fold lower KM for soluble starch. Interestingly, the mutant no longer exhibited sigmoidal kinetics, suggesting that allosteric communication between the putative SBS and the active site was disrupted. These results confirm the unusual structure and function of this widespread enzyme, and suggest that our understanding of starch degradation in plants is incomplete.

SUBMITTER: Monroe JD

PROVIDER: S-EPMC6102588 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Quaternary Structure, Salt Sensitivity, and Allosteric Regulation of β-AMYLASE2 From <i>Arabidopsis thaliana</i>.

Monroe Jonathan D JD Pope Lauren E LE Breault Jillian S JS Berndsen Christopher E CE Storm Amanda R AR

Frontiers in plant science 20180814

The β-amylase family in <i>Arabidopsis thaliana</i> has nine members, four of which are both plastid-localized and, based on active-site sequence conservation, potentially capable of hydrolyzing starch to maltose. We recently reported that one of these enzymes, BAM2, is catalytically active in the presence of physiological levels of KCl, exhibits sigmoidal kinetics with a Hill coefficient of over 3, is tetrameric, has a putative secondary binding site (SBS) for starch, and is highly co-expressed ...[more]

PMID: 30154813

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Project description:Plant growth promoting rhizobacteria (PGPR) are a functionally diverse group of microbes having immense potential as biostimulants and stress alleviators. Their exploitation in agro-ecosystems as an eco-friendly and cost-effective alternative to traditional chemical inputs may positively affect agricultural productivity and environmental sustainability. The present study describes selected rhizobacteria, from a range of origins, having plant growth promoting potential under controlled conditions. A total of 98 isolates (ectophytic or endophytic) from various crop and uncultivated plants were screened, out of which four endophytes (n, L, K and Y) from Phalaris arundinacea, Solanum dulcamara, Scorzoneroides autumnalis, and Glycine max, respectively, were selected in vitro for their vegetative growth stimulating effects on Arabidopsis thaliana Col-0 seedlings with regard to leaf surface area and shoot fresh weight. A 16S rRNA gene sequencing analysis of the strains indicated that these isolates belong to the genera Pseudomonas, Bacillus, Mucilaginibacter and Rhizobium. Strains were then further tested for their effects on abiotic stress alleviation under both Petri-plate and pot conditions. Results from Petri-dish assay indicated strains L, K and Y alleviated salt stress in Arabidopsis seedlings, while strains K and Y conferred increases in fresh weight and leaf area under osmotic stress. Results from subsequent in vivo trials indicated all the isolates, especially strains L, K and Y, distinctly increased A. thaliana growth under both normal and high salinity conditions, as compared to control plants. The activity of antioxidant enzymes (ascorbate peroxidase, catalase and peroxidase), proline content and total antioxidative capacity also differed in the inoculated A. thaliana plants. Furthermore, a study on spatial distribution of the four strains, using either conventional Petri-plate counts or GFP-tagged bacteria, indicated that all four strains were able to colonize the endosphere of A. thaliana root tissue. Thus, the study revealed that the four selected rhizobacteria are good candidates to be explored as plant growth stimulators, which also possess salt stress mitigating property, partially by regulating osmolytes and antioxidant enzymes. Moreover, the study is the first report of Scorzoneroides autumnalis (fall dandelion) and Solanum dulcamara (bittersweet) associated endophytes with PGP effects.

Dataset Information

Quaternary Structure, Salt Sensitivity, and Allosteric Regulation of ?-AMYLASE2 From Arabidopsis thaliana.

Publications

Quaternary Structure, Salt Sensitivity, and Allosteric Regulation of β-AMYLASE2 From <i>Arabidopsis thaliana</i>.

Similar Datasets

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