Abstract
ADP-glucose pyrophosphorylase (AGPase), a key allosteric enzyme involved in higher plant starch biosynthesis, is composed of pairs of large (LS) and small subunits (SS). Current evidence indicates that the two subunit types play distinct roles in enzyme function. Recently the heterotetrameric structure of potato AGPase has been modeled. In the current study, we have applied the molecular mechanics generalized born surface area (MM-GBSA) method and identified critical amino acids of the potato AGPase LS and SS subunits that interact with each other during the native heterotetrameric structure formation. We have further shown the role of the LS amino acids in subunit-subunit interaction by yeast two-hybrid, bacterial complementation assay and native gel. Comparison of the computational results with the experiments has indicated that the backbone energy contribution (rather than the side chain energies) of the interface residues is more important in identifying critical residues. We have found that lateral interaction of the LS-SS is much stronger than the longitudinal one, and it is mainly mediated by hydrophobic interactions. This study will not only enhance our understanding of the interaction between the SS and the LS of AGPase, but will also enable us to engineer proteins to obtain better assembled variants of AGPase which can be used for the improvement of plant yield.
Highlights
ADP-glucose pyrophosphorylase (AGPase) is a key regulatory allosteric enzyme involved in starch biosynthesis in higher plants
To determine the critical amino acid residues of the potato AGPase LS that interact with potato AGPase SS, we performed molecular mechanics generalized born surface area (MM-GBSA) method which calculates the binding free energy and decomposes the energy at the amino acid level
These results suggested that altering amino acid residues of hot spots of the LS disturbed the heterotetrameric AGPase assemblies in E. coli
Summary
ADP-glucose pyrophosphorylase (AGPase) is a key regulatory allosteric enzyme involved in starch biosynthesis in higher plants. SS was shown to have both catalytic and regulatory functions whereas LS is mainly responsible for regulating the allosteric properties of SS [9,10,11,12] These results were supported by the studies that showed LS was incapable of assembling into a catalytically active oligomeric structure, whereas SS was able to form a homotetramer with catalytic properties [9,13]. Using chimeric maize/potato small subunits, Cross et al [19] found a polymorphic motif in the SS which is critical for subunit interaction They have concluded that a 55-amino acid region between the residues 322–376 directly interacts with LS and significantly contributes to the overall enzyme stability
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