Modelling and comparison of in silico mutagenesis generated ADP-glucose pyrophosphorylase large subunits in rice (Oryza sativa L.) with Rev6 mutant of maize (Zea mays L.)
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Date
2013
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CCSHAU
Abstract
ADP – Glucose Phosphorylase (AGPase) is a key enzyme for starch synthesis in plant kingdom. It is a heterotetrameric enzyme consisting of two large subunits (LS) encoded by Shrunken-2 (Sh2) and two small subunit by Brittle-2 (Bt2). Manipulation of AGPase is a prime target to increase starch production and in turns crop yield as observed in maize where Rev6 mutated structure of AGPase resulted in increase in starch content & kernal weight. In spite of 93% identity in amino acid sequences of AGPase LS in maize and rice, the crop yield increase was not observed in rice with same insertion as in Rev6 mutant of maize. In this study, in silico mutagenesis in normal LS sequence of AGPase in rice was carried out using Mutation Pressure Simulator. The mutation in Sh2 gene (LS) involved substitution mutations in sequence SDHPEE and insertion of 6 nucleotides in Sh2 gene, thereby insertion of two amino acid residues Ser and Tyr at specific position. Then, the homology modeling of the normal and 9 mutated LS from rice was done using MODELLER9.11. The models were refined for energy minimization using GROMOS force field and verified with various structure refinement programs at WHATIF Server. The models were further evaluated using PROCHECK, VERIFY3D and ERRAT from SAVES. All the models were visualized in UCSF Chimera and in normal LS of rice AGPase, secondary structure accounted by six amino acids (SDHPEE) sequence just before the site of insertion of additional amino acid (S,Y) was observed. This secondary structure was also present in S490T, D491N and D491E mutants but it got disappeared similar to Rev6 mutant of maize in S490A, P493A, E494Q, E495D and E495Q mutants. These mutated models of rice were compared with normal LS of rice and Rev6 mutant of maize using Superpose Version1.0. The RMSD was found to be < 2 Å for all mutants with respect to normal LS whereas only P493A had RMSD 1.76Å (below 2Å) with respect to Rev6 mutant. The surface topography analysis using CASTp Server showed that the number of binding pockets increased (81 -90) in all mutants as compared to normal LS (69) except P493A in which it reduced to 65. Lastly, the surface topology of the rice mutants was compared with Rev6 mutant to find the most similar mutant. The only rice mutant that displayed almost similar changes in surface topology like Rev6 mutant of maize was observed to be P493A. As P493A mutant of rice was found to be the most similar to Rev6 mutant as evident by 1.76Å RMSD and quite similar structural and topology differences so it may be expected to behave in rice similar to the Rev6 mutant of maize.
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Keywords
ADP-glucose pyrophosphorylase, In silico mutagenesis, Homology modeling, Structural superimposition, Surface topography