| Summary: | 碩士 === 國立臺灣大學 === 植物科學研究所 === 103 === Histone Deacetylases (HDACs) play a role to remove acetyl group on the histone in the cell and alter interaction between histone and DNA in chromatin to condense for regulating the gene expression, which is a kind of suppressors to repressthe transcription of genes.
Arabidopsis thaliana histone deacetylase 5 (AtHDA5), a homolog of yeast RPD3 class II, contains the histone deacetylase (HD) conserved domain. The N terminal of AtHDA5 from 10th -26th was predicted as nuclear localization signal (NLS) and the C terminal showed highly variable region. Since the plant histone deacetylases remained undiscovered in molecular levels, it worths to study the molecular function and protein structures.
In this study, we aimed to construct the recombinant AtHDA5 with a strategy from MBP (Maltose binding protein) fusion tag for protein expression and purification. Interestingly, AtHDA5 showed two different sizes of polysome and monomer form after the analyses of size-exclusion chromatography and dynamic light scattering. To confirm the enzymatic function, Boc-Lys(Ac)-AMC substrate was used to check the activity of polysome and monomer form. To identify the phosphorylation sites, the mutants of AtHDA5 ND26, AtHDA5 S7/8A and AtHDA5 S7A were prepared and showed the enzymatic activity decreased with 26%-33% compared to wildtype. The mutant with mimic phosphorylation S7/8D could rescue the 15% activity from S7/8A. The results showed that phosphorylation in AtHDA5 would regulate the enzyme activity. Truncated C-terminal (residues 385-661) caused the loss of enzyme activity of histone deacetylase. Subcelluar localization confirmed AtHDA5 located at cytoplasm.
Transmission electron microscopy was used to reconstruct a low resolution of 3D model of AtHDA5. The preliminary structure of MBP-AtHDA5 shows a monomeric form (~18 Å). A homology modeling of AtHDA5 was generated with the template of human HDAC4 (pdbid: 2VQO). Furthermore, the AtHDA5 model was used to reveal the substrate Boc-Lys(Ac)-AMC in the binding pocket of AtHDA5. The structural and functional analyses would provide the insight into the molecular mechanisms for regulating gene expression of plants.
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