阿拉伯芥組蛋白去乙醯基酶HDA15之蛋白質結構、多聚體形成及酵素活性分析

Crystal Structure, Oligomerization and Enzymatic Analyses of Arabidopsis Histone Deacetylase 15

許哲誠(Jhe-Cheng Hsu)

指導教授 ： 鄭貽生

國立臺灣大學/生命科學院/植物科學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201602321

摘要

植物組蛋白去乙醯基酶(Histone deacetylases, HDAs)參與調控植物的生長與發育。組蛋白去乙醯基酶主要作用於組蛋白上的離胺酸(Lysine)的末端，當組蛋白失去了乙醯基的修飾後，染色質濃縮，進而造成該區基因無法表現。HDA15是屬於第二群組蛋白去乙醯基酶，HDA15可與PIF3(Pytochrome interacting factor 3)交互作用並去除組蛋白H3與H4上的乙醯基修飾，進而調控葉綠素的生合成路徑。目前部分的訊息調控雖已被研究，但其酵素作用之分子機制仍不清楚。
本研究利用大腸桿菌異源系統，表達植物第二群組蛋白去乙醯基酶的去乙醯基功能區(HDA5 HD、HDA15 HD與HDA18 HD)之重組蛋白質。在蛋白質純化過程中發現HDA15 HD為四聚體且具有較高的酵素活性，經管柱液相層析實驗及生化分析，測定HDA15 HD四聚體與單體的酵素活性，同時比較HDA5與HDA18 的去乙醯基功能區，結果顯示HDA15四聚體活性最高。若將HDA15的N端123胺基酸補回HDA15 HD，其酵素活性可提高近五倍；由膠體過濾層析與高效液相層析實驗發現，HDA15 HD四聚體在加回N端後會轉變成三聚體，顯示N端具有影響酵素活性及多聚體形成的能力。若以膠體過濾層析與生化實驗分析HDA15.1與HDA15.2同形蛋白(isoforms)，結果顯示C端具有抑制酵素活性功能，並可促進HDA15多聚體的形成。HDA15 HD之結晶結構顯示為四聚體，各個單體交互作用區接近酵素活性中心，具有穩定活性中心胺基酸的能力，促使酵素催化活性上升。利用HD功能區結構比對，雖然HD核心結構相似，但HDA15 HD多聚體的形成方式多變，其影響酵素活性上升的方式有其獨特性，推測HDA15去乙醯基酶活性受其C端與其他調控因子的影響。本篇研究解析植物第一個組蛋白去乙醯基酶晶體結構，其酵素活性與多聚體形成有關，藉由此結構與功能分析，將有助於明瞭去乙醯基酶的分子調控機制。

關鍵字

組蛋白去乙醯基酶 ； 多聚體 ； 蛋白質結構 ； 酵素活性

並列摘要

Histone deacetylases (HDAs) involved in growth and developmental processes in plants. HDAs remove the acetyl group from Lysine residue on histone tail and cause chromatin condensation for suppressing gene expression. HDA15 is a member of class II HDA. HDA15 interacts with PIF3, a transcription factor, to repress biosynthesis of chlorophyll by reomoving acetyl modification on H3 and H4 histones. Although several study reveal HDA15 how to control physiology regulation, but its molecular mechanism is still unknown.
In this sudy, an overexpression system of Escherichia coli was used to produce recombinant protein of Histone deacetylase domain (HD domain) of all class II HDAs, including HDA5, HDA15, and HDA18. During protein purification, HDA15 HD showed much higher enzyme activity. Gel filtration chromatography revealed that HDA15 HD contained two forms of tetramer and monomer in solution, but HDA5 HD and HDA18 HD showed only monomer. In biochemical analyses, HDA15 HD tetramer showed the highest enzyme activity than those of HDA5 HD monomer, HDA15 HD monomer and HDA18 HD monomer. For restoring 123 residues of N terminus to HD of HDA15, it became to trimer and its enzyme activity was 5-fold higher than that of HDA15 HD tetramer. The results demonstated the Nt of HDA15 would affect the enzymatic activity and its oligomerization. Since two isoforms HDA15.1 and HDA15.2 with slightly difference of C-terminus existed in plants, the results from gel filtration chromatography and HPLC revealed that HDA15.1 and HDA15.2 contained no deacetylase activity and fromed aggregation. It showed HDA15 C-terminus could regulate the deacetylase activity and affect its aggregation. X-ray crystallography showed crystal structure of HDA15 HD is tetramer. The interaction interface of tetramer is nearby active site and then stabilizes the residues of active site. Finally, tetramer form could enhance its enzyme activity. By superimposition of HD structures, the overall structures of HD are similar. However, HDA15 shows its various oligomerization and regulates the enzyme activity with N-terminus and C-terminus. This is the first structure of histone deacetylase domain from plant. Its oligomerizaion showed correlation with its enzymatic activity. From structural and fuctional studies, it will provide a new insight into molecular mechanism in histone deacetylases.

並列關鍵字

Histone deacetylase ； Oligomerization ； Protein structure ； Enzyme activity

參考文獻

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