Understanding the pivotal step in cytokinin synthesis: crystal structure, substrate specificity and catalytic mechanism of adenylate isopentenyltransferase from H. lupulus
博士 === 臺灣大學 === 生化科學研究所 === 98 === Cytokinins are important plant hormones, and their biosynthesis most begins with the transfer of isopentenyl group from dimethylallyl diphosphate (DMAPP) to the N6-amino group of adenine by either adenylate isopentenyltransferase (AIPT) or tRNA-IPT. Plant AIPTs use...
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ndltd-TW-098NTU051030022015-10-13T13:40:02Z http://ndltd.ncl.edu.tw/handle/72718540522522540915 Understanding the pivotal step in cytokinin synthesis: crystal structure, substrate specificity and catalytic mechanism of adenylate isopentenyltransferase from H. lupulus 植物細胞分裂素生成的關鍵步驟:啤酒花腺苷酸異戊二烯基轉移酵素其晶體結構、受質特異性、與催化機制之研究 Hsing-Mao Chu 朱鑫懋 博士 臺灣大學 生化科學研究所 98 Cytokinins are important plant hormones, and their biosynthesis most begins with the transfer of isopentenyl group from dimethylallyl diphosphate (DMAPP) to the N6-amino group of adenine by either adenylate isopentenyltransferase (AIPT) or tRNA-IPT. Plant AIPTs use ATP/ADP as an isopentenyl acceptor and bacterial AIPTs prefer AMP, whereas tRNA-IPTs act on specific sites of tRNA. The crystal structure of an AIPT-ATP complex from Humulus lupulus (HlAIPT) presented here is similar to the previous structures of Agrobacterium AIPT and yeast tRNA-IPT. The enzyme is structurally homologous to the NTP-binding kinase family of proteins but forms a solvent-accessible channel that binds to the donor substrate DMAPP, which is directed toward the acceptor substrate ATP/ADP. When measured with isothermal titration calorimetry, some nucleotides displayed different binding affinities to HlAIPT with an order of ATP > dATP ~ ADP > GTP > CTP > UTP. However, further measurements of activity using these nucleotides showed that the order becomes ATP > ADP > dATP > CTP >> GTP ≈ UTP ≈ 0. In addition to DMAPP (C5), HlAIPT also showed remarkable activity for GPP (C10) as the donor substrate. Two basic residues Lys275 and Lys220 in HlAIPT interact with the β and γ-phosphate of ATP. By contrast, the interactions are absent in Agrobacterium AIPT because they are replaced by the acidic residues Asp221 and Asp171. Despite its structural similarity to the yeast tRNA-IPT, HlAIPT has evolved with a different binding strategy for adenylate. Finally, by screening a series of dinucleotide polyphosphates, it was found, surprisingly, that the binding affinities of some diadenosine polyphosphates (A(p)4A, A(p)5A and A(p)6A) are even higher than those of the original substrates (ATP and ADP). These results may imply that novel substrates of HlAIPT have been found in plant. 王惠鈞 2010 學位論文 ; thesis 92 en_US |
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博士 === 臺灣大學 === 生化科學研究所 === 98 === Cytokinins are important plant hormones, and their biosynthesis most begins with the transfer of isopentenyl group from dimethylallyl diphosphate (DMAPP) to the N6-amino group of adenine by either adenylate isopentenyltransferase (AIPT) or tRNA-IPT. Plant AIPTs use ATP/ADP as an isopentenyl acceptor and bacterial AIPTs prefer AMP, whereas tRNA-IPTs act on specific sites of tRNA. The crystal structure of an AIPT-ATP complex from Humulus lupulus (HlAIPT) presented here is similar to the previous structures of Agrobacterium AIPT and yeast tRNA-IPT. The enzyme is structurally homologous to the NTP-binding kinase family of proteins but forms a solvent-accessible channel that binds to the donor substrate DMAPP, which is directed toward the acceptor substrate ATP/ADP. When measured with isothermal titration calorimetry, some nucleotides displayed different binding affinities to HlAIPT with an order of ATP > dATP ~ ADP > GTP > CTP > UTP. However, further measurements of activity using these nucleotides showed that the order becomes ATP > ADP > dATP > CTP >> GTP ≈ UTP ≈ 0. In addition to DMAPP (C5), HlAIPT also showed remarkable activity for GPP (C10) as the donor substrate. Two basic residues Lys275 and Lys220 in HlAIPT interact with the β and γ-phosphate of ATP. By contrast, the interactions are absent in Agrobacterium AIPT because they are replaced by the acidic residues Asp221 and Asp171. Despite its structural similarity to the yeast tRNA-IPT, HlAIPT has evolved with a different binding strategy for adenylate. Finally, by screening a series of dinucleotide polyphosphates, it was found, surprisingly, that the binding affinities of some diadenosine polyphosphates (A(p)4A, A(p)5A and A(p)6A) are even higher than those of the original substrates (ATP and ADP). These results may imply that novel substrates of HlAIPT have been found in plant.
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author2 |
王惠鈞 |
author_facet |
王惠鈞 Hsing-Mao Chu 朱鑫懋 |
author |
Hsing-Mao Chu 朱鑫懋 |
spellingShingle |
Hsing-Mao Chu 朱鑫懋 Understanding the pivotal step in cytokinin synthesis: crystal structure, substrate specificity and catalytic mechanism of adenylate isopentenyltransferase from H. lupulus |
author_sort |
Hsing-Mao Chu |
title |
Understanding the pivotal step in cytokinin synthesis: crystal structure, substrate specificity and catalytic mechanism of adenylate isopentenyltransferase from H. lupulus |
title_short |
Understanding the pivotal step in cytokinin synthesis: crystal structure, substrate specificity and catalytic mechanism of adenylate isopentenyltransferase from H. lupulus |
title_full |
Understanding the pivotal step in cytokinin synthesis: crystal structure, substrate specificity and catalytic mechanism of adenylate isopentenyltransferase from H. lupulus |
title_fullStr |
Understanding the pivotal step in cytokinin synthesis: crystal structure, substrate specificity and catalytic mechanism of adenylate isopentenyltransferase from H. lupulus |
title_full_unstemmed |
Understanding the pivotal step in cytokinin synthesis: crystal structure, substrate specificity and catalytic mechanism of adenylate isopentenyltransferase from H. lupulus |
title_sort |
understanding the pivotal step in cytokinin synthesis: crystal structure, substrate specificity and catalytic mechanism of adenylate isopentenyltransferase from h. lupulus |
publishDate |
2010 |
url |
http://ndltd.ncl.edu.tw/handle/72718540522522540915 |
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