Molecular cloning, expression, and characterization of adenylate isopentenyltransferase from hop (Humulus lupulus L.)
Molecular cloning, expression, and characterization of adenylate isopentenyltransferase from hop (Humulus lupulus L.)
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DOI：
10.1016/j.phytochem.2004.08.006
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发表时间：
2004-09-01
期刊：
影响因子：
3.8
通讯作者：
Abe, I
中科院分区：
文献类型：
--
作者：
Sakano, Y;Okada, Y;Abe, I
从啤酒花（HumuluslupulusL.）使用基于拟南芥AIPT同工酶（AtIPT 1、AtIPT 3、AtIPT 4、AtIPT 5、AtIPT 6、AtIPT 7和AtIPT 8）保守序列的寡核苷酸引物进行RT-PCR。全长cDNA含有一个990 ×的开放阅读框，编码分子量为36，603 Da的329个氨基酸的蛋白质。此外，基因组DNA的DNA测序显示框架中不存在内含子。Southern杂交分析表明，在H. RT-PCR分析表明，该基因在植物的根、茎、叶和球花中均有表达。推导的氨基酸序列与A.拟南芥AtIPT。在大肠杆菌中表达的重组酶催化异戊烯基转移反应，从二甲基烯丙基二磷酸（DMAPP）到腺苷单磷酸（AMP），腺苷二磷酸（ADP）和腺苷三磷酸（ATP）的N-6氨基。相反，其他核苷酸;鸟苷一磷酸（GMP），肌苷一磷酸（IMP），胞嘧啶一磷酸（CMP），尿苷一磷酸（UMP），不被接受为底物。有趣的是，稳态动力学分析表明，ADP和ATP的异戊烯化比AMP更有效，如以前报道的A。拟南芥AtIPT 4.最后，对H.与其它已知的异戊烯基转移酶一样，啤酒花AIPT在N-末端含有推定的ATP/GTP结合基序。一个保守的Asp 62，位于后的ATP/GTP结合基序，与Ala的定点突变导致酶活性的完全丧失。(C)2004爱思唯尔有限公司保留所有权利。
A cDNA encoding adenylate isopentenyltransferase (AIPT) was cloned and sequenced from cones of hop (Humulus lupulus L.) by RT-PCR using oligonucleotide primers based on the conserved sequences of Arabidopsis thaliana AIPT isozymes (AtIPT1, AtIPT3, AtIPT4, AtIPT5, AtIPT6, AtIPT7 and AtIPT8). A full-length cDNA contained a 990-by open reading frame encoding a molecular mass of 36,603 Da protein with 329 amino acids. Further, DNA sequencing of genomic DNA revealed absence of introns in the frame. On Southern blot analysis, a single AIPT gene was detected in H. lupulus, while RT-PCR analyses demonstrated that the gene was equally expressed in almost all tissues in the plant including roots, stems, leaves and cones. The deduced amino acid sequence shares 38-51% identity to those of A. thaliana AtIPTs. A recombinant enzyme expressed in Escherichia coli catalyzed isopentenyl transfer reaction from dimethylallyldiphosphate (DMAPP) to the N-6 amino group of adenosine monophosphate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP), respectively. In contrast, other nucleotides; guanosine monophosphate (GMP), inosine monophosphate (IMP), cytosine monophosphate (CMP), uridine monophosphate (UMP), were not accepted as a substrate. Interestingly, steady-state kinetic analyses revealed that the isopentenylation of ADP and ATP were more efficient than that of AMP as previously reported for A. thaliana AtIPT4. Finally, H. lupulus AIPT contains the putative ATP/GTP binding motif at the N-terminal as in the case of other known isopentenyltransferases. Site-directed mutagenesis of a conserved Asp62, located right after the ATP/GTP binding motif, with Ala resulted in complete loss of enzyme activity. (C) 2004 Elsevier Ltd. All rights reserved.