[1]王昱堃,魏弘宜,陆海*. 毛果杨腺苷酸合成酶基因ACS-1的克隆和酶学分析[J].西北林学院学报,2014,29(05):78-83.[doi:doi:10.3969/j.issn.1001-7461.2014.05.14]
　WANG Yu-kun,WEI Hong-yi,LU Hai*. Cloning and Enzymatic Analysis of ACS-1 from Adenylate-forming Enzymes Superfamily in Populus trichocarpa Torr. & Gray[J].JOURNAL OF NORTHWEST FORESTRY UNIVERSITY,2014,29(05):78-83.[doi:doi:10.3969/j.issn.1001-7461.2014.05.14]
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 毛果杨腺苷酸合成酶基因ACS-1的克隆和酶学分析()
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《西北林学院学报》[ISSN:1001-7461/CN:61-1202/S]

卷:
第29卷
期数:
2014年05期
页码:
78-83
栏目:
出版日期:
2014-09-30

文章信息/Info

Title:
 Cloning and Enzymatic Analysis of ACS-1 from Adenylate-forming Enzymes Superfamily in Populus trichocarpa Torr. & Gray
文章编号:
1001-7461(2014)05-0078-06
作者:
 王昱堃; 魏弘宜; 陆海*
 （北京林业大学 生物科学与技术学院，北京 100083）
Author(s):
 WANG Yu-kun; WEI Hong-yi; LU Hai*
 (Biological Science and Technology, Beijing Forestry University, Beijing 100083, China)
关键词:
 ; 毛果杨; ACS; 4CL; 原核表达; 酶活性
Keywords:
 ; Populus trichocarpa Torr. &;  Gray;  ACS;  4CL;  prokaryotic expression;  enzymatic activity
分类号:
S718.43
DOI:
doi:10.3969/j.issn.1001-7461.2014.05.14
文献标志码:
A
摘要:
 乙酰辅酶A连接酶(ACS)属于腺苷酸合成酶超基因家族，与4-香豆酸辅酶A连接酶(4CL)有着紧密的进化关系。通过对毛果杨数据库中4CL基因的同源检索，克隆获得了毛果杨基因Prt4CL8的序列(命名ACS-1，Genebank登录号XM_002329288.1)。序列分析表明，ACS-1具有ACS的关键残基以及C端末尾的PTS1序列；而4CL的高度保守结构域box Ⅰ和box Ⅱ在该蛋白中不保守。利用PCR技术克隆序列，构建表达载体pET30a，转化大肠杆菌并表达重组蛋白。对其进行酶学活性分析，结果证明该蛋白不能催化4CL常见底物，而对ACS底物乙酸钠具有明显催化活性：Km为(0.25±0.02)mmol·L-1；Vm为(8.63±0.63)mmol·L-1。结果表明，ACS-1是ACS家族成员，为腺苷酸合成酶超基因家族成员的鉴定与功能分析提供了一定的理论基础。
Abstract:
 Acetyl-CoA synthase (ACS) family is a subfamily of adenylate-forming enzymes superfamily, which has a close evolutionary relationship with 4-coumarate: CoA ligase (4CL) family. We cloned Prt4CL8 (renamed ACS-1, Genebank：XM_002329288.1) via BLAST in the database JGI of Populus trichocarpa. Sequence analysis showed that ACS-1 had the key residues of ACS and PTS1 at the end of C-terminal sequence. While the 4CL conservative domains which called box I and box II of ACS-1 weren’t similar with the other 4CLs. The ACS-1 gene was isolated by PCR and transformed into Escherichia coli by the individual expression construct pET30a and purified the recombinant protein. Enzymatic analysis showed that the recombinant protein had none catalytic activity to the substrates of 4CL, while had remarkable catalytic activity to sodium acetate, the substrates of ACS. As Km was (0.25±0.02) mmol·L-1 and Vm was (8.63±0.63) mmol·L-1. The Results demonstrated that ACS-1 was one of the ACS families, and supplied theoretical foundation in identifying and functional analysis of members from adenylate-forming enzymes superfamily.

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备注/Memo

备注/Memo:
 收稿日期：2014-01-03修回日期：2014-03-07
基金项目：国家自然科学基金(31170574，30671697)。
作者简介：王昱堃，女，在读硕士，研究方向：植物生物化学与分子生物学。E-mail：abby777903@163.com
*通信作者：陆海，男，教授，研究方向：植物生物化学与分子生物学。E-mail：luhai1974@bjfu.edu.cn
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