Characterization of mutants of a tyrosine ammonia-lyase from Rhodotorula glutinis
Characterization of mutants of a tyrosine ammonia-lyase from Rhodotorula glutinis
复制标题
粘红酵母酪氨酸解氨酶突变体的表征
DOI：
10.1007/s00253-016-7672-8
复制
发表时间：
2016-07
影响因子：
5
通讯作者：
Li Huazhong
中科院分区：
文献类型：
--
作者：
Zhou Shenghu;Liu Peiran;Chen Jian;Du Guocheng;Zhou Jingwen;Li Huazhong
在苯丙烷生产过程中，对香豆酸是最重要的中间代谢物。人们普遍认为，将L酪氨酸转化为p-香豆酸的酪氨酸解氨酶(TAL)的活性代表着限速步骤。因此，采用容易出错的基于随机突变的聚合酶链式反应策略来筛选具有较高催化活性的突变体。经过几轮筛选，获得了三种变异酶，其产率分别为41.2%、37.1%和38.0%。通过定点突变，发现与TAL与L酪氨酸(E518V)亲和力增强、表达水平提高(S9N)、催化效率提高(A11T)相关的突变体是有益的氨基酸替换。结合所有有益的氨基酸取代，获得了催化活性最高的突变体MT-S9N/-A11T/-E518V。MT-S9N/-A11T/-E518V的Km值比野生型降低了25.4%，活性、kcat/Km和p-香豆酸产率分别提高了36.5%、31.2%和65.9%。此外，还对MT-S9N基因5‘端的二级结构和MT-E518V的三维蛋白质结构进行了模拟。因此，推测了两种可能的机制：(1)简化的mRNA5‘端二级结构促进了TAL的表达；(2)锚定柔性环区(Glu325-Arg336)以保持活性部位的口袋开口，确保了L酪氨酸容易进入活性部位，从而提高了p-香豆酸的产量。
In the phenylpropanoid production process, p-coumaric acid is the most important intermediate metabolite. It is generally accepted that the activity of tyrosine ammonia-lyase (TAL), which converts l-tyrosine to p-coumaric acid, represents the rate-limiting step. Therefore, an error-prone PCR-based random mutagenesis strategy was utilized for screening variants with higher catalytic activity. After rounds of screening, three variant enzymes were obtained, exhibiting improved production rates of 41.2, 37.1, and 38.0 %, respectively. Variants associated with increased expression level (S9N), improved catalytic efficiency (A11T), and enhanced affinity between TAL and L-tyrosine (E518V) were identified as beneficial amino acid substitutions by site-directed mutagenesis. Combining all of the beneficial amino acid substitutions, a variant, MT-S9N/-A11T/-E518V, exhibiting the highest catalytic activity was obtained. The Km value of MT-S9N/-A11T/-E518V decreased by 25.4 % compare to that of wild-type, while the activity, kcat/Km, and p-coumaric-acid yield were improved by 36.5, 31.2, and 65.9 %, respectively. Furthermore, the secondary structure of the 5′-end of MT-S9N mRNA and the three-dimensional protein structure of MT-E518V were modeled. Therefore, two potential mechanisms were speculated: (1) a simplified mRNA 5′-end secondary structure promotes TAL expression and (2) anchoring the flexible loop region (Glu325–Arg336) to maintain the active-site pocket opening ensures easy access by the l-tyrosine to the active site and thus improves p-coumaric acid yields.
登录
查看更多内容
影响因子：
3.7
作者：
Wu J;Zhou T;Du G;Zhou J;Chen J
通讯作者：
Chen J
影响因子：
7.8
作者：
Cobb, Ryan E.;Si, Tong;Zhao, Huimin
通讯作者：
Zhao, Huimin
影响因子：
2.9
作者：
Kaufmann, Kristian W.;Lemmon, Gordon H.;DeLuca, Samuel L.;Sheehan, Jonathan H.;Meiler, Jens
通讯作者：
Meiler, Jens
AutoDock4(Zn)：改进的 AutoDock 力场，用于小分子与锌金属蛋白的对接。
影响因子：
5.6
作者：
Santos-Martins D;Forli S;Ramos MJ;Olson AJ
通讯作者：
Olson AJ
影响因子：
4.4
作者：
Jendresen, Christian Bille;Stahlhut, Steen Gustav;Nielsen, Alex Toftgaard
通讯作者：
Nielsen, Alex Toftgaard