Luciferase from Vibrio campbellii is more thermostable and binds reduced FMN better than its homologues
Luciferase from Vibrio campbellii is more thermostable and binds reduced FMN better than its homologues
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DOI：
10.1093/jb/mvm155
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发表时间：
2007-10-01
影响因子：
2.7
通讯作者：
Chaiyen, Pimchai
中科院分区：
文献类型：
--
作者：
Suadee, Chutintorn;Nijvipakul, Sarayut;Chaiyen, Pimchai
克隆了一种新的坎贝尔氏弧菌荧光素酶(Lux_VC)，并在大肠杆菌中进行了表达和纯化。虽然Lux_ve的氨基酸序列和催化反应与哈维氏弧菌荧光素酶(Lux_VH)高度相似，但两种酶对还原的FMN(FMNH-)的亲和力不同。在4℃、pH为8的条件下，用停流吸收光谱和荧光光谱研究了Lux_VC和Lux_VH的催化反应。在4℃时测得FMNH-与Lux_VC的结合K-d为1.8u M，而Lux_VH的结合K-d为11µM。LUX_VE的t(1/2)为1020分钟，而LUX_VH的t(1/2)为201分钟(37℃)。FMNH-优异的热稳定性和更紧密的结合使LUX_VC成为比LUX_VH更易于控制的荧光素酶，用于进一步的结构和功能研究，以及更适合某些应用。这里报道的动力学结果揭示了荧光素酶反应的瞬时状态，这是以前没有文献记载的。
A new luciferase from V. campbellii (Lux_Vc) was cloned and expressed in Escherichia coli and purified to homogeneity. Although the amino acid sequences and the catalytic reactions of Lux_Ve are highly similar to those of the luciferase from V. harveyi (Lux_Vh), the two enzymes have different affinities toward reduced FMN (FMNH-). The catalytic reactions of Lux_Vc and Lux Vh were monitored by stopped-flow absorbance and luminescence spectroscopy at 4 degrees C and pH 8. The measured K-d at 4 degrees C for the binding of FMNH- to Lux_Vc was 1.8 mu M whereas to Lux_Vh, it was 11 mu M. Another difference between the two enzymes is that Lux_Vc is more stable than Lux_Vh over a range of temperatures; Lux_Ve has t(1/2) Of 1020 min while Lux_Vh has t(1/2) of 201 min at 37 degrees C. The superior thermostability and tighter binding of FMNH- make Lux_Vc a more tractable luciferase than Lux_Vh for further structural and functional studies, as well as a more suitable enzyme for some applications. The kinetics results reported here reveal transient states in the reaction of luciferase that have not been documented before.