拟南芥乙酰羟酸合成酶(AHAS)点突变原核表达与活性测定

Expression and Determination of Activity of the Point Mutated Arabidopsis Acetohydroxyacid Synthase

拟南芥乙酰羟酸合成酶(AHAS)参与支链氨基酸合成。为考察AHAS不同结构域对支链氨基酸合成的影响,分别对其大小亚基上特定位点进行点突变后进行原核表达,体外重组后对其全酶活性进行测定,并对其终端产物之一——缬氨酸对AHAS全酶活性的影响进行探讨。结果显示:AHAS小亚基G88D突变将解除其终端产物的反馈抑制作用,而大亚基E305D与E482D的突变降低AHAS全酶活性,且2种不同突变大亚基对AHAS全酶活性影响存在差异。AHAS大亚基E482D突变较E305D突变影响更大。研究结果表明:AHAS大小亚基间存在着相互作用,且大小亚基不同结构域突变对AHAS全酶活性具有不同的影响。

Acetohydroxyacid synthase（AHAS） is involved in the synthesis of branched-chain amino acids （BCAAs） in ArabidoIosis. To investigate the effects of various domains of AHAS on the BCAAs synthesis, the point mutations harboring in the specific sites of the large and small units of AHAS were introduced by site-directed mutagenesis. The mutagened histidine-tagged units of AHAS were expressed individually in the bacterial hosts and the recombinant proteins were purified using Ni heads. The point mutated large and small units were reconstituted in vitro and the activities of holoenzymes were determined. Moreover, the effects of valine,which is one of the final end products of AHAS,on the activities of the mutated hoioen- zymes were also examined. The results showed that the G88D mutation in the small unit of AHAS abol- ished the final end product inhibition and the E305D or E482D mutation in the large unit decreased the activity of AHAS holoenzyme. The two mutations in the large unit displayed difference in the activity of AHAS and the E482D mutation presents the more effects than the E305D on the activity of AHAS. The resuits in this study suggest that the large unit interacts with the small unit in the AHAS and the various do- mains in the units of AHAS exhibit distinct functions.