一种新型酰基转移酶GPAT的克隆、表达与酶学性质研究

Cloning,Expression and Characterization of a Novel Acyltransferase GPAT

Lithocarpins为分离自海洋真菌Phomopsis lithocarpus的聚酮类新骨架化合物,具有开发成为新型抗肿瘤药物先导化合物的潜力。基于基因组测序和生物信息学分析预测了lithocarpins的生物合成基因簇,对其中的未知功能基因g7779进行扩增,并在大肠杆菌中进行表达,利用镍亲和层析柱进行纯化,通过质谱测序、生物信息学分析及酶活力检测等方法,对纯化的蛋白进行了功能分析。结果表明:基因g7779表达的蛋白是一个兼具较强的酰基转移酶活性和一定的谷丙转氨酶活性的新型双功能酶(glutamic-pyruvic transaminase-acyltransferase,GPAT),纯度达到98.6%。结构分析显示,该蛋白由326个氨基酸残基组成,其分子量为36 kD,其二级结构包括55.52%α-螺旋、29.45%无规则卷曲、9.82%延伸链和5.21%β-转角;酰基转移酶活性分析表明,GPAT对底物乙酰辅酶A的最适反应温度为35℃,当pH为7.0时酶活最高,在40℃时的热稳定性较好,在此温度下处理2 h后,残留酶活力仍大于80%,而在50℃下热稳定性较差,处理30 min后残留酶活力低于10%;其酶动力学常数Km=761.57μmol/L,最大反应速率Vmax=29370μmol/(mg·min)。研究结果将为后续阐明lithocarpins生物合成途径提供分子生物学依据。

Lithocarpins,a new polyketide skeleton compound isolated from marine fungus Phomopsis littocarpus,has the potential to be developed as lead compound of novel antitumor drugs.Based on genome sequencing and bioinformatics analysis,the biosynthetic gene cluster of lithocarpins was predicted.The unknown functional gene g7779 was amplified and successfully expressed in Escherichia coli.The recombinant protein was purified by Ni-NTA affinity chromatography.The function of the purified protein was analyzed by mass spectrometry,bioinformatics analysis combined with enzyme activity detection.The results showed that g7779 encoded a novel bi-functional enzyme(glutamicpyruvic transaminase-acyltransferase,GPAT)with strong acyltransferase activity and certain glutamic pyruvic transaminase activity,its purity was 98.6%.Structural analysis showed that the protein was composed of 326 amino acids with a molecular weight of 36 kD.The secondary structure of GPAT consisted of 55.52%α-helix,29.45%random coil,9.82%extend strand and 5.21%β-turn.The activity of acyltransferase demonstrated that the optimal reaction temperature of GPAT for acetyl CoA was 35℃,and the highest enzyme activity was at pH 7.0.GPAT showed good thermal stability at 40℃,and the residual enzyme activity was>80%after 2 h treatment at this temperature;but the thermal stability was poor at 50℃,and the residual enzyme activity was<10%after 30 min.The values of Km and Vmax of GPAT towards acetyl CoA were 761.57μmol/L and 29370μmol/(mg·min),respectively.The results may provide molecular biological basis for the elucidation of lithocarpins biosynthesis pathway.