一种新型酰基转移酶GPAT的克隆、表达与酶学性质研究

doi:10.13560/j.cnki.biotech.bull.1985.2020-1508

摘要/Abstract

摘要：

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K_m = 761.57 μmol/L,最大反应速率V_max= 29 370 μmol/（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。

关键词: 海洋真菌, Phomopsis lithocarpus, 异源表达, 酰基转移酶, 酶学性质, 生物信息学

Abstract:

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（glutamic-pyruvic 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 K_m and V_max of GPAT towards acetyl CoA were 761.57 μmol/L and 29 370 μmol/（mg·min）,respectively. The results may provide molecular biological basis for the elucidation of lithocarpins biosynthesis pathway.

Key words: marine fungus, Phomopsis lithocarpus, heterologous expression, acyltransferase, enzymatic properties, bioinformatics

刘珊, 叶伟, 朱牧孜, 李赛妮, 邓张双, 章卫民. 一种新型酰基转移酶GPAT的克隆、表达与酶学性质研究[J]. 生物技术通报, 2021, 37(11): 257-266.

LIU Shan, YE Wei, ZHU Mu-zi, LI Sai-ni, DENG Zhang-shuang, ZHANG Wei-min. Cloning,Expression and Characterization of a Novel Acyltransferase GPAT[J]. Biotechnology Bulletin, 2021, 37(11): 257-266.

图/表 9

图1 基因g7779的PCR验证 A:基因g7779的克隆;B:菌液PCR验证。M:DNA marker 2K Plus;1:基因g7779;P:阳性对照;2-8:pET28a-g7779阳性单克隆

Fig.1 PCR verification of gene g7779 A:Cloning of gene g7779. B:PCR verification of bacterial solution. M:DNA marker 2K Plus. 1:Gene g7779. P:Positive control. 2-8:Positive monoclonal of pET28a-g7779

图2 蛋白GPAT的纯化与Western-Blot检测 A:蛋白GPAT表达条件的优化,M:Blue Plus Ⅱ protein marker;1、3、5、7、9、11:条件①、②、③、④、⑤、⑥的上清;2、4、6、8、10、12:条件①、②、③、④、⑤、⑥ 的沉淀。B:蛋白GPAT的纯化,M:Blue Plus Ⅱ protein marker;1:未诱导总蛋白;2:诱导后上清总蛋白;3-5:流出液;6:68 mmol/L咪唑洗脱液;7:116 mmol/L 咪唑洗脱液。C:蛋白GPAT的Western-Blot检测,M:EasySee Western marker;1:未诱导总蛋白;2:诱导后上清;3:流出液;4:68 mmol/L咪唑洗脱液;5:116 mmol/L 咪唑洗脱液

Fig.2 Purification and Western-Blot analysis of GPAT A:Optimization of the expression condition of GPAT. M:Blue Plus II protein marker. 1, 3, 5, 7, 9 and 11:Supernatant of condition ①, ②, ③, ④, ⑤ and ⑥. 2, 4, 6, 8, 10 and 12:Sediment of condition ①, ②, ③, ④, ⑤ and ⑥. B:Purification of GPAT. M:Blue Plus Ⅱ protein marker. 1:Uninduced total protein. 2:Total protein of supernatant after induction. 3-5:Binding buffer. 6:68 mmol/L imidazole eluent. 7:116 mmol/L imidazole eluent. C:Western-Blot analysis of GPAT. M:EasySee Western marker. 1:Uninduced total protein. 2:Total protein of supernatant after induction. 3:Binding buffer. 4:68 mmol/L imidazole eluent. 5:116 mmol/L imidazole eluent

图3 蛋白GPAT中代表性肽段的MALDI-TOF结果

Fig.3 MALDI-TOF result of representative peptides in GPAT

图4 蛋白GPAT与质谱检测结果的匹配情况红色部分为可匹配部分

Fig.4 Matching of GPAT with mass spectrometry result The red part is the matching part

图5 GPAT的酶学性质表征 A:GPAT的最适反应温度;B:GPAT的最适反应pH;C:GPAT的热稳定性;D:GPAT的酶动力学

Fig.5 Characterization of enzymatic properties of GPAT A:Optimal reaction temperature of GPAT. B:Optimal reaction pH of GPAT. C:Thermostability of GPAT. D:Enzyme kinetics of GPAT

图6 蛋白GPAT的亲水性/疏水性分析

Fig.6 Hydrophilicity/hydrophobicity analysis of GPAT

图7 蛋白GPAT的二级结构分析竖线从长到短依次为:α-螺旋（蓝色）、延伸链（红色）、β-转角（绿色）和无规则卷曲（紫色）

Fig.7 Secondary structure analysis of GPAT The order of vertical lines from long to short are:α - helix（blue）,extend strand（red）,β - turn（green）and random coil（purple）

图8 蛋白GPAT的磷酸化位点预测

Fig.8 Prediction of phosphorylation sites of GPAT

图9 GPAT在lithocarpins生源途径中的功能推测

Fig.9 Functional inference of GPAT in the biosynthetic pathway of lithocarpins

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