嗜盐古菌Haloferax volcanii WFD11中龙胆酸1,2-双加氧酶HagA的研究

Characterization of gentisate 1,2-dioxygenase Hag A from Haloferax volcanii WFD11

【目的】研究嗜盐古菌Haloferax volcanii WFD11菌株以不同芳香酸作为碳源的生长情况;鉴定其通过龙胆酸途径代谢芳香酸过程中的开环酶龙胆酸1,2-双加氧酶的基因,并对其进行生化水平的研究;初步揭示古菌和细菌代谢芳香酸的可能差异。【方法】分别以4 mmol/L的6种不同芳香酸为唯一碳源培养菌株WFD11,利用全自动生长曲线分析仪测定菌株生长情况并绘制生长曲线;利用高效液相色谱检测菌株WFD11代谢3-羟基苯甲酸的中间产物;对菌株WFD11的基因组进行生物信息学分析,寻找潜在的龙胆酸1,2-双加氧酶编码基因,并在Haloferax volcanii H1424中异源表达;通过快速纯化系统(采用Ni2+-NTA亲和层析柱)纯化异源表达的蛋白,以龙胆酸为底物通过紫外分光光度计检测粗酶液和纯化后的龙胆酸1,2-双加氧酶和相关酶学特性;通过实时定量PCR观察hag A的表达类型。【结果】菌株WFD11能以4 mmol/L的3-羟基苯甲酸和3-羟基苯丙酸为唯一碳源和能源生长;高效液相色谱检测证明菌株WFD11通过龙胆酸代谢3-羟基苯甲酸(3HBA);克隆和异源表达了龙胆酸1,2-双加氧酶基因hag A;Hag A粗酶液和纯化蛋白均具龙胆酸1,2-双加氧酶的活性,催化龙胆酸开环生成顺丁二酸单酰丙酮酸;Hag A的龙胆酸1,2-双加氧酶比活力为0.024 8 U/mg,且其活性不依赖于Fe2+;荧光定量PCR实验结果证明hag A是组成型表达。【结论】嗜盐古菌H.volcanii WFD11可能是通过龙胆酸途径代谢芳香酸类物质,为进一步研究古菌和细菌代谢芳香酸的可能差异打下了基础。

[Objective] We studied the growth characteristics of Haloferax volcanii WFD11 cultivated on a number of aromatic acids as carbon sources to reveal the possible difference between bacteria and archaea in aromatic acids degradation. [Methods] The putative gentisate 1,2-dioxygenase gene was cloned and the enzyme heterologously expressed, purified and functionally identified before determining its key enzyme parameters. We measured the growth of//. volcanii WFD11 in halophile growth medium minimal medium containing one of the six aromatic acids （4 mmol/L） separately as sole carbon source. The intermediate from 3-hydroxybenzoic acid catabolism was determined using high performance liquid chromatography. The putative gentisate 1,2-dioxygenase gene, designated hagA, was heterologously expressed in H. volcanii H1424, and HagA was purified by affinity chromatography on an Ni2＋ chelating column of rapid purification system. HagA was functionally identified based on its specific activity to catalyze the ring cleavage of gentisate using ultraviolet spectrophotometer. The expression type of hagA was revealed by real-time PCR. [Results] Strain WFDll could use 4 mmol/L 3-hydroxybenzoic acid （3HBA） and 3-hydroxyphenylpropionic acid （3HPP） as sole carbon source for growth, but not on 4 mmol/L benzoic acid （BA）, 2-hydroxybenzoic acid （2HBA）, 4-hydroxybenzoic acid （4HBA）, 3-phenylpropionic （3PP）. Gentisate was determined as the intermediate during 3HBA catabolism. The cell extract of 11. volcanii WFD11 catalyzed the ring cleavage of gentisate to maleylpyruvate. Purified HagA exhibited a specific activity of 0.024 8 U/mg of gentisate 1,2-dioxygenase, without Fe2＋. Real-time PCR proved that hagA was constitutively expressed in strain WFD 11. [Conclusion] This study provides the basis to further explore the difference between bacteria and archaea in aromatic acids degradation.