嗜碱盐单胞菌X3中异化型硝酸盐还原酶编码基因簇的功能验证及蛋白结构预测

Function Verification and Protein Structure Prediction of Gene Cluster narGYJV Encoding Dissimilatory Nitrate Reductase in Halomonas alkaliphila X3

细菌的氮代谢推动环境的氮循环,硝酸盐还原反应是氮代谢途径中重要步骤之一。本文运用酶活测定、qRTPCR和生物信息学软件分析的方法,对嗜碱盐单胞菌(Halomonas alkaliphila)X3中编码细菌异化型硝酸盐还原酶(Dissimilatory nitrate reductase,Nar)不同亚基的基因簇narGYJV进行了功能验证、蛋白结构预测和系统发育分析。研究表明,X3菌株中存在narGYJV基因簇并具有表达活性,测得Nar的酶活为每克蛋白21.415U;预测到的narGYJV编码蛋白功能区域中1～1246氨基酸属于NarG超家族,编码Nar的α亚基;1261～1752氨基酸属于DMSOR-beta-like超家族,编码Nar的β亚基;2061～2279氨基酸编码γ亚基,1802～2018氨基酸编码δ亚基;Nar的二级结构中无规卷曲占37.59%,α螺旋占34.43%,延伸链占18.33%;NarG、NarY和NarV的3D结构与PDB中大肠杆菌(Escherichia coli)的1Q16 3D结构最接近,覆盖率96%～100%。系统发育树显示,菌株X3中NarG与同属菌的遗传距离较近,在不同菌属间虽然功能相似,其同源性较低;NarY与大肠杆菌中的氨基酸序列的同源关系较近,说明异化型硝酸盐还原酶Nar中不同亚基的系统发育地位不同。研究结果表明,Halomonas alkaliphila X3菌株中存在编码Nar的基因簇narGYJV,编码蛋白具有独特的3D结构,不同亚基的系统发育地位不同。研究结果为进一步研究该菌的氮代谢通路选择和调控机制提供了依据。

The nitrogen metabolism of bacteria promotes the environmental nitrogen cycle,and the nitrate reduction reaction is one of the most important steps in nitrogen metabolism pathway.With enzyme activity assay,qRT-PCR and bioinformatic tools,the functional verification,protein structure prediction and phylogenetic analysis were carried out for the gene cluster narGYJV encoding different subunits of dissimilatory nitrate reductase(Nar)in Halomonas alkaliphila X3.The results showed that the enzyme activity of the dissimilatory nitrate reductase was 21.415 U per gram of protein,and qRT-PCR amplified the target fragments,thus verified the authenticity of the function of narGYJV.It was predicted that 1~1 246 amino acids(aa)of the narGYJV encoding protein belonged to the NarG superfamily and formed theαsubunit of Nar while 1 261~1 752 aa belonged to the DMSOR-beta-like superfamily and formed theβsubunit of Nar,2 061~2 279 aa formed theγsubunit,and 1 802~2 018 aa formed theδsubunit.In the secondary structure of the alfa subunit of Nar,random coil accounted for 37.59%,α-helix accounted for 34.43%and extended chain accounted for 18.33%.The 3D structure of NarG,NarY and NarV were the closest with that of Escherichia coli(1Q16)in PDB with 96%~100%query coverage.The phylogenetic trees showed that the genetic distance of NarG in X3 was closest to those in the samegenus,but far from those in other genus although they possessed the same function.NarY was closely related to that of E.coli in amino acid sequence,but low in similarity.These findings proved that the genetic evolution of the subunits in the NAR of alienated nitrate reductase was different.Our results provided the basis for the next step in studying the nitrogen metabolism pathway selection and regulation mechanism of the bacterium.