BACTERIAL METABOLISM OF NAPHTHALENE - CONSTRUCTION AND USE OF RECOMBINANT BACTERIA TO STUDY RING CLEAVAGE OF 1,2-DIHYDROXYNAPHTHALENE AND SUBSEQUENT REACTIONS
BACTERIAL METABOLISM OF NAPHTHALENE - CONSTRUCTION AND USE OF RECOMBINANT BACTERIA TO STUDY RING CLEAVAGE OF 1,2-DIHYDROXYNAPHTHALENE AND SUBSEQUENT REACTIONS
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DOI：
10.1128/jb.174.23.7542-7554.1992
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发表时间：
1992-12-01
影响因子：
3.2
通讯作者：
CHAPMAN, PJ
中科院分区：
文献类型：
--
作者：
EATON, RW;CHAPMAN, PJ
利用从质粒NAH 7中克隆的基因重组细菌，对萘代谢为水杨酸的反应进行了重新研究。当携带编码前三种酶的途径的DNA片段的铜绿假单胞菌PAO 1的完整细胞与萘一起孵育时，它们形成了双加氧酶催化的1，2-二羟基萘环裂解的产物。产物经Sephadex G-25柱层析分离，H-1和C-13核磁共振谱和气相色谱-质谱鉴定为2-羟基苯并吡喃-2-羧酸酯（HCCA）和反式-邻羟基苯亚甲基丙酮酸酯（tHBPA）。通过其特征UV光谱检测到HCCA作为这些孵育混合物中的第一反应产物，该光谱缓慢变化为指示HCCA和tHBPA的平衡混合物的光谱。两种纯化产物的异构化缓慢且自发地发生，得到组成基本相同的平衡混合物。tHBPA也可通过质粒NAH 7编码的异构酶的作用由HCCA形成。将编码该酶的基因nahD克隆在1.95 kb Kpn I-BglII片段上。携带该片段的大肠杆菌JM 109的提取物催化HCCA和tHBPA的快速平衡。tHBPA通过水合和羟醛切割代谢为水杨醛，由1-kb MluI-StuI限制性片段编码的单一酶催化。提出了水合酶-醛缩酶催化反应的机理。将水杨醛脱氢酶基因nahF克隆在2.75-kb BamHI片段上，该片段还携带萘二氢二醇脱氢酶基因nahB。根据对不同克隆编码的酶的鉴定，nah操纵子的基因顺序为p、A、B、F、C、E、D。
The reactions involved in the bacterial metabolism of naphthalene to salicylate have been reinvestigated by using recombinant bacteria carrying genes cloned from plasmid NAH7. When intact cells of Pseudomonas aeruginosa PAO1 carrying DNA fragments encoding the first three enzymes of the pathway were incubated with naphthalene, they formed products of the dioxygenase-catalyzed ring cleavage of 1,2-dihydroxynaphthalene. These products were separated by chromatography on Sephadex G-25 and were identified by H-1 and C-13 nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry as 2-hydroxychromene-2-carboxylate (HCCA) and trans-o-hydroxybenzylidenepyruvate (tHBPA). HCCA was detected as the first reaction product in these incubation mixtures by its characteristic UV spectrum, which slowly changed to a spectrum indicative of an equilibrium mixture of HCCA and tHBPA. Isomerization of either purified product occurred slowly and spontaneously to give an equilibrium mixture of essentially the same composition. tHBPA is also formed from HCCA by the action of an isomerase enzyme encoded by plasmid NAH7. The gene encoding this enzyme, nahD, was cloned on a 1.95-kb KpnI-BglII fragment. Extracts of Escherichia coli JM109 carrying this fragment catalyzed the rapid equilibration of HCCA and tHBPA. Metabolism of tHBPA to salicylaldehyde by hydration and aldol cleavage is catalyzed by a single enzyme encoded by a 1-kb MluI-StuI restriction fragment. A mechanism for the hydratase-aldolase-catalyzed reaction is proposed. The salicylaldehyde dehydrogenase gene, nahF, was cloned on a 2.75-kb BamHI fragment which also carries the naphthalene dihydrodiol dehydrogenase gene, nahB. On the basis of the identification of the enzymes encoded by various clones, the gene order for the nah operon was shown to be p, A, B, F, C, E, D.