Zylstra G J, Gibson D T
Department of Microbiology, University of Iowa, Iowa City 52242.
J Biol Chem. 1989 Sep 5;264(25):14940-6.
The nucleotide sequence of the todC1C2BADE genes which encode the first three enzymes in the catabolism of toluene by Pseudomonas putida F1 was determined. The genes encode the three components of the toluene dioxygenase enzyme system: reductaseTOL (todA), ferredoxinTOL (todB), and the two subunits of the terminal dioxygenase (todC1C2); (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase (todD); and 3-methylcatechol 2,3-dioxygenase (todE). Knowledge of the nucleotide sequence of the tod genes was used to construct clones of Escherichia coli JM109 that overproduce toluene dioxygenase (JM109(pDT-601]; toluene dioxygenase and (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase (JM109(pDTG602]; and toluene dioxygenase, (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase, and 3-methylcatechol 2,3-dioxygenase (JM109(pDTG603]. The overexpression of the tod-C1C2BADE gene products was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The three E. coli JM109 strains harboring the plasmids pDTG601, pDTG602, and pDTG603, after induction with isopropyl-beta-D-thiogalactopyranoside, oxidized toluene to (+)-cis-(1S, 2R)-dihydroxy-3-methylcyclohexa-3,5-diene, 3-methylcatechol, and 2-hydroxy-6-oxo-2,4-heptadienoate, respectively. The tod-C1C2BAD genes show significant homology to the reported nucleotide sequence for benzene dioxygenase and cis-1,2-dihydroxycyclohexa-3,5-diene dehydrogenase from P. putida 136R-3 (Irie, S., Doi, S., Yorifuji, T., Takagi, M., and Yano, K. (1987) J. Bacteriol. 169, 5174-5179). In addition, significant homology was observed between the nucleotide sequences for the todDE genes and the sequences reported for cis-1,2-dihydroxy-6-phenylcyclohexa-3,5-diene dehydrogenase and 2,3-dihydroxybiphenyl-1,2-dioxygenase from Pseudomonas pseudoalcaligenes KF707 (Furukawa, K., Arimura, N., and Miyazaki, T. (1987) J. Bacteriol. 169, 427-429).
测定了恶臭假单胞菌F1中编码甲苯分解代谢前三种酶的todC1C2BADE基因的核苷酸序列。这些基因编码甲苯双加氧酶系统的三个组分：还原酶TOL（todA）、铁氧化还原蛋白TOL（todB）以及末端双加氧酶的两个亚基（todC1C2）；（+）-顺式-（1S，2R）-二羟基-3-甲基环己-3,5-二烯脱氢酶（todD）；以及3-甲基儿茶酚2,3-双加氧酶（todE）。利用tod基因的核苷酸序列知识构建了过量产生甲苯双加氧酶的大肠杆菌JM109克隆（JM109(pDT - 601]）；甲苯双加氧酶和（+）-顺式-（1S，2R）-二羟基-3-甲基环己-3,5-二烯脱氢酶（JM109(pDTG602]）；以及甲苯双加氧酶、（+）-顺式-（1S，2R）-二羟基-3-甲基环己-3,5-二烯脱氢酶和3-甲基儿茶酚2,3-双加氧酶（JM109(pDTG603]）。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳检测到tod - C1C2BADE基因产物的过表达。用异丙基-β-D-硫代半乳糖苷诱导后，携带质粒pDTG601、pDTG602和pDTG603的三株大肠杆菌JM109菌株分别将甲苯氧化为（+）-顺式-（1S，2R）-二羟基-3-甲基环己-3,5-二烯、3-甲基儿茶酚和2-羟基-6-氧代-2,4-庚二烯酸。tod - C1C2BAD基因与恶臭假单胞菌136R - 3中报道的苯双加氧酶和顺式-1,2-二羟基环己-3,5-二烯脱氢酶的核苷酸序列具有显著同源性（Irie，S.，Doi，S.，Yorifuji，T.，Takagi，M.，和Yano，K.（1987）《细菌学杂志》169，5174 - 5179）。此外，在todDE基因的核苷酸序列与恶臭假单胞菌KF707中报道的顺式-1,2-二羟基-6-苯基环己-3,5-二烯脱氢酶和2,3-二羟基联苯-1,2-双加氧酶的序列之间也观察到显著同源性（Furukawa，K.，Arimura，N.，和Miyazaki，T.（1987）《细菌学杂志》169，427 - 429）。
