Furukawa K, Hirose J, Suyama A, Zaiki T, Hayashida S
Department of Agricultural Chemistry, Kyushu University, Fukuoka, Japan.
J Bacteriol. 1993 Aug;175(16):5224-32. doi: 10.1128/jb.175.16.5224-5232.1993.
bph operons coding for biphenyl-polychlorinated biphenyl degradation in Pseudomonas pseudoalcaligenes KF707 and Pseudomonas putida KF715 and tod operons coding for toluene-benzene metabolism in P. putida F1 are very similar in gene organization as well as size and homology of the corresponding enzymes (G. J. Zylstra and D. T. Gibson, J. Biol. Chem. 264:14940-14946, 1989; K. Taira, J. Hirose, S. Hayashida, and K. Furukawa, J. Biol. Chem. 267:4844-4853, 1992), despite their discrete substrate ranges for metabolism. The gene components responsible for substrate specificity between the bph and tod operons were investigated. The large subunit of the terminal dioxygenase (encoded by bphA1 and todC1) and the ring meta-cleavage compound hydrolase (bphD and todF) were critical for their discrete metabolic specificities, as shown by the following results. (i) Introduction of todC1C2 (coding for the large and small subunits of the terminal dioxygenase in toluene metabolism) or even only todC1 into biphenyl-utilizing P. pseudoalcaligenes KF707 and P. putida KF715 allowed them to grow on toluene-benzene by coupling with the lower benzoate meta-cleavage pathway. Introduction of the bphD gene (coding for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase) into toluene-utilizing P. putida F1 permitted growth on biphenyl. (ii) With various bph and tod mutant strains, it was shown that enzyme components of ferredoxin (encoded by bphA3 and todB), ferredoxin reductase (bphA4 and todA), and dihydrodiol dehydrogenase (bphB and todD) were complementary with one another. (iii) Escherichia coli cells carrying a hybrid gene cluster of todClbphA2A3A4BC (constructed by replacing bphA1 with todC1) converted toluene to a ring meta-cleavage 2-hydroxy-6-oxo-hepta-2,4-dienoic acid, indicating that TodC1 formed a functional multicomponent dioxygenase associated with BphA2 (a small subunit of the terminal dioxygenase in biphenyl metabolism), BphA3, and BphA4.
在假产碱假单胞菌KF707和恶臭假单胞菌KF715中编码联苯 - 多氯联苯降解的bph操纵子，以及在恶臭假单胞菌F1中编码甲苯 - 苯代谢的tod操纵子，在基因组织以及相应酶的大小和同源性方面非常相似（G. J. 齐尔斯特拉和D. T. 吉布森，《生物化学杂志》264:14940 - 14946，1989；平良健一、广濑纯、林田修和古川和男，《生物化学杂志》267:4844 - 4853，1992），尽管它们的代谢底物范围不同。研究了bph和tod操纵子之间负责底物特异性的基因组成部分。末端双加氧酶的大亚基（由bphA1和todC1编码）和环间位裂解化合物水解酶（bphD和todF）对它们各自不同的代谢特异性至关重要，如下列结果所示。（i）将todC1C2（编码甲苯代谢中末端双加氧酶的大亚基和小亚基）甚至仅将todC1导入利用联苯的假产碱假单胞菌KF707和恶臭假单胞菌KF715中，使它们能够通过与较低的苯甲酸间位裂解途径偶联而在甲苯 - 苯上生长。将bphD基因（编码2 - 羟基 - 6 - 氧代 - 6 - 苯基己 - 2,4 - 二烯酸水解酶）导入利用甲苯的恶臭假单胞菌F1中，使其能够在联苯上生长。（ii）利用各种bph和tod突变菌株表明，铁氧化还原蛋白（由bphA3和todb编码）、铁氧化还原蛋白还原酶（bphA4和todA）和二氢二醇脱氢酶（bphB和todD）的酶组分彼此互补。（iii）携带todClbphA2A3A4BC杂交基因簇（通过用todC1替换bphA1构建）的大肠杆菌细胞将甲苯转化为环间位裂解产物2 - 羟基 - 6 - 氧代 - 庚 - 2,4 - 二烯酸，表明TodC1与BphA2（联苯代谢中末端双加氧酶的小亚基）、BphA3和BphA4形成了功能性多组分双加氧酶。
