Zylstra G J, Olsen R H, Ballou D P
Cellular and Molecular Biology Program, University of Michigan Medical School, Ann Arbor 48109-0620.
J Bacteriol. 1989 Nov;171(11):5907-14. doi: 10.1128/jb.171.11.5907-5914.1989.
The genes for the alpha and beta subunits of the enzyme protocatechuate 3,4-dioxygenase (EC 1.13.11.3) were cloned from the Pseudomonas cepacia DBO1 chromosome on a 9.5-kilobase-pair PstI fragment into the broad-host-range cloning vector pRO2317. The resultant clone was able to complement protocatechuate 3,4-dioxugenase mutations in P. cepacia, Pseudomonas aeruginosa, and Pseudomonas putida. Expression studies showed that the genes were constitutively expressed and subject to catabolite repression in the heterologous host. Since the cloned genes exhibited normal induction patterns when present in P. cepacia DBO1, it was concluded that induction was subject to negative control. Regulatory studies with P. cepacia wild-type and mutant strains showed that protocatechuate 3,4-dioxygenase is induced either by protocatechuate or by beta-carboxymuconate. Further studies of P. cepacia DBO1 showed that p-hydroxybenzoate hydroxylase (EC 1.14.13.2), the preceding enzyme in the pathway, is induced by p-hydroxybenzoate and that beta-carboxymuconate lactonizing enzyme, which catalyzes the reaction following protocatechuate 3,4-dioxygenase, is induced by both p-hydroxybenzoate and beta-ketoadipate.
从洋葱伯克霍尔德菌DBO1染色体上的一个9.5千碱基对的PstI片段中克隆出原儿茶酸3,4 - 双加氧酶（EC 1.13.11.3）的α和β亚基基因，并将其插入广宿主范围的克隆载体pRO2317中。所得克隆能够互补洋葱伯克霍尔德菌、铜绿假单胞菌和恶臭假单胞菌中原儿茶酸3,4 - 双加氧酶的突变。表达研究表明，这些基因在异源宿主中组成性表达并受到分解代谢物阻遏。由于克隆基因在洋葱伯克霍尔德菌DBO1中存在时表现出正常的诱导模式，因此得出诱导受负调控的结论。对洋葱伯克霍尔德菌野生型和突变株的调控研究表明，原儿茶酸3,4 - 双加氧酶可由原儿茶酸或β - 羧基粘康酸诱导。对洋葱伯克霍尔德菌DBO1的进一步研究表明，该途径中的前一种酶对羟基苯甲酸羟化酶（EC 1.14.13.2）由对羟基苯甲酸诱导，而催化原儿茶酸3,4 - 双加氧酶之后反应的β - 羧基粘康酸内酯化酶由对羟基苯甲酸和β - 酮己二酸诱导。
