Nikodem Patricia, Hecht Volker, Schlömann Michael, Pieper Dietmar H
Department of Environmental Microbiology, GBF-German Research Center for Biotechnology, D-38124 Braunschweig, Germany.
J Bacteriol. 2003 Dec;185(23):6790-800. doi: 10.1128/JB.185.23.6790-6800.2003.
Pseudomonas sp. strain MT1 is capable of degrading 4- and 5-chlorosalicylates via 4-chlorocatechol, 3-chloromuconate, and maleylacetate by a novel pathway. 3-Chloromuconate is transformed by muconate cycloisomerase of MT1 into protoanemonin, a dominant reaction product, as previously shown for other muconate cycloisomerases. However, kinetic data indicate that the muconate cycloisomerase of MT1 is specialized for 3-chloromuconate conversion and is not able to form cis-dienelactone. Protoanemonin is obviously a dead-end product of the pathway. A trans-dienelactone hydrolase (trans-DLH) was induced during growth on chlorosalicylates. Even though the purified enzyme did not act on either 3-chloromuconate or protoanemonin, the presence of muconate cylcoisomerase and trans-DLH together resulted in considerably lower protoanemonin concentrations but larger amounts of maleylacetate formed from 3-chloromuconate than the presence of muconate cycloisomerase alone resulted in. As trans-DLH also acts on 4-fluoromuconolactone, forming maleylacetate, we suggest that this enzyme acts on 4-chloromuconolactone as an intermediate in the muconate cycloisomerase-catalyzed transformation of 3-chloromuconate, thus preventing protoanemonin formation and favoring maleylacetate formation. The maleylacetate formed in this way is reduced by maleylacetate reductase. Chlorosalicylate degradation in MT1 thus occurs by a new pathway consisting of a patchwork of reactions catalyzed by enzymes from the 3-oxoadipate pathway (catechol 1,2-dioxygenase, muconate cycloisomerase) and the chlorocatechol pathway (maleylacetate reductase) and a trans-DLH.
假单胞菌属菌株MT1能够通过一条新途径，经由4-氯儿茶酚、3-氯粘康酸和马来酰乙酸降解4-和5-氯水杨酸酯。如先前对其他粘康酸环异构酶所显示的那样，MT1的粘康酸环异构酶将3-氯粘康酸转化为原白头翁素，这是一种主要反应产物。然而，动力学数据表明，MT1的粘康酸环异构酶专门用于3-氯粘康酸的转化，无法形成顺式二烯内酯。原白头翁素显然是该途径的终产物。在以氯水杨酸酯为生长底物时，诱导产生了一种反式二烯内酯水解酶（trans-DLH）。尽管纯化后的该酶对3-氯粘康酸或原白头翁素均无作用，但粘康酸环异构酶和trans-DLH共同存在时，与单独存在粘康酸环异构酶相比，原白头翁素浓度显著降低，且由3-氯粘康酸形成的马来酰乙酸量更多。由于trans-DLH也作用于4-氟粘康内酯，形成马来酰乙酸，我们认为该酶作用于4-氯粘康内酯，作为粘康酸环异构酶催化3-氯粘康酸转化过程中的中间体，从而防止原白头翁素的形成，促进马来酰乙酸的形成。以这种方式形成的马来酰乙酸被马来酰乙酸还原酶还原。因此，MT1中氯水杨酸酯的降解通过一条新途径发生，该途径由3-氧代己二酸途径（儿茶酚1,2-双加氧酶、粘康酸环异构酶）和氯儿茶酚途径（马来酰乙酸还原酶）的酶催化的一系列反应以及一种trans-DLH拼凑而成。
