Growth of Pseudomonas putida F1 on styrene requires increased catechol-2,3-dioxygenase activity, not a new hydrolase

Abstract

Pseudomonas putida F1 cannot grow on styrene despite being able to degrade it through the toluene degradation (tod) pathway. Previous work had suggested that this was because TodF, the meta-fission product (MFP) hydrolase, was unable to metabolize the styrene MFP 2-hydroxy-6-vinylhexa-2,4-dienoate. Here we demonstrate via kinetic and growth analyses that the substrate specificity of TodF is not the limiting factor preventing F1 from growing on styrene. Rather, we found that the metabolite 3-vinylcatechol accumulated during styrene metabolism and that micromolar concentrations of this intermediate inactivated TodE, the catechol-2, 3-dioxygenase (C23O) responsible for its cleavage. Analysis of cells growing on styrene suggested that inactivation of TodE and the subsequent accumulation of 3-vinylcatechol resulted in toxicity and cell death. We found that simply overexpressing TodE on a plasmid (pTodE) was all that was necessary to allow F1 to grow on styrene. Similar results were also obtained by expressing a related C23O, DmpB from Pseudomonas sp. CF600, in tandem with its plant-like ferredoxin, DmpQ (pDmpQB). Further analysis revealed that the ability of F1 (pDmpQB) and F1 (pTodE) to grow on styrene correlated with increased C23O activity as well as resistance of the enzyme to 3-vinylcatechol-mediated inactivation. Although TodE inactivation by 3-halocatechols has been studied before, to our knowledge, this is the first published report demonstrating inactivation by a 3-vinylcatechol. Given the ubiquity of catechol intermediates in aromatic hydrocarbon metabolism, our results further demonstrate the importance of C23O inactivation as a determinant of growth substrate specificity.

Strategy of Pseudomonas pseudoalcaligenes C70 for effective degradation of phenol and salicylate

“…The assumption that catechols have an inhibitory effect on C23Os has been confirmed by several groups [ 11 , 50 , 52 ], but the role of HMS in the degradation of aromatic compounds has received little attention. Muñoz et al .…”

“…Catechol degradation intermediates are also able to inactivate the dioxygenases responsible for their cleavage [ 12 , 52 , 56 ]. Cerdan et al .…”

“…For example, a mutant strain of P . putida F1 gained the ability to degrade styrene due to a single base pair mutation in toluene dioxygenase that resulted in attenuated activity of the enzyme and decreased the rate of 3-vinylcatechol production compared to the wt strain [ 52 ]. Another possibility to avoid catechol toxicity is to increase the rate of catechol-consuming reactions through duplication of catechol dioxygenase genes.…”