Enzymes involved in the anaerobic degradation of phthalates
Biochemistry
Final Report Abstract
Phthalic acid esters (PAEs) are produced annually in the 5-8 million ton scale and are most commonly incorporated as plasticizers in high molecular weight polymers. They are non-covalently bound and can easily leach into the environment during manufacture, use and disposal. Several studies on the potential adverse health effects of phthalates suggest an endocrine disrupting activity. Biodegradation by microorganisms is considered to be the main remediation process. The initial steps of PAE degradation, the hydrolysis to phthalate and alcohols, are identical under oxic and anoxic conditions. In aerobic microorganisms, the biodegradation of phthalate is characterized by ring-hydroxylating or ring-cleaving oxygenases, and hence, the anaerobic degradation of phthalate must follow completely different principles. In 2016, the group of the applicant demonstrated a previously unobserved capacity of numerous well-studied denitrifying model organisms that utilize aromatic compounds in the absence of oxygen. In vitro assays showed the succinyl-CoA-dependent activation of phthalate to phthaloyl-CoA by a highly specific class III CoA transferase (SCPCT), followed by the decarboxylation of phthaloyl-CoA to the central intermediate benzoyl-CoA by a specific phthaloyl-CoA decarboxylase (PCD). The latter was proposed to belong to the UbiD- family of decarboxylases that contain a unique prenylated flavin cofactor (prFMN). Its maturation requires auxiliary enzymes such as a UbiX prenyltransferase, which adds a fourth ring to a reduced FMNH2 cofactor. The main objective of the research project was the first isolation, biochemical, functional and structural characterization of the previously unknown enzymes involved in phthalate degradation in nitrate- and sulphate-respiring anaerobic bacteria. During the project, a prFMN containing PCD and a SCPCT were isolated and biochemically characterized in the phthalate-degrading Thauera chlorobenzoica. A variant of this pathway involving an ATP-dependent phthalate CoA ligase was identified in the sulfate-respiring Desulfosarcina cetonica. Furthermore, a hybrid phthalate degradation pathway was identified in T. chlorobenzoica, which uses the newly identified anaerobic pathway for the conversion of phthalate to benzoyl-CoA in combination with an oxygen-dependent pathway for benzoyl-CoA degradation. These results were summarized in an invited review. In a side project, a novel and highly efficient dibutyl phthalate-degrading organism (Paenarthrobacter species) was isolated and the degradation pathway was elucidated. In collaboration with Uli Ermler (MPI Biophysics, Frankfurt) and Matthias Ullmann (University of Bayreuth) the cryo-EM structures of the 360 kDa PCD were solved at 2.1 Å in the substrate-free, benzoyl-CoA and inhibitor-bound state providing unexpected insights into the mechanism of phthaloyl-CoA decarboxylation at an active site prFMN cofactor. Finally, analysis of the maturation of the prFMN revealed that the prenyltransferase UbiX, which catalyzes prenylation of FMN with dimethylallyl pyrophosphate (DMAPP), and apoPCD form a stable complex in which the precursor of prFMN is delivered from UbiX and inserted into apoPCD followed by a final oxidative maturation step. This part requires some final experiments, which are currently being carried out by master students and a DAAD-funded doctoral researcher. The investigation of other UbiD-like (de)carboxylases was not pursued further, as other groups have started research on this topic.
Publications
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Phthaloyl‐coenzyme A decarboxylase from Thauera chlorobenzoica : the prenylated flavin‐, K + ‐ and Fe 2+ ‐dependent key enzyme of anaerobic phthalate degradation. Environmental Microbiology, 19(9), 3734-3744.
Mergelsberg, Mario; Willistein, Max; Meyer, Heike; Stärk, Hans‐Joachim; Bechtel, Dominique F.; Pierik, Antonio J. & Boll, Matthias
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Evolution of a xenobiotic degradation pathway: formation and capture of the labile phthaloyl‐CoA intermediate during anaerobic phthalate degradation. Molecular Microbiology, 108(6), 614-626.
Mergelsberg, Mario; Egle, Valentin & Boll, Matthias
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Enzymes involved in phthalate degradation in sulphate‐reducing bacteria. Environmental Microbiology, 21(10), 3601–3612.
Geiger, Robin Alexander; Junghare, Madan; Mergelsberg, Mario; Ebenau‐Jehle, Christa; Jesenofsky, Vivien Jill; Jehmlich, Nico; von Bergen, Martin; Schink, Bernhard & Boll, Matthias
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Microbial degradation of phthalates: biochemistry and environmental implications. Environmental Microbiology Reports, 12(1), 3-15.
Boll, Matthias; Geiger, Robin; Junghare, Madan & Schink, Bernhard
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An Aerobic Hybrid Phthalate Degradation Pathway via Phthaloyl-Coenzyme A in Denitrifying Bacteria. Applied and Environmental Microbiology, 86(11).
Ebenau-Jehle, Christa; Soon, Christina I. S. L.; Fuchs, Jonathan; Geiger, Robin & Boll, Matthias
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Degradation of dibutyl phthalate by Paenarthrobacter sp. Shss isolated from Saravan landfill, Hyrcanian Forests, Iran. Biodegradation, 33(1), 59-70.
Shariati, S.; Ebenau-Jehle, C.; Pourbabaee, A. A.; Alikhani, H. A.; Rodriguez-Franco, M.; Agne, M.; Jacoby, M.; Geiger, R.; Shariati, F. & Boll, M.
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Expanding prenylated flavin cofactor catalysis for phthaloyl-coenzyme A decarboxylation, a key reaction of phthalate plasticiser degradation (draft). In: PhD thesis Robin Geiger University of Freiburg.
Kayastha K., Geiger R., Reinhardt J.K., Ullmann M. & Boll, M.
