Characterization of the key enzymes involved in anaerobic degradation of phthalate

Abstract

Anaerobic phthalate degradation was assumed to proceed through initial decarboxylation of phthalate (ortho) to benzoate. However, the intermediates and enzymes involved in anaerobic phthalate decarboxylation were largely unknown. The aim of this dissertation was to investigate the biochemistry of anaerobic phthalate degradation, especially the steps and enzymes involved in the decarboxylation of phthalate to benzoate. In particular, the study focused on the enrichment and isolation of anaerobic phthalate-degrading bacteria under nitrate-reducing, sulfate-reducing and fermenting conditions.Strain PA01 was isolated and purified from an enrichment culture that degrades phthalate coupled to nitrate reduction. 16S rRNA gene sequencing suggested that strain PA01 is a member of the genus Azoarcus that is known for aromatic compound degradation. Azoarcus sp. strain PA01 could degrade a wide variety of aromatic compounds, including phthalate and benzoate coupled to nitrate reduction. No growth was observed with isophthalate or terephthalate. To gain detailed insights into the biochemistry of phthalate degradation, strain PA01 was genome sequenced. The draft genome of strain PA01 possesses the gene clusters for degradation of aromatic compounds, i.e. for benzoate degradation. Differential two-dimensional protein profiling of phthalate- versus benzoate-grown cells identified the specific proteins induced with phthalate.The phthalate-induced protein-coding genes were found to constitute a single gene cluster in the genome of Azoarcus sp. strain PA01. Phthalate-induced proteins included a transporter, two CoA-transferases, and UbiX-like/UbiD-like decarboxylases. It was concluded that o-phthalate is first activated to o-phthalyl-CoA by a succinyl-CoA dependent succinyl-CoA:o-phthalate CoA-transferase (PhtSa and PhtSb), and is subsequently decarboxylated to benzoyl-CoA by an o-phthalyl-CoA decarboxylase (PhtDa and PhtDb). In vitro enzyme assays with cell-free extracts of phthalate-grown cells of Azoarcus sp. strain PA01 demonstrated the formation of o-phthalyl-CoA, specifically with o-phthalate and succinyl-CoA as the CoA donor, and established its subsequent decarboxylation to benzoyl-CoA using LC-MS analysis. Neither free CoA nor acetyl-CoA served as the CoA donor. Isophthalyl-CoA and terephthalyl-CoA were not decarboxylated. Phylogenetic analysis of phthalate-induced PhtSa and PhtSb proteins of strain PA01 revealed that they shared high sequence homology to the known enzyme succinyl-CoA:(R)-benzylsuccinate CoA-transferase involved in toluene activation in denitrifying T. aromatica. PhtDa and PhtDb proteins showed high similarity to the recently discovered enzyme family of UbiD-like and UbiX-like decarboxylases that function in ubiquinone synthesis in a wide range of bacteria.Furthermore, cloning and heterologous expression of the PhtDa and PhtDb proteins in host E. coli verified that these proteins together indeed decarboxylate phthalyl-CoA to benzoyl-CoA. PhtDb is a 8 | P a g e flavin mononucleotide (FMN)-binding protein homologous to known FMN-binding UbiX-like of E. coli, which itself does not possess decarboxylase activity. Instead, it generates a modified-FMN cofactor that is required by PhtDa for decarboxylase activity. Multiple sequence alignments and structure modelling of both proteins suggested that only PhtDb has a binding site for a FMN ligand. This strongly indicates that PhtDb bound FMN plays an essential role in the decarboxylation of o-phthalyl-CoA. Further, it is assumed that FMN functions as a potential electron shuttle between the phthalate ring and the enzyme complex (PhtDa and PhtDb) for facilitating the anaerobic phthalate decarboxylation. Additionally, structural modelling based on known structures of UbiX/UbiD-like enzymes suggested that PhtDb (22 kDa) forms a dodecamer and PhtDa (60 kDa) a homodimer that together build an enzyme complex of about 400 kDa. Native gel analysis of cell-free extract from Azoarcus sp. strain PA01 showed a protein band with an approximate molecular size of 380-400 kDa from which only PhtDa and PhtDb proteins were identified by MS analysis. These results were further supported by native gel analysis of recombinant the PhtDa and PhtDb proteins together which showed a single protein band of molecular size in the same range (380 - 400 kDa).A mixed culture (KOPA) degrading phthalate was enriched under sulfate-reducing conditions. Isolation and purification of bacteria from the mixed culture resulted in the identification of a novel benzoate-degrading bacterium Desulfoprunum benzoelyticum gen. nov., sp. nov. D. benzoelyticum could not degrade phthalate, but bacterial community analysis of the KOPA culture revealed that, it is a predominant bacterium in the enrichment culture. Other potential phthalate degraders include members of the family Desulfobulbaceae. The enrichment culture could also simultaneously be adapted for benzoate utilization, indicating that phthalate degradation occurs through phthalate decarboxylation to benzoate. Addition of 5 mM molybdate completely inhibited phthalate degradation, suggesting that sulfate reducers are key phthalate degrading bacteria in the enrichment. We assumed that in sulfate-reducing/fermenting bacteria, phthalate is activated to phthalyl-CoA by CoA-transferase possibly with acetyl-CoA as CoA donor, analogous to the denitrifying Azoarcus sp. strain PA01 in which succinyl-CoA acts as CoA donor to form phthalyl-CoA which is then decarboxylated to benzoyl-CoA.