Enzymes involved in the anaerobic degradation of phthalates

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Ortho-phthalic acid esters are annually produced in the million tonscale and are classified as environmentally relevant xenobiotics. Theirbiodegradation by microorganisms is generally initiated by theenzymatic hydrolysis to alcohols and o-phthalate. The furtheranaerobic biodegradation of o-phthalate in denitrifying or sulfatereducing bacteria has mainly been studied by the applicant andinvolves (i) the activation of o-phthalate by transferases or ATPdependentligases to the extremely unstable phthaloyl-CoA, followedby (ii) the decarboxylation of the latter by a benzoyl-CoA-forming,oxygen-labile enzyme. In the preceding project we have isolated andpreliminarily characterized the phthaloyl-CoA forming anddecarboxylating enzymes and identified that their balanced synthesisis crucial for the capture of the instable phthaloyl-CoA intermediate.The decarboxylase belongs to the UbiD enzyme family and containsan only recently identified active site prenylated flavin cofactor.Synthesis of the latter requires the prenyltransferase UbiX and adimethylallyl-monophosphate (DMAP) forming hydrolase. In theproposed project we aim to understand the catalytic function andmaturation of the key enzyme of anaerobic phthalate degradation,phthaloyl-CoA decarboxylase, on the molecular level. Structural andkinetic studies are planned to elucidate the unknown mechanism ofphthaloyl-CoA decarboxylase. By the use of the heterologouslyproduced UbiX and the DMAP forming enzymes, we attempt toestablish a general one-step in vitro reconstitution procedure forphthaloyl-CoA decarboxylases and other UbiD enzyme familymembers. Finally we aim to use the phthalate-degrading denitrifyingThauera chlorobenzoica as in vivo production platform forbiochemically inaccessible UbiD-like (de)carboxylases. Theanticipated results not only will shed light on the enzymology involvedin an only recently evolved xenobiotic degradation pathway, but willalso open the door for the production of biotechnologically relevant(de)carboxylases of the UbiD-enzyme family.