Enzymes involved in the anaerobic degradation of phthalates

邻苯二甲酸酯的年产量达到百万吨，被归类为与环境有关的外源药物。微生物对它们的生物降解通常是由酶水解成醇和邻苯二甲酸酯引起的。在反硝化或硫酸盐还原细菌中邻苯二甲酸酯的进一步厌氧生物降解主要由申请人研究，涉及(i)通过转移酶或atp依赖连接酶将邻苯二甲酸酯激活为极不稳定的邻苯二甲酸辅酶a，然后（ii）由苯甲酰辅酶a形成的氧不稳定酶将后者脱羧。在之前的项目中，我们已经分离并初步表征了苯二甲酰辅酶a形成和脱羧酶，并确定了它们的平衡合成对于捕获不稳定的苯二甲酰辅酶a中间体至关重要。该脱羧酶属于UbiD酶家族，含有一种最近才发现的活性位点戊酰化黄素辅因子。后者的合成需要戊烯基转移酶UbiX和二甲基烯丙基单磷酸（DMAP）形成水解酶。在这个项目中，我们的目标是在分子水平上了解厌氧邻苯二甲酸酯降解的关键酶，邻苯二甲酸辅酶a脱羧酶的催化功能和成熟。我们计划通过结构和动力学研究来阐明邻苯二甲酰辅酶a脱羧酶的未知机制。利用异源产生的UbiX和DMAP形成酶，我们试图建立一个通用的一步体外重组程序，用于邻苯二甲酰辅酶a脱羧酶和其他UbiD酶家族成员。最后，我们的目标是利用邻苯二甲酸酯降解反硝化的绿藻作为生物化学上不可接近的ubd样羧化酶的体内生产平台。预期的结果不仅将阐明最近进化的外源降解途径所涉及的酶学，而且还将为生产生物技术相关的ubid酶家族（去）羧化酶打开大门。

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.