Enzymes involved in the anaerobic degradation of phthalates

邻苯二甲酸酯的年产量以百万吨为单位，被归类为与环境有关的外来生物。微生物对它们的生物降解一般是由酶解醇和邻苯二甲酸酯引起的。邻苯二甲酸酯在反硝化或硫酸盐还原细菌中的进一步厌氧生物降解主要是由申请人研究的，包括(I)由转移酶或依赖于ATP的连接酶将邻苯二甲酸酯活化为极不稳定的邻苯二甲酰辅酶A，然后(Ii)后者通过形成苯甲酰辅酶A的不稳定氧的酶进行脱羧基反应。在前面的项目中，我们已经分离并初步表征了邻苯二甲酰辅酶A的形成和脱羧酶，并鉴定了它们的平衡合成对于捕获不稳定的邻苯二甲酰辅酶A中间体是至关重要的。脱羧酶属于UbiD酶家族，只包含最近发现的活性部位预烯基化黄素辅因子。后者的合成需要戊基转移酶UbiX和形成水解酶的二甲基烯丙基单磷酸(DMAP)。在本项目中，我们旨在从分子水平上了解邻苯二甲酸酯厌氧降解的关键酶--邻苯二甲酰辅酶A脱羧酶的催化功能和成熟过程。计划进行结构和动力学研究，以阐明邻苯二甲酰辅酶A脱羧酶的未知机制。通过利用异源产生的UbiX和DMAP形成酶，我们试图建立一个通用的一步法体外重建邻苯二甲酰辅酶A脱羧酶和其他UbiD酶家族成员的程序。最后，我们的目标是使用邻苯二甲酸盐降解脱硝化的氯苯甲酸作为体内生产平台，以生产生化上无法达到的UbiD样羧基酶。预期的结果不仅将阐明一种最近进化的外源降解途径所涉及的酶学，而且还将为生产UbiD-酶家族的生物技术相关(去)羧基酶打开大门。

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.