Enzymes involved in the anaerobic degradation of phthalates

参与邻苯二甲酸盐厌氧降解的酶

• 批准号: 352196571
• 负责人: Professor Dr. Matthias Boll
• 金额: --
• 依托单位: Forschungsschwerpunkt Mikrobiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/352196571?language=en
• 关键词: Enzymes; involved; anaerobic; degradation; phthalates

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.

邻苯二甲酸酯的年产量为百万吨，被列为与环境相关的异生物质。它们的微生物降解通常是通过酶水解成醇类和邻苯二甲酸酯来启动的。申请人主要研究了邻苯二甲酸酯在反硝化细菌或硫酸盐还原细菌中的进一步厌氧生物降解，包括（i）通过转移酶或ATP依赖性连接酶将邻苯二甲酸酯活化为极不稳定的邻苯二甲酰-CoA，然后（ii）通过形成苯甲酰-CoA的氧不稳定酶使后者脱羧。在前一个项目中，我们分离并初步鉴定了邻苯二甲酰辅酶A形成和脱羧酶，并确定它们的平衡合成对于捕获不稳定的邻苯二甲酰辅酶A中间体至关重要。脱羧酶属于UbiD酶家族，含有最近发现的活性位点异戊烯基黄素辅因子。后者的合成需要异戊烯基转移酶UbiX和二甲基烯丙基单磷酸（DMAP）形成水解酶。本课题旨在从分子水平上研究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。结构和动力学研究计划阐明未知的机制的邻苯二甲酰辅酶A脱羧酶。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酯降解菌Thauera chlorobenzoica作为生物化学不可及的UbiD样（de）羧化酶的体内生产平台。预期的结果不仅将阐明在最近才进化的异生物质降解途径中所涉及的酶学，而且还将为UbiD-酶家族的生物技术相关的（去）羧化酶的生产打开大门。