Junghare Madan, Spiteller Dieter, Schink Bernhard
Konstanz Research School of Chemical Biology, University of Konstanz, Konstanz, D-78457, Germany.
Department of Biology, Microbial Ecology, University of Konstanz, Konstanz, D-78457, Germany.
Environ Microbiol. 2016 Sep;18(9):3175-88. doi: 10.1111/1462-2920.13447. Epub 2016 Aug 3.
The pathway of anaerobic degradation of o-phthalate was studied in the nitrate-reducing bacterium Azoarcus sp. strain PA01. Differential two-dimensional protein gel profiling allowed the identification of specifically induced proteins in o-phthalate-grown compared to benzoate-grown cells. The genes encoding o-phthalate-induced proteins were found in a 9.9 kb gene cluster in the genome of Azoarcus sp. strain PA01. The o-phthalate-induced gene cluster codes for proteins homologous to a dicarboxylic acid transporter, putative CoA-transferases and a UbiD-like decarboxylase that were assigned to be specifically involved in the initial steps of anaerobic o-phthalate degradation. We propose that o-phthalate is first activated to o-phthalyl-CoA by a putative succinyl-CoA-dependent succinyl-CoA:o-phthalate CoA-transferase, and o-phthalyl-CoA is subsequently decarboxylated to benzoyl-CoA by a putative o-phthalyl-CoA decarboxylase. Results from in vitro enzyme assays with cell-free extracts of o-phthalate-grown cells demonstrated the formation of o-phthalyl-CoA from o-phthalate and succinyl-CoA as CoA donor, and its subsequent decarboxylation to benzoyl-CoA. The putative succinyl-CoA:o-phthalate CoA-transferase showed high substrate specificity for o-phthalate and did not accept isophthalate, terephthalate or 3-fluoro-o-phthalate whereas the putative o-phthalyl-CoA decarboxylase converted fluoro-o-phthalyl-CoA to fluoro-benzoyl-CoA. No decarboxylase activity was observed with isophthalyl-CoA or terephthalyl-CoA. Both enzyme activities were oxygen-insensitive and inducible only after growth with o-phthalate. Further degradation of benzoyl-CoA proceeds analogous to the well-established anaerobic benzoyl-CoA degradation pathway of nitrate-reducing bacteria.
在硝酸盐还原菌偶氮弧菌属菌株PA01中研究了邻苯二甲酸的厌氧降解途径。二维差异蛋白质凝胶图谱分析能够鉴定出与苯甲酸培养的细胞相比，在邻苯二甲酸培养的细胞中特异性诱导表达的蛋白质。编码邻苯二甲酸诱导蛋白的基因存在于偶氮弧菌属菌株PA01基因组中的一个9.9 kb基因簇中。邻苯二甲酸诱导基因簇编码的蛋白质与二羧酸转运蛋白、假定的辅酶A转移酶和一种类泛素D脱羧酶同源，这些蛋白被认为特别参与厌氧邻苯二甲酸降解的初始步骤。我们提出，邻苯二甲酸首先由一种假定的琥珀酰辅酶A依赖性琥珀酰辅酶A：邻苯二甲酸辅酶A转移酶激活为邻苯二甲酰辅酶A，随后邻苯二甲酰辅酶A由一种假定的邻苯二甲酰辅酶A脱羧酶脱羧生成苯甲酰辅酶A。用邻苯二甲酸培养的细胞的无细胞提取物进行的体外酶分析结果表明，以邻苯二甲酸和琥珀酰辅酶A作为辅酶A供体可形成邻苯二甲酰辅酶A，随后其脱羧生成苯甲酰辅酶A。假定的琥珀酰辅酶A：邻苯二甲酸辅酶A转移酶对邻苯二甲酸表现出高底物特异性，不接受间苯二甲酸、对苯二甲酸或3-氟邻苯二甲酸，而假定的邻苯二甲酰辅酶A脱羧酶将氟邻苯二甲酰辅酶A转化为氟苯甲酰辅酶A。未观察到间苯二甲酰辅酶A或对苯二甲酰辅酶A的脱羧酶活性。两种酶活性均对氧不敏感，且仅在以邻苯二甲酸生长后才可诱导。苯甲酰辅酶A的进一步降解类似于硝酸盐还原菌中已确立的厌氧苯甲酰辅酶A降解途径。
