Graduate Program in Environmental Toxicology, Institute for Comparative and Environmental Toxicology, Cornell University, Ithaca, NY 14850, USA.
Department of Microbiology, B53A Wing Hall, Cornell University, Ithaca, NY 14850, USA.
Microbiology (Reading). 2013 Mar;159(Pt 3):621-632. doi: 10.1099/mic.0.062273-0. Epub 2013 Jan 17.
Sphingomonas Ibu-2 has the unusual ability to cleave the acid side chain from the pharmaceutical ibuprofen and related arylacetic acid derivatives to yield corresponding catechols under aerobic conditions via a previously uncharacterized mechanism. Screening a chromosomal library of Ibu-2 DNA in Escherichia coli EPI300 allowed us to identify one fosmid clone (pFOS3G7) that conferred the ability to metabolize ibuprofen to isobutylcatechol. Characterization of pFOS3G7 loss-of-function transposon mutants permitted identification of five ORFs, ipfABDEF, whose predicted amino acid sequences bore similarity to the large and small units of an aromatic dioxygenase (ipfAB), a sterol carrier protein X (SCPx) thiolase (ipfD), a domain of unknown function 35 (DUF35) protein (ipfE) and an aromatic CoA ligase (ipfF). Two additional ORFs, ipfH and ipfI, which encode putative ferredoxin reductase and ferredoxin components of an aromatic dioxygenase system, respectively, were also identified on pFOS3G7. Complementation of a markerless loss-of-function ipfD deletion mutant restored catechol production as did complementation of the ipfF Tn mutant. Expression of subcloned ipfABDEF alone in E. coli did not impart full metabolic activity unless coexpressed with ipfHI. CoA ligation followed by ring oxidation is common to phenylacetic acid pathways. However, the need for a putative SCPx thiolase (IpfD) and DUF35 protein (IpfE) in aerobic arylacetic acid degradation is unprecedented. This work provides preliminary insights into the mechanism behind this novel arylacetic acid-deacylating, catechol-generating activity.
硫单胞菌 Ibu-2 具有独特的能力，可在有氧条件下通过一种以前未被描述的机制，从药物布洛芬和相关芳基乙酸衍生物中切断酸侧链，生成相应的儿茶酚。在大肠杆菌 EPI300 中筛选 Ibu-2 DNA 的染色体文库，使我们能够鉴定出一个能够代谢布洛芬生成异丁基儿茶酚的 fosmid 克隆（pFOS3G7）。pFOS3G7 功能丧失转座子突变体的特征描述允许鉴定出五个 ORF，ipfABDEF，其预测的氨基酸序列与芳香二氧酶的大、小单位（ipfAB）、固醇载体蛋白 X（SCPx）硫解酶（ipfD）、未知功能域 35（DUF35）蛋白（ipfE）和芳香 CoA 连接酶（ipfF）具有相似性。另外两个 ORF，ipfH 和 ipfI，分别编码芳香二氧酶系统的假定铁氧还蛋白还原酶和铁氧还蛋白成分，也在 pFOS3G7 上被鉴定。ipfD 缺失突变体的无标记功能丧失的互补恢复了儿茶酚的产生，ipfF Tn 突变体的互补也恢复了儿茶酚的产生。除非与 ipfHI 共同表达，否则单独表达亚克隆的 ipfABDEF 不会赋予大肠杆菌完全的代谢活性。CoA 连接随后的环氧化是苯乙酸途径所共有的。然而，有氧芳基乙酸降解中需要假定的 SCPx 硫解酶（IpfD）和 DUF35 蛋白（IpfE）是前所未有的。这项工作为这种新型芳基乙酸脱酰基、儿茶酚生成活性的机制提供了初步的见解。
