Bohuslavek J, Payne J W, Liu Y, Bolton H, Xun L
School of Molecular Biosciences, Washington State University, Pullman, Washington 99164, USA.
Appl Environ Microbiol. 2001 Feb;67(2):688-95. doi: 10.1128/AEM.67.2.688-695.2001.
EDTA is a chelating agent, widely used in many industries. Because of its ability to mobilize heavy metals and radionuclides, it can be an environmental pollutant. The EDTA monooxygenases that initiate EDTA degradation have been purified and characterized in bacterial strains BNC1 and DSM 9103. However, the genes encoding the enzymes have not been reported. The EDTA monooxygenase gene was cloned by probing a genomic library of strain BNC1 with a probe generated from the N-terminal amino acid sequence of the monooxygenase. Sequencing of the cloned DNA fragment revealed a gene cluster containing eight genes. Two of the genes, emoA and emoB, were expressed in Escherichia coli, and the gene products, EmoA and EmoB, were purified and characterized. Both experimental data and sequence analysis showed that EmoA is a reduced flavin mononucleotide-utilizing monooxygenase and that EmoB is an NADH:flavin mononucleotide oxidoreductase. The two-enzyme system oxidized EDTA to ethylenediaminediacetate (EDDA) and nitrilotriacetate (NTA) to iminodiacetate (IDA) with the production of glyoxylate. The emoA and emoB genes were cotranscribed when BNC1 cells were grown on EDTA. Other genes in the cluster encoded a hypothetical transport system, a putative regulatory protein, and IDA oxidase that oxidizes IDA and EDDA. We concluded that this gene cluster is responsible for the initial steps of EDTA and NTA degradation.
乙二胺四乙酸（EDTA）是一种螯合剂，广泛应用于许多行业。由于其具有螯合重金属和放射性核素的能力，它可能成为一种环境污染物。已在菌株BNC1和DSM 9103中纯化并鉴定了启动EDTA降解的EDTA单加氧酶。然而，编码这些酶的基因尚未见报道。通过用从单加氧酶N端氨基酸序列生成的探针探测菌株BNC1的基因组文库，克隆了EDTA单加氧酶基因。对克隆的DNA片段进行测序，发现了一个包含八个基因的基因簇。其中两个基因emoA和emoB在大肠杆菌中表达，对基因产物EmoA和EmoB进行了纯化和鉴定。实验数据和序列分析均表明，EmoA是一种利用还原型黄素单核苷酸的单加氧酶，EmoB是一种NADH：黄素单核苷酸氧化还原酶。该双酶系统将EDTA氧化为乙二胺二乙酸（EDDA），将次氮基三乙酸（NTA）氧化为亚氨基二乙酸（IDA），并产生乙醛酸。当BNC1细胞在EDTA上生长时，emoA和emoB基因共转录。该基因簇中的其他基因编码一种假定的转运系统、一种推定的调节蛋白以及氧化IDA和EDDA的IDA氧化酶。我们得出结论，该基因簇负责EDTA和NTA降解的初始步骤。
