Haigler B E, Suen W C, Spain J C
AL/EQ-OL, Tyndall Air Force Base, Florida 32403-5323, USA.
J Bacteriol. 1996 Oct;178(20):6019-24. doi: 10.1128/jb.178.20.6019-6024.1996.
4-Methyl-5-nitrocatechol (MNC) is an intermediate in the degradation of 2,4-dinitrotoluene by Burkholderia sp. strain DNT. In the presence of NADPH and oxygen, MNC monooxygenase catalyzes the removal of the nitro group from MNC to form 2-hydroxy-5-methylquinone. The gene (dntB) encoding MNC monooxygenase has been previously cloned and characterized. In order to examine the properties of MNC monooxygenase and to compare it with other enzymes, we sequenced the gene encoding the MNC monooxygenase and purified the enzyme from strain DNT. dntB was localized within a 2.2-kb ApaI DNA fragment. Sequence analysis of this fragment revealed an open reading frame of 1,644 bp with an N-terminal amino acid sequence identical to that of purified MNC monooxygenase from strain DNT. Comparison of the derived amino acid sequences with those of other genes showed that DntB contains the highly conserved ADP and flavin adenine dinucleotide (FAD) binding motifs characteristic of flavoprotein hydroxylases. MNC monooxygenase was purified to homogeneity from strain DNT by anion exchange and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein with a molecular weight of 60,200, which is consistent with the size determined from the gene sequence. The native molecular weight determined by gel filtration was 65,000, which indicates that the native enzyme is a monomer. It used either NADH or NADPH as electron donors, and NADPH was the preferred cofactor. The purified enzyme contained 1 mol of FAD per mol of protein, which is also consistent with the detection of an FAD binding motif in the amino acid sequence of DntB. MNC monooxygenase has a narrow substrate specificity. MNC and 4-nitrocatechol are good substrates whereas 3-methyl-4-nitrophenol, 3-methyl-4-nitrocatechol, 4-nitrophenol, 3-nitrophenol, and 4-chlorocatechol were not. These studies suggest that MNC monooxygenase is a flavoprotein that shares some properties with previously studied nitrophenol oxygenases.
4-甲基-5-硝基邻苯二酚（MNC）是伯克霍尔德氏菌属DNT菌株降解2,4-二硝基甲苯过程中的一种中间体。在烟酰胺腺嘌呤二核苷酸磷酸（NADPH）和氧气存在的情况下，MNC单加氧酶催化从MNC上去除硝基，形成2-羟基-5-甲基醌。编码MNC单加氧酶的基因（dntB）先前已被克隆和表征。为了研究MNC单加氧酶的特性并将其与其他酶进行比较，我们对编码MNC单加氧酶的基因进行了测序，并从DNT菌株中纯化了该酶。dntB位于一个2.2 kb的ApaI DNA片段内。对该片段的序列分析揭示了一个1644 bp的开放阅读框，其N端氨基酸序列与从DNT菌株纯化的MNC单加氧酶的序列相同。将推导的氨基酸序列与其他基因的序列进行比较表明，DntB含有黄素蛋白羟化酶特有的高度保守的腺苷二磷酸（ADP）和黄素腺嘌呤二核苷酸（FAD）结合基序。通过阴离子交换和凝胶过滤色谱法从DNT菌株中纯化出了均一的MNC单加氧酶。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示有一条分子量为60200的单一蛋白质条带，这与从基因序列确定的大小一致。通过凝胶过滤测定的天然分子量为65000，这表明天然酶是单体。它可以使用烟酰胺腺嘌呤二核苷酸（NADH）或NADPH作为电子供体，而NADPH是首选的辅因子。纯化的酶每摩尔蛋白质含有1摩尔FAD，这也与在DntB氨基酸序列中检测到的FAD结合基序一致。MNC单加氧酶具有较窄的底物特异性。MNC和4-硝基邻苯二酚是良好的底物，而3-甲基-4-硝基苯酚﹑3-甲基-4-硝基邻苯二酚﹑4-硝基苯酚﹑3-硝基苯酚和4-氯邻苯二酚则不是。这些研究表明，MNC单加氧酶是一种黄素蛋白，与先前研究的硝基苯酚加氧酶具有一些共同特性。
